Analysis of Secreted Enzymes, Metabolism and Virulence in the Bat Fungal Pathogen Pseudogymnoascus Destructans

Analysis of Secreted Enzymes, Metabolism and Virulence in the Bat Fungal Pathogen Pseudogymnoascus destructans

Chapman N. Beekman

B.A., Wheaton College (MA), 2010

Thesis

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Division of Biology of Medicine at Brown University

PROVIDENCE, RHODE ISLAND May, 2019

This dissertation by Chapman N. Beekman is accepted in its present form by the Department of Molecular Biology, Cell Biology and Biochemistry as satisfying the dissertation requirement for the degree of Doctor of Philosophy.

Date______Dr. Richard J. Bennett, Advisor

Recommended to the Graduate Council

Date______Dr. Mark Johnson, Reader

Date______Dr. Alison DeLong, Reader

Date______Dr. Peter Belenky, Reader

Date______Dr. Robert A. Cramer, Jr, Reader

Approved by the Graduate Council

Date______Dr. Andrew Campbell, Dean of the Graduate School

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Acknowledgements

I would not have gotten to this point without the help of many people along the way. Firstly, I would like to thank my advisor Richard Bennett for pushing me to be the best scientist I can be while also allowing me the freedom to pursue my scientific curiosities throughout my PhD. Your guidance, advice and support has been instrumental throughout this project. I would also like to thank my committee members: Alison DeLong, Mark Johnson and Peter Belenky for your advice and perspective throughout our meetings and discussions and for your genuine interest in seeing me succeed. I would also like to thank my former mentors, especially Robert Obar from whom I learned an incredible amount during my time as a research assistant. I am also extremely grateful to Anthony O’Donoghue and Giselle Knudsen, whom have served as collaborators, friends, and mentors during this time. They brought not only their expertise but also a level of energy and positivity that has inspired and motivated me throughout my PhD. I am especially grateful for the time they took to train and mentor me during my visit to their labs. A special thanks goes to Gigi for showing me around San Francisco, Mission street and introducing me to the best burrito I have ever had in my life. Thank you also to the MCB graduate program, Mark, Judith, Elaine, Ashley and Ray as well as the MMI department, Cheny, Sam, Denise and Michelle for all of your work which makes mine and other’s research at Brown possible. I would also like to thank the members of the Bennett lab both past and present as well as others in MMI and MCB who have been great friends and colleagues. Jules Ene especially, who has been a great friend and second mentor throughout my time in lab. I would also like to thank my mentee Eleanor Kim for her work and contribution to this project.

I would also like to thank all my friends who have been a constant source of support and entertainment. I truly feel blessed to have all of you in my life and I sincerely doubt I would have made it through the challenges of graduate school without you. A special thanks to my best man Jeff, our wedding officiants Ben and Nick, and Amy whom have been there for me and Currie since the first year of college at Wheaton, as well as Andy and Lyvia who have always kept a spot for us to crash when visiting Boston …or now Maine. I would also like to thank skateboarding, which as my second (or first) passion has provided me with a physical and creative outlet throughout my PhD and through which I have met many awesome people including Luke and Mason whom have been great friends since elementary school.

I would also like to thank my family for their love and unwavering support throughout my life. My Parents especially, who have always supported me in whatever goal I set forth and have always been willing to listen and help in any way possible with whatever challenges or decisions I am facing. You are also the hardest working people I know and you have been a source of inspiration for me throughout my PhD and my life. Lastly, I would like to thank my wife Currie. Currie, you have been my best friend and partner throughout grad school and well before, and you have helped keep me sane during the most challenging times. You are the first person I come to whether I have good news or bad. You have always been there for me and made me feel incredibly loved like the several times you have stayed with me in the lab until 3 or 4 in the morning just to keep me company while I try and finish an experiment. I wouldn’t have been able to get to this point without you, so thank you!

Table of Contents

Signature Page…………………………………………………………………………………..iii

Acknowledgements……………………………………………………………………………...iv

Table of Contents………………………………………………………………………………...v

List of Figures and Tables………………………………………………………………………ix

CHAPTER 1: INTRODUCTION ...... 1 The threat of emerging fungal pathogens ...... 2 Saprophytic growth and its connection to pathogenicity ...... 3 Chytridiomycosis: a recent example of the destructive capability of invasive fungi ...... 4 Pathogenesis of chytridiomycosis and Snake Fungal disease: devastating cutaneous infections ...... 5 Secreted proteins and virulence in mammalian fungal pathogens ...... 7 Secreted peptidases in mammalian fungal pathogens ...... 11 The emergence and impact of White-nose Syndrome in bats ...... 16 The Pathology of WNS...... 17 The role of host immune responses during WNS ...... 18 Differences in host susceptibility to WNS: resistance vs. tolerance ...... 20 Origins of P. destructans and WNS: An invasive fungal pathogen ...... 22 Morphology, growth and niche-range of P. destructans...... 23 Phylogenic history of P. destructans and cross-species comparisons ...... 24 Putative virulence factors in P. destructans ...... 26 Existing tools to study virulence in P. destructans (this will be quick) ...... 27 Overview ...... 27 Figures ...... 29 References ...... 32 CHAPTER 2: Characterization of PdCP1, a Serine Carboxypeptidase from Pseudogymnoascus destructans, the Causal Agent of White-nose Syndrome ..... 46 Abstract ...... 48 Introduction ...... 49 Results ...... 52

Secretome analysis in P. destructans and related species ...... 52 Recombinant expression and purification of PdCP1 ...... 53 Analysis of PdCP1 substrate specificity ...... 55 Characterization of PdCP1 activity using AMC substrates ...... 56 Discussion ...... 58 Materials and Methods ...... 63 Strains and culture media ...... 63 Secretome generation ...... 63 Generation of recombinant PdCP1 expression constructs ...... 64 Transformation of P. pastoris ...... 64 PdCP1 expression and purification ...... 65 SDS-PAGE ...... 66 Fluorescent peptidase assays ...... 66 MSP-MS assay ...... 67 Mass Spectrometry ...... 67 Acknowledgements ...... 70 Figures ...... 71 Supplementary Figures ...... 78 References ...... 82 CHAPTER 3: Galleria mellonella as an insect model forP. destructans, the cause of White-nose Syndrome in bats ...... 87 Abstract ...... 89 Introduction ...... 90 Results ...... 92 Evaluation of G. mellonella as a suitable host for P. destructans ...... 92 Lethal infections require live spores ...... 93 Virulence of P. destructans is increased by pre-germinating fungal spores ...... 94 Antifungal drug screen with Phenotype Microarray (PM) plates ...... 94 Evaluation of PM drug screen hits ...... 96 Inhibitory compounds can block killing of G. mellonella larvae by P. destructans ...97 Discussion ...... 98 Materials and Methods ...... 103 Strains and culture conditions ...... 103 G. mellonella virulence assays ...... 103 Histology ...... 104

Colony Forming Units (CFUs) in Infected Larvae ...... 104 Phenotype Microarray (PM) screen ...... 105 Comparison of P. destructans and C. albicans PM data ...... 105 Determination of minimum inhibitory concentrations (MICs) ...... 106 Evaluation of compounds for fungicidal activity...... 106 Acknowledgements ...... 106 Funding ...... 106 Figures and Tables ...... 107 References ...... 114 CHAPTER 4: Analysis of Primary Metabolism in P. destructans and Non- Pathogenic Relatives ...... 118 Introduction ...... 119 P. destructans exhibits limited carbon source utilization ...... 120 Deficiencies in carbohydrate metabolism ...... 120 Is P. destructans more metabolically active than non-pathogenic relatives? ...... 123 Is P. destructans more dependent on mitochondrial function than non-pathogenic relatives? ...... 124 Discussion ...... 127 Carbon metabolism and implications for pathogenicity in P. destructans ...... 127 Mitochondrial function in fungi and implications for pathogenicity ...... 129 Methods ...... 130 Strains and culture conditions ...... 130 Biolog Phenotype Microarray for carbon source utilization...... 131 Biolog Dye assays, ETC inhibitors and SOD experiments ...... 131 Figures and Tables ...... 133 References: ...... 138 CHAPTER 5: Discussion ...... 143 Overview ...... 144 Similarities in secretome composition ...... 145 The P. destructans secretome and pathogenicity: is less more? ...... 146 Secreted peptidases: conservation of important virulence factors? ...... 147 Galleria mellonella: A new model system to evaluate virulence in P. destructans . 148 Conclusion ...... 150 Figures ...... 152 References ...... 153

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APPENDIX A: Destructin-1 is a Collagen-Degrading Endopeptidase Secreted by P. destructans, the Causative Agent of White-Nose Syndrome ...... 158 Abstract ...... 160 Significance Statement ...... 161 Introduction ...... 162 Results ...... 164 Discussion ...... 172 Materials and Methods ...... 176 Acknowledgements ...... 185 Figures ...... 187 References ...... 192 Supplementary Figures and Tables ...... 198 Supplementary References ...... 207 APPENDIX B: Supplementary proteomic Tables for Chapter ...... 209 APPENDIX C: Supplementary Data Table for Chapter 3 ...... 227 APPENDIX D: Supplementary Data Tables for Chapter 4 ...... 235 Appendix E: Genetic evaluation of a putative LxrA homolog in Pseudogymnoascus sp...... 245 Results ...... 246 Methods ...... 248 Generation of “LxrA ortholog” expression constructs ...... 248 Transformation of Agrobacterium tumefaciens ...... 248 ATMT transformation of P. destructans ...... 249 Solid medium carbon-source growth assay ...... 249 Figures and Tables ...... 250 References ...... 253

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List of Tables and Figures

CHAPTER 1: INTRODUCTION Figure 1. The growing threat of invasive fungi ...... 29 Figure 2. The geographic range of WNS ...... 30 Figure 3. Schematic tree showing relative relationships of different Pseudogymnoascus species ...... 31 CHAPTER 2: Characterization of PdCP1, a Serine Carboxypeptidase from Pseudogymnoascus destructans, the Causal Agent of White-nose Syndrome Figure 1. LC-MS/MS analysis of secretomes from P. destructans and four non- pathogenic relatives ...... 71 Figure 2: Recombinant expression and purification of PdCP1, a putative serine carboxypeptidase ...... 72 Figure 3: PdCP1 activity vs IQ substrate ...... 73 Figure 4: Characterization of PdCP1 substrate specificity by MSP-MS assay ...... 74 Figure 5: Screening of AMC substrates and characterization of PdCP1 activity ...... 75 Figure 6: Screening peptidase inhibitors against PdCP1 ...... 76 Table 1: A list of putative peptidases detected in the P. destructans secretome ...... 77 Supplementary Figure 1. SDS-PAGE analysis of PdCP1 ...... 78 Supplementary Figure 2. Amino acid alignment of PdCP1 ...... 79 Supplementary Figure 3. Number of detected (MSP-MS) PdCP1-generated cleavages containing the indicated Amino acids at the P1’ position ...... 80 Supplementary Figure 4. MSP-MS analysis of PdCP1 at pH 6.2 ...... 81 CHAPTER 3: Galleria mellonella as an insect model for P. destructans, the cause of White-nose Syndrome in bats Figure 1. Inoculation of G. mellonella larvae with P. destructans spores leads to larval killing in an inoculum- and temperature-dependent manner ...... 107 Figure 2. Effective killing of larvae requires live P. destructans spores ...... 108 Figure 3. Pre-germinated P. destructans spores kill larvae more effectively than non- germinated spores ...... 109 Figure 4. Identification and evaluation of anti-P. destructans compounds ...... 110 Figure 5. Evaluation of anti-P. destructans compounds using the G. mellonella infection model ...... 111 Figure 6. Evaluation of antifungal treatments administered after infection with P. destructans ...... 112

Table 1. Selected anti-P. destructans compounds from PM drug screen...... 113 CHAPTER 4: Analysis of Primary Metabolism in P. destructans and Non- Pathogenic Relatives Figure 1. Carbon source utilization in P. destructans and non-pathogenic relatives . 133 Figure 2. Genomic alignment of putative LxrA ortholog in Pseudogymnoascus species ...... 134 Figure 3. Fungal arabinose and alternative galactose catabolic pathways ...... 135 Figure 4. Carbon source utilization in presence of a metabolic indicator ...... 136 Figure 5. Metabolic features relevant to Biolog dye reduction ...... 137 CHAPTER 5: Discussion Figure 1. Evaluation of peptidase inhibitor treatments and virulence of multiple Pseudogymnoascus species using Galleria mellonella larvae ...... 152 APPENDIX A: Destructin-1 is a Collagen-Degrading Endopeptidase Secreted by P. destructans, the Causative Agent of White-Nose Syndrome Figure 1. Analysis of the secretome of P. destructans...... 187 Figure 2. Peptidase substrate specificity from P. destructans conditioned medium . 188 Figure 3. Purification of a serine S8 peptidase, Destructin-1, from P. destructans conditioned medium...... 189 Figure 4. Characterization of recombinant Destructin-1 activity ...... 190 Figure 5. Inhibition of Destructin-1 reveals the presence of other peptidases in the P. destructans secretome ...... 191 Figure S1. Protein alignment of Destructin-1, -2 and -3 with serine endopeptidases from other fungi ...... 198 Figure S2. Identification of the cleavage sites in IQ8 and IQ12 substrates using MALDI-TOF MS ...... 199 Figure S3. Determination of purity and pH Optimum for Destructin-1 ...... 200 Figure S4. Homology model of Destructin-1 ...... 201 Table S1. Protein Identifications by LC-MS/MS in the P. destructans secretome .... 202 APPENDIX B: Supplementary proteomic Tables for Chapter Supplementary Table 1. Proteins identified in P. destructans secretome by LC-MS/MS ...... 210 Supplementary Table 2. Proteins identified in P. verrucosus secretome by LC-MS/MS ...... 212 Supplementary Table 3. Proteins identified in P. sp. 03VT05 secretome by LC-MS/MS ...... 213 Supplementary Table 4. Proteins identified in P. sp. 05NY08 secretome by LC-MS/MS ...... 215

Supplementary Table 5. Proteins identified in P. sp. WSF3629 secretome by LC- MS/MS ...... 217 Supplementary Table 6. Proteins in P. destructans secretome functionally annotated using PANNZER ...... 218 Supplementary Table 7. Proteins in P. verrucosus secretome functionally annotated using PANNZER ...... 220 Supplementary Table 8. Proteins in P. sp. 03VT05 secretome functionally annotated using PANNZER ...... 221 Supplementary Table 9. Proteins in P. sp. 05NY08 secretome functionally annotated using PANNZER ...... 223 Supplementary Table 10. Proteins in P. sp. WSF3629 secretome functionally annotated using PANNZER ...... 225 APPENDIX C: Supplementary Data Table for Chapter 3 Supplementary Table 1. Biolog chemical inhibitor screen ...... 234 APPENDIX D: Supplementary Data Tables for Chapter 4 Supplementary Table 1. Results of PM carbon source analysis at 13°C (without Biolog Dye)...... 238 Supplementary Table 2. Results of PM carbon source analysis at 18°C (without Biolog Dye)...... 241 Supplementary Table 3. Results of PM carbon source analysis at 13°C with Biolog Dye present ...... 244 APPENDIX E: Genetic evaluation of a putative LxrA homolog in Pseudogymnoascus sp. Figure 1. Cloning strategy for expression of putative LxrA orthologs in P. destructans . . 250

Figure 2. Growth of P. destructans strains transformed with LxrA ortholog constructs . . 251

Abstract

Pseudogymnoascus destructans is the invasive fungal pathogen responsible for White-Nose Syndrome (WNS), a devastating disease affecting North American bats. WNS has led to the loss of approximately 6 million bats over the past decade and is currently threatening at least one species with extinction. While infection by P. destructans has a lethality rate over 90% in some bats, closely related Pseudogymnoascus species are generally non-pathogenic saprophytes. Here I conducted comparative analyses of P. destructans and related fungi to identify differences associated with virulence. As secreted enzymes are important virulence factors in many fungal pathogens, I analyzed the secretomes of several Pseudogymnoascus species using liquid chromatography and tandem mass spectrometry (LC-MS/MS). This revealed features unique to the P. destructans secretome including a predicted peptidase, PdCP1. This enzyme was recombinantly expressed, purified and characterized using fluorescent reporter substrates and an LC-MS/MS-based substrate profiling assay, confirming it acts as a carboxypeptidase with broad specificity. The P. destructans secretome also contained a smaller set of proteins compared to non-pathogens and was lacking enzymes likely important for saprophytic growth. To assess the saprophytic capability of P. destructans, I analyzed its utilization of 190 different carbon sources within the Biolog Phenotype Microarray. These experiments revealed that P. destructans possesses a limited carbon metabolism relative to non-pathogenic Pseudogymnoascus species. P. destructans was unable to use many carbohydrates but utilized peptide and fatty acid carbon sources abundant in host tissue. These results suggest that pathogenicity in P. destructans may be linked to metabolic specialization. I also established a novel infection model using Galleria mellonella larvae. My experiments demonstrate that P. destructans readily kills G. mellonella larvae and that mortality is dependent on a live infection. Additionally, I demonstrate the feasibility of this model for screening chemical inhibitors in vivo. In conjunction with efforts to develop gene-targeting methods, this infection model will also enable future evaluation of secreted enzymes, metabolism and other putative virulence factors in P. destructans. Together these studies shed light on the development of pathogenicity in P. destructans and establish a novel tool for studies going forward.

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CHAPTER 1: INTRODUCTION

The threat of emerging fungal pathogens

Fungi make up one of the most diverse kingdoms of life on earth with as many as

5 million (estimated) extant species 1. Along with this species diversity, fungi occupy a

wide range of ecological niches and lifestyles. Fungi can be found in virtually any habitat on the planet, including marine environments, deserts and artic permafrost as well as in association with plant, animal and microbial hosts 2,3. Individual fungal species often also

display impressive versatility, capable of occupying multiple and disparate environmental

and/or host niches. A large number of fungal species exist primarily as saprophytes,

extracting essential nutrients from dead organic material 2. Yet fungi can also exist as

obligate symbionts, commensals or parasites. Fungal interactions with plants are well

documented, with Fungi serving roles ranging from highly beneficial endosymbionts to

devastating parasites 2. Fungi can also colonize animal species ranging from arthopods4, fish5, and reptiles6 to mammals7. Fungal colonization of animals most often takes the form

of a parasitic relationship and in some cases results in severe disease.

The introduction of fungal species into new ecosystems often results in the

development of new and devastating pathogenic interactions. Fungal pathogens have

demonstrated a fairly unique ability to drive extinctions in their host and are responsible

for 60-70% of all recorded infection-driven extinctions (Figure 1A).8 Invasive fungi are

well-recognized as a threat to agricultural crops as well as endemic plant species. Famous

examples include the loss of millions of Dutch Elm and Chestnut trees in Europe and North

America due to Ophiostoma ulmi and Cryphonectria parasitica, respectively 9,10. Fungi are

also responsible for huge losses to major food crops such as rice, corn and wheat each

year, with associated costs in the billions of dollars 11,12. Recently, fungal animal pathogens have also become recognized as a major threat to global biodiversity (Figure

1B) 8,13. Fungal pathogens have caused recent extinctions or are currently threatening

extinction in various animal species including fish5, reptiles14, amphibians15 and bats.

Saprophytic growth and its connection to pathogenicity

The propensity for fungal pathogens to drive extinction in their hosts is likely a result of multiple factors. However, one crucial factor is the natural versatility of fungi and

their ability to colonize multiple niches. Fungal pathogens of animals are rarely obligate

pathogens and can typically occupy environmental niches outside of the host. Most known animal-infecting fungi also exist as saprophytes in soil, aquatic, and other environments.

Prominent examples include human pathogens, Aspergillus fumigatus16, Cryptococcus neoformans17, and Histoplasma capsulatum18. This ability of fungal pathogens to exist outside of their host can contribute significantly to disease outcomes. Environmental reservoirs serve as sources of infection and reinfection, increasing pathogen load and enabling continued spread even after host killing 8. The ability to grow outside the host

also releases selective pressure for co-evolution towards reduced virulence as these fungi

do not depend on a host for propagation. Accordingly, many fungal pathogens of animals

can maintain extreme levels of virulence, with host mortality rates approaching 100% in

some cases 7,19.

In addition to promoting disease transmission, saprophytic life stages in fungal

pathogens may contribute directly to the development of virulence traits. Selective

pressures in soil and other environmental niches may drive adaptations that can be useful

during host colonization. Growth of A. fumigatus in compost heaps has driven high

thermotolerance which is thought to contribute to its ability to colonize the human host 20.

Competition against soil-associated microbes may also drive virulence-relevant

adaptations in fungi. The cell wall capsule of C. neoformans may have developed to resist

predatory amoebae within the environment, but also enables survival in mammalian

phagocytes 21. Secretion of secondary metabolites from fungi such as gliotoxin, produced

by A. fumigatus may also have developed to help fungi resist competitive microbes in the

environment, but can promote immune-inhibition and tissue damage during host

colonization 20.

Saprophytic growth also requires a high level of metabolic flexibility to efficiently

utilize nutrients as they become available. Fungi are renowned for their production of

secreted enzymes with diverse specificities. These enzymes enable the break-down of a

wide range of biopolymers, releasing cell-diffusible carbon, nitrogen and micronutrient

sources. While important for saprophytic growth, these enzymes can also promote

infection. In addition to breaking down host polymers for energy and growth, secreted

lipases, and peptidases can promote tissue invasion and immune-evasion by damaging

host tissues and immune system components 22. Together, the ability to thrive outside of the host and the extensive collection of potential virulence traits likely play a significant role in the ability of some fungal species to drive host extinctions.

Chytridiomycosis: a recent example of the destructive capability of

invasive fungi

One of the most destructive examples of invasive fungi is seen in the closely-

related chytrid species, Batrachochytrium dendrobatidis and B. salamandrivorans, causal

agents of chytridiomycosis in amphibians. These two fungi have already driven massive

population declines and extinctions in hundreds of species 23,24. First discovered in 1998,

B. dendrobatidis has spread across all 5 continents and is believed to be a major

contributor to an overall decline in amphibians world-wide 25. Currently over a third of

amphibian species are threatened and about 7 percent of species at immediate risk of

extinction 26. It has been estimated that the current rate of amphibian population declines

is ~200 times the historical average 27. While it is believed that human activities have contributed to the spread of chytridiomycosis the mechanism of its global dispersal is not fully understood 28. Recent genomic-based phylogenic analyses suggest a complex

relationship between geographic location and strain background but supports B. dendrobatidis as a recently-introduced invasive pathogen in many locations 29. B. dendrobatidis is considered a generalist pathogen with a wide host range and has been confirmed to colonize at least 500 different amphibian species 30. While the persistence of

B. dendrobatids in the environment remains an open research question, it is known to

spend part of its life cycle outside of the host in the form of zoospores. Phylogenic analyses

demonstrate that B. dendrobatidis and B. salamandrivorans both evolved from a clade of

mostly saprophytic fungi raising the possibility that these pathogens retain the ability for

saprophytic growth 31,32. Mathematical modeling also demonstrates that a saprophytic life

stage may contribute to the pathogen’s ability to drive extinction, though the ability of B.

dendrobatidis to exist as a saprophyte has not been confirmed 33. Environmental

transmission does play a key role in the spread of chytridiomycosis however, as aquatic

zoospores are highly infectious.

Pathogenesis of chytridiomycosis and Snake Fungal disease: devastating

cutaneous infections

Colonization of amphibians by B. dendrobaditis and B. salamandrivorans takes the form of a cutaneous infection, with the fungus invading epidermal layers. Skin invasion leads to severe tissue damage and disruption of the natural epidermal barrier. The amphibian epidermis is crucial for maintaining electrolyte balance and disruption of this

function is believed to be the main cause of death in chytridiomycosis 34. The ability of B. dendrobatidis and B. salamandrivorans to invade cutaneous host tissue and the overall evolution of virulence in these two species has been strongly linked to the production of secreted factors. The genomes of each of these fungal pathogens demonstrate a marked expansion of secreted proteins in comparison to closely related non-pathogenic species

32. Specific gene expansions in B. dendrobatidis and B. salamandrivorans secreted

peptidases and lipases are of particular interest as these classes of enzymes have been

well studied as virulence factors in other fungi. The B. dendrobatidis genome shows particular expansion in serine, aspartyl, and metallopeptidase gene families, indicative of recent gene duplication events 31. Given the evolutionary history of these two pathogens,

these enzymes likely developed to support saprophytic growth, however their specific

expansion in B. dendrobatidis and elevated expression during infection32 suggests they

serve important roles in host colonization. Experimental evidence has also demonstrated

that secretions from B. dendrobatidis are capable of rapidly breaking down intercellular

junctions within amphibian skin. The same study confirmed the presence of multiple

secreted peptidases and lipases within these secretions, supporting a functional role for

these enzymes in pathogenesis 35. Secreted enzymes from B. dendrobatidis may also promote immune evasion during infection. A subtilisin-like serine peptidase isolated from this fungus can rapidly degrade host antimicrobial peptides potentially blocking an important host immune response. Secretions from B. dendrobatidis can also specifically

inhibit lymphocyte proliferation and cause apoptotic death in these cells 36. This ability may explain an observed lack of robust cellular immune responses in infected amphibians 37.

Taken together these findings suggest a crucial role for secreted factors from B.

dendrobatidis and B. salamandrivorans in the pathogenesis of chytridiomycosis by

promoting tissue invasion and immune-evasion.

Ophidiomyces ophiodiicola is the causative agent of snake fungal disease, an

emerging threat to various snake species in N. America. This fungus has been shown to

infect over 30 different species14 and is currently threatening at least 2 species with extinction 38,39. The pathology of snake fungal disease also involves infection and invasion

of host cutaneous tissue in many cases resulting in severe and debilitating deep-seated

lesions. Given that O. ophiodiicola was only confirmed as the casual pathogen of snake

fungal disease in 2015, almost nothing is known regarding molecular virulence factors.

However, O. ophiodiicola does secrete a variety of enzymatic activities including

peptidases which could contribute to the invasion and degradation of host tissue that is

associated with this disease 6.

Secreted proteins and virulence in mammalian fungal pathogens

Elevated body temperature and immune function are thought to limit the

susceptibility of mammals to invasive fungal infections. Accordingly, most human fungal

pathogens primarily impact patients who are immunocompromised and serious fungal infections in healthy individuals are rare. However, once established in a susceptible host, human fungal pathogens can be deadly, killing an estimated 1.5 million people every year with mortality rates as high as 90% for some pathogens / populations40. A small number

of species are responsible for most invasive fungal infections in humans41 and several of these including Aspergillus and Cryptococcus species and H. capsulatum can also exist as saprophytes in the environment. Understanding the traits that enable these species to cause disease and set them apart from strictly saprophytic fungi has been a focus of much research and many factors contributing to virulence in mammalian fungal pathogens have been identified.

In mammalian fungal pathogens, like all fungi, secreted enzymes serve a crucial

function by breaking down extracellular substrates and releasing nutrients to support

growth. However, the position of secreted enzymes at the host-pathogen interface enables

many of them to serve additional roles during infection of the mammalian host. Secreted

proteins are recognized as key virulence factors in many mammalian fungal pathogens

including A. fumigatus42, C. neoformans43, C. albicans44 and H. capsulatum45 where they facilitate adhesion, tissue invasion and immune evasion. In A. fumigatus, colonization of the mouse lung is associated with an upregulation of ~18% of the predicted secretome 46.

Expression of many secreted proteins is also increased in Blastomyces dermatitidis during infection 47. The transition of H. capsulatum from the saprophytic hyphal form to the infectious yeast form is associated with changes in secretome composition as well48.

Together these results suggest that fungal secretome components play important roles

during host colonization. This hypothesis is supported in Paracoccidioides lutzii where

chemical inhibition of protein secretion blocks infection of mammalian cells 49. Likewise, a

genetic screen in C. neoformans identified a number of secreted proteins that are required

for infectivity of the mouse lung 50. Differences in secretome composition may also

contribute to variation in virulence within species as hypervirulent strains of C. gattii/neoformans contain a higher proportion of hydrolytic enzymes 51.

C. neoformans and C. gattii, infect the lungs and central nervous system (CNS) in

immunocompromised humans and many enzymes they secrete serve established

functions in pathogenicity 43. Cryptococcal urease, an enzyme catalyzing the conversion

of urea to ammonia and carbonic acid, contributes to penetration of the blood-brain

barrier52 and also enables lung colonization by altering the host immune response52.

Urease is also required for full virulence in Coccidiodes posadasii, another fungal pathogen causing human lung infections 53. The associated generation of ammonia which

alters tissue pH and illicits cytotoxic effects on host cells is believed to be the primary

mechanism by which urease induces these effects 54. In addition to urease, Cryptococcus

species also secrete large amounts of DNAses, phosphatases and ROS-scavenging

enzymes 43. Extracellular DNAse could function to break down DNA within neutrophil

extracellular traps (NETs), an important host immune response to fungal pathogens 55.

However, the role of DNAses in Cryptococcal virulence has not been directly tested.

Phosphatases secreted by C. neoformans play an important role in pathogenicity by mediating adhesion to host tissue and their deletion significantly reduces virulence in both

G. mellonella and mouse infection models. Extracellular phosphatases are also produced by C. albicans56, C. parapsilosis57 and A. fumigatus58 where they similarly enable adhesion to host cells, suggesting that this function is conserved in mammalian fungal pathogens.

ROS-scavenging redox enzymes are also abundant in the secretomes of mammalian fungal pathogens and protect against host-generated oxygen radicals.

Superoxide dismutase (SOD), which converts toxic superoxide radicals to H2O2 plays a

particularly important role in mediating interactions with host phagocytes. In C.

neoformans, SOD1 is required for full growth within macrophages and virulence in mice

59. Extracellular SOD enzymes from C. albicans60 and H. capsulatum61 also provide

protection against macrophage killing by neutralizing ROS produced during the NADPH oxidase-dependent oxidative burst. Deletion of SOD enzymes in these species similarly increased susceptibility to macrophages, and reduced virulence in mouse infection models, again confirming the importance of ROS resistance during infection. SOD genes are also upregulated in A. fumigatus during infection of the mammalian lung, raising the

possibility that they serve a similar role in this species62. Catalase and thioredoxin two additional ROS-neutralizing enzymes widely present in fungal secretomes42,45,63 can also contribute to virulence in some cases. In H. capsulatum catalase provides resistance to

phagocyte-mediated killing similarly to SOD. Thioredoxin contributes to virulence in C.

albicans64 during systemic infection and in C. neoformans it appears to be essential for

general viability65. Together, these studies demonstrate a conserved role for secreted

redox enzymes in pathogenicity and indicate that resistance to ROS is important for

successful colonization of a mammalian host.

Lipolytic enzymes (lipases and phospholipases) secreted by mammalian fungal pathogens also have established roles in virulence. The break-down of host lipids by these enzymes not only provides a source of energy and carbon, but can promote damage, adherence and invasion of host tissues. Candida albicans possesses a notably large gene family of secreted lipases (LIP1-10) 66. Many of the C. albicans secreted lipases are expressed upon hyphae formation, a morphological transition associated with increased invasiveness in this species 67. Additionally, several are expressed during infection, further supporting a role in pathogenicity. At least one lipase from C. albicans (LIP8) has a confirmed role in virulence as its deletion completely blocks host mortality and greatly reduces fungal burden during systemic infection 68. Secreted phospholipases are also

present in C. albicans and increased phospholipase activity across clinical isolates has

been associated with increased virulence 69. These enzymes, capable of hydrolyzing membrane lipids, contribute to cellular invasion and are required for full virulence during systemic infection by C. albicans 70,71. In C. neoformans Phospholipase B1 (PLB1) enables metabolism of intracellular lipids and survival during phagocytosis by macrophages72 and

also promotes invasion of host tissue and systemic dissemination during lung infection73.

Secreted phospholipase D also contributes to virulence during lung infection by A. fumigatus where it is required for intracellular invasion of epithelial cells 74. Lipolytic

enzymes are also believed to be essential to the biology and virulence of Malassezia

species, specialized fungal pathogens of mammalian skin. Malassezia genomes are

among some of the smallest of any free-living fungi, however, at the same time they show

marked expansion in lipolytic enzymes 75,76. The expansion of lipase genes in Malassezia

is consistent with a noted specialization for lipid-based carbon sources. Malassezia are

also some of the only fungal species known to lack the ability to synthesize fatty acids and

therefore depend on exogenous lipids for growth 75. Their colonization of mammalian skin

is therefore tied to the production of secreted lipases.

Secreted peptidases in mammalian fungal pathogens

Perhaps the most widely recognized class of secreted virulence factors produced by mammalian fungal pathogens are peptidases. Secreted peptidases are ubiquitous throughout the fungal kingdom63 and likely serve important roles in saprophytic fungi as well. However, most niches within the mammalian host are rich in protein, providing the opportunity for secreted peptidases to contribute substantially to pathogenicity. Most mammalian fungal pathogens secrete a variety of peptidases 22,77,78. The prevalence of

secreted peptidases suggests an important role in fungal biology, however the genetic

redundancy of these enzymes in many fungi has also complicated efforts to identify

functions of individual peptidases. Nevertheless, secreted peptidases have been shown

to contribute to pathogenicity of mammalian fungal pathogens in multiple ways including

mediating adhesion, damage and invasion of host tissues and by degradation of host

immune effectors.

Peptidases are enzymes which act to cleave the peptide bonds holding together

large proteins and smaller peptide molecules. While all peptidases serve this same core

function, many types exist and have evolved independent mechanisms for peptide bond

cleavage. Peptidases are first categorized by the amino acid residues / chemical group

within the peptidase structure that directly catalyzes peptide bond cleavage. For example,

prominent peptidase classes found in fungi are serine, aspartyl and metallopeptidases. In

addition, peptidases are also classified by the position of the peptide bond that they cleave

(scissile bond). Peptidases can cleave peptide bonds located at the amino (N) or carboxyl

the largest known class of peptidases79 and share a conserved catalytic triad of Ser, His,

Asp residues that participate directly in cleavage of the scissile bond 80. Peptide bond

catalysis by serine peptidases is initiated by nucleophilic attack of the carbonyl group

adjacent to the scissile bond by the catalytic Ser. Histidine assists by deprotonating and

activating the catalytic Ser and is in turn stabilized by hydrogen bonding with the third

catalytic residue, Asp 81. Catalysis by aspartyl peptidases uses two adjacent Asp residues

which form hydrogen bonds with a water molecule in order to facilitate its nucleophilic attack on the substrate carbonyl group and subsequent peptide cleavage 82.

Metallopeptidases use a variety of amino acids to bind a divalent metal ion. The bound

metal ion can also vary between individual enzymes, however in all cases it is believed to

function by binding and enabling a water molecule to act in the nucleophilic attack of the

substrate carbonyl group to initiate cleavage 83. While all peptidases within a class, such

as serine peptidases share a common catalytic mechanism, substrate specificities can

vary greatly with endo and exopeptidases present in most classes. Peptidases within a

single class can also vary greatly in the preferred amino acid composition of substrates.

Substrate specificity is likely determined by larger structural features including the shape

of the active-site cleft, as well as the composition of auxiliary amino acids close to the

catalytic site capable of interacting with substrate residues 82-84.

In C. albicans, a family of at least 10 different secreted aspartyl endopeptidases

(SAPs) has received much attention. SAPs have been implicated in the degradation of multiple host epithelial proteins85 and in tissue damage during infection of reconstituted

human oral epithelia 86. Furthermore, SAPS are implicated in the degradation of E-

cadherin, an important cell-cell adhesion protein in epithelial cells, and this is function is

associated with tissue invasion in reconstituted human epithelia 87. Adhesion to mammalian keratinocytes is also likely dependent on SAP activity88. In addition to

mediating tissue invasion and damage, SAPs are also able to degrade multiple host

immune proteins including antimicrobial peptides89, IgG90 and complement proteins91. The

degradation of these proteins suggests that SAPs may also contribute significantly to

immune evasion. Consistent with these putative roles during infection, multiple SAP genes

are expressed during human mucosal infection. However, the same study found that

deletion of single and even multiple SAP enzymes results in compensatory upregulation

of other SAPs. Accordingly, few studies have demonstrated a substantial phenotypic

difference upon deletion of a single SAP gene. Most studies, including those described

here have relied on pepstatin, a broad aspartyl peptidase inhibitor, or the deletion of

multiple SAP genes to demonstrate their importance. The genetic and functional

redundancy of the SAP family has likely also complicated efforts to determine their

ultimate requirement for virulence. Two studies have demonstrated that deletion of

SAPs1-392 or SAPs4-693 results in attenuated virulence in different systemic infection models. However additional studies have found conflicting results demonstrating no difference in tissue invasion94 or host killing95 between wild-type and SAP1-3 or 4-6 mutants. Differences in experimental conditions as well as pathogen and host strains used in these studies may have also contributed to their conflicting results. However, while the requirement of SAPs for systemic infection remains controversial, given the various functions for these enzymes in models of mucosal/epithelial infection it is highly likely they make substantial contributions to pathogenicity within this niche.

Secreted peptidases have also been identified in C. neoformans and H.

capsulatum. While much less is known regarding secreted peptidases in these species at

least a few of them have been implicated in virulence-related processes. In H. capsulatum,

multiple peptidase enzymes are secreted during the pathogenic yeast phase 48. At least

one of these, (DPPIVB) is able to digest a host chemokine peptide, implying it could impact

the host immune response 96. A variety of active peptidases are present within the

secretome of C. neoformans97 including serine98, aspartyl99,100 and metallopeptidases101,102. Cryptococcus peptidases are also able to degrade several

different mammalian immune molecules including IgG and complement proteins 97. In addition, clear roles in virulence have been defined for some Cryptococcal secreted peptidases. The metallo endopeptidase Mpr1 is required for full virulence during infection of mouse lung tissue and mediates penetration of the blood brain barrier and brain- dissemination of C. neoformans in this model 102. A secreted aspartyl peptidase from C.

neoformans, May1, is also required for virulence in a mouse infection model 99. Cells

lacking May1 are also unable to grow under acidic conditions or proliferate within

macrophages, providing a potential mechanism by which this peptidase contributes to

virulence.

A. fumigatus also secretes various serine, aspartyl and metallopeptidases with the

ability to degrade host proteins 103,104. Peptidases within the A. fumigatus secretome are

able to induce cellular damage, inflammation105 and disrupt focal adhesions in mammalian epithelial cell, thereby implicating these enzymes in the damage and invasion of host tissues. Alp1, a serine endopeptidase from A. fumigatus is also able to degrade host complement proteins106 drawing a similarity to peptidases from C. albicans and C. neoformans. However, direct evaluation of the contribution of A. fumigatus peptidases to virulence has revealed few conclusive results. This is again at least partially due to genetic

and functional redundancy, as A. fumigatus possesses as many as 100 individual genes

encoding secreted peptidases 107. Alp1 is at least one peptidase required for virulence in

a neutropenic mouse model of Aspergillosis 108. However, even this result is dependent

on the infection model as this enzyme is not required for virulence in non-neutropenic mice

106.

Secreted peptidases are especially important to the biology and pathogenicity of dermatophytes, specialized fungal pathogens of mammalian skin. Dermatophytes including Trichoderma rubrum, Microsporum canis and Arthroderma benhamiae possess gene expansions in multiple secreted protease families76. Proteomic analysis of

dermatophyte secretomes has confirmed the presence of various activities including

serine endo and carboxypeptidases, aspartyl endopeptidases and metallo amino and endopeptidases 109-111. These species are believed to utilize endo and exopeptidases

synergistically to degrade large keratin polymers into short peptides and amino acids for

subsequent internalization and metabolization 77. Subtilisin serine endopeptidases which

are specifically expanded in dermatophytes, have a demonstrated ability to digest keratin

substrates 110. At least one subtilisin gene from A. benhamiae was upregulated during

infection, suggesting these peptidases may be important for host colonization. In addition

to breaking down keratin for growth, multiple subtilisins also contribute to the adherence

of dermatophytes to host tissue 112,113. Together the prevalence and functions of

peptidases in dermatophytes suggest that these enzymes are crucial for the colonization

of mammalian dermal tissue. Interestingly, another group of fungi specialized for growth

on mammalian skin, Malassezia, also possess an expanded gene family of aspartyl

peptidases, though the role of these enzymes during infection has not been established

76.

The emergence and impact of White-nose Syndrome in bats

White-nose Syndrome (WNS) is fungal disease currently threatening bat species across North America. Initial discovery of WNS occurred in 2007 in a bat hibernation site in upstate New York where researchers encountered a sudden and catastrophic loss of bats over the course of a single hibernation season. Where a healthy and numerous bat population had existed, researchers encountered hundreds of carcasses and virtually no surviving bats. Affected bats were observed to possess a characteristic white-fluffy growth around the muzzle for which the disease was subsequently named. This observation along with the presence of white powdery growth in the wings and histological examination of infected tissue implicated a novel fungal species Pseudogymnoascus (formerly

Geomyces) destructans in WNS 7. However a study in 2011 was the first to conclusively demonstrate P. destructans as the causal agent in WNS by experimental infection of naïve bats 114. Since this initial discovery, WNS has proven to be one of the most destructive

and rapidly spreading fungal diseases ever recorded. WNS has now been confirmed in 33

U.S. states and 7 Canadian provinces with its western expansion recently extending as

far as the state of Washington (Figure 2) 115. P. destructans appears to act as a generalist

pathogen of bats and has been isolated from 16 different bat species native to N. America

116. Mortality rates due to WNS vary drastically depending on host species but lethality can

approach 100% for some bats. As a result, the spread of WNS is estimated to have caused

the deaths of over 6 million individual bats to date. One of the most susceptible hosts, the

little brown bat (Myotis lucifugus) was once the most highly populated species in N.

America. However, WNS has driven the most rapid decline ever observed in a wild

population and is now threatening M. lucifugus with regional extinction 117. Following the

discovery of WNS in N. America, P. destructans was also isolated from various bat species in Europe and Asia. Though European and Asian bats seem to show varying levels of

WNS-like symptoms, significant mortality has not been observed. This along with other

evidence (discussed later) strongly indicates that P. destructans in an invasive species

endemic to Eurasia and recently introduced to N. America.

Most bats impacted by WNS are insectivorous and are known to serve a crucial

role as controllers of insect populations. The presence of bats can reduce insect numbers

by as much as 84% in forested regions which in turn greatly supports plant health by

reducing insect herbivory 118,119. The impact of losing these species on their native

ecosystems is therefore likely to be drastic and unpredictable. The loss of bats will likely

also have significant economic impacts via the benefit that insect predation provides to

agriculture. One study in corn demonstrated that exclusion of bats led to a 59% increase

in larval pests and a correlating increase in overall crop damage. This study estimated the

value of bats to corn alone to be as high as $1 billion USD per year 120. An independent analysis estimated the overall value of bats to N. American agriculture to be ~$3.7 billion

USD/year 121. Given the ecological and economic importance of bats, the impacts of WNS

may extend well beyond its host species placing substantial burden on native ecosystems

and even humans.

The Pathology of WNS

Like chytridiomycosis and snake fungal disease, WNS manifests as a cutaneous infection. Upon colonization by P. destructans, fungal hyphae penetrate epidermal layers, with noted colonization of hair follicles and sebaceous glands. Hyphal invasion can reach the basement membrane leading to separation of epidermal and subdermal tissues 122. In

the wing, which is particularly impacted during WNS, P. destructans is able to invade the

collagen and elastin-rich connective tissue layer and hyphae can even pass through the

entire width of the wing membrane 123. Tissue invasion is associated substantial cellular

damage and leads to the formation of ulcerative lesions containing necrotic tissue. Despite

invasive growth in cutaneous tissue, post-mortem examination of bats killed by WNS show

no signs of systemic infection. Due to the important role of batwing tissue in gas exchange and thermoregulation124, it has been hypothesized that wing damage may be the key driver

of mortality in WNS. Physiological studies indicate that wing damage is linked to an

increase in blood CO2 and acidification as well as electrolyte imbalances and increased dehydration due to evaporative water loss, thereby supporting this hypothesis 125,126.

Infected bats seem to respond to tissue damage by increasing their overall metabolic rate

and fat utilization during hibernation 127. Infected bats also demonstrate abnormal behaviors during WNS including increased arousals from torpor and daytime flights and the severity of infection is correlated with the frequency of these arousals 128,129. This

increased energy expenditure is believed to be the ultimate driver of death in WNS as

susceptible bats use up energy stores prematurely during hibernation. This hypothesis is

consistent with observations that bats in terminal stages of WNS appear emaciated and

devoid of fat stores 7.

The role of host immune responses during WNS

As WNS impacts bats strictly during hibernation, the role of the host immune response presents an interesting and highly complex question. The torpor state in hibernating mammals is generally associated with a broad reduction in immune function including lower circulation of neutrophils and lymphocytes, as well as lower antibody and complement levels 130,131. Transcriptomic responses to WNS in M. lucifugus also suggest a lack of lymphocyte and neutrophil activation in wing tissue consistent with observations in other hibernating mammals 132. Therefore, bats in hibernation may be in what is

essentially an immunocompromised state. However, the same study also detected

substantial activation of pro-inflammatory cytokines and upregulation of innate pattern-

recognition receptors (PRRs), indicating that some innate immune responses remain

functional in hibernating bats. Additional studies also support an activation of pro-

inflammatory responses to P. destructans in bats, demonstrating increased transcription

of IL-6, and IL-17a in wing in lymph tissues 133. This pro-inflammatory response appears to be systemic as increased transcripts for IL-23 and TNF-alpha were also elevated in lung tissue from WNS-affected bats 134.

Multiple studies have also indicated an increase in circulating leukocytes during

WNS,125,135 seemingly contradictory to findings in other hibernating mammals (mentioned

above). However, these findings may be partially explained by alterations in hibernation

patterns associated with WNS. Because arousal from torpor is associated with at least

partial restoration in immune function136, the increased arousal frequency observed in bats at terminal stages of WNS may cause an overall increase in cellular immune responses.

In studies reporting an increase in circulating immune cells, many of the bats were in fact in a state of arousal or even at euthermic body temperature at the time of sampling. As immune functions are energetically costly, the increased immune activity associated with arousal may be detrimental to hibernating bats, further accelerating the exhaustion of

energy stores.

However, some evidence suggests that P. destructans may also be capable of

directly inhibiting the recruitment of immune cells. Multiple histological studies have noted

the presence of other superficial fungal and bacterial infections present in both healthy

and WNS-affected M. lucifugus. These minor infections are often associated with local

inflammation and recruitment of neutrophils. The same studies found a striking absence

of inflammation and phagocyte recruitment to regions specifically containing P.

destructans hyphae 123,132,137. This suggests that hibernating bats remain capable of

mounting a cellular immune response against some microbes and that P. destructans may

possess mechanisms to evade or inhibit this response. Yet torpor does also have a clear impact on the response to P. destructans, as WNS-positive bats that survive winter and return to a permanent euthermic body temperature initiate a massive inflammatory response to the infection. This inflammation causes a rapid and severe worsening of tissue damage that may be a secondary cause of mortality 138. This over-active immune response

may be due to a sudden recovery of full immune function and subsequent recognition of

extensive colonization by P. destructans. However, as the euthermic bat body temperature

(~37 °C) is well above that permissible for P. destructans, it is also possible that the sustained euthermic temperature causes fungal lysis and subsequent exposure of immunogenic fungal molecules that are normally hidden.

Differences in host susceptibility to WNS: resistance vs. tolerance

While P. destructans can colonize a wide range of bat species the outcomes vary widely. Bats such as M. lucifugus are highly susceptible to WNS, but other N. American bats show almost no mortality upon contact with P. destructans 139 One such example is the Large Brown Bat (Eptesicus fuscus). Field studies show that E. fuscus populations have not declined since the discovery of WNS and have even expanded at some locations

140. Additionally, E. fuscus is not extensively colonized upon the introduction of P.

destructans, exhibiting a lower pathogen load with fewer and in some cases, none of the necrotic lesions that are associated with WNS 137,140. Together these findings suggest that

E. fuscus possesses a natural resistance to WNS, yet the mechanisms determining this

resistance remain an open research question. While differential immune responses to P.

destructans may play a role, this has yet to be investigated. However, other physiological

and behavioral responses likely contribute to the ability of E. fuscus to resist infection by

P. destructans. One area of focus has been on variations in lipid composition between the

skin of E. fuscus and M. lucifugus. Some evidence suggests that the skin of E. fuscus

contains higher levels of specific fatty acids and other lipid molecules which can inhibit

growth of P. destructans in vitro 141,142. However, the impact of these differences during

infection has not been directly tested and an independent study found that M. lucifugus

and E. fuscus have similar fatty acid profiles 143. Energy stores and metabolic rate during

hibernation may also drive differences in WNS survival. E. fuscus utilizes fat reserves at

a lower rate than M. lucifugus 144. Interestingly, E. fuscus also appears to increase torpor duration in response to P. destructans in contrast to the shortened torpor and increased arousal frequency seen in M. lucifugus.137 As depletion of energy stores is believed to be a major driver of mortality in M. lucifugus, these may in fact be important differences determining overall survival. Behavioral and environmental factors may also contribute to

differences in WNS susceptibility across species that roost in different microclimates as

hibernation sites with higher temperature and relative humidity accelerate disease progression 139,145.

In Europe and Asia where P. destructans is likely endemic, the fungus has also

been shown to colonize a large number of bat species, but without significant host mortality

146. In contrast to the resistance exhibited by E. fuscus, however, several bat species

native to Europe and Russia show pathogen loads similar to those seen in susceptible N.

American bats 147,148. Additionally, bats in Europe and Asia often develop cupping lesions

and tissue damage upon infection. In some European species the number and severity of

WNS-associated lesions matches those seen in N. American bats 149,150. For at least one

European species, Myotis myotis, infection and tissue damage by P. destructans even results in the some of the same physiological impacts thought to drive mortality in M. lucifugus, including a reduction in body mass index, and mild metabolic acidosis 126,151.

However, these physiological impacts appear to be less severe and less sensitive to

pathogen load. These observations indicate that many Eurasian bats have developed

tolerance to infection by P. destructans. The mechanism(s) enabling this tolerance are

unknown, however it is consistent with other evidence indicating P. destructans is native

to Europe and may share significant co-evolutionary history with bats in these regions.

Origins of P. destructans and WNS: An invasive fungal pathogen

Multiple lines of evidence support the theory that P. destructans is a novel and invasive pathogen in N. America originating from Europe. Firstly, all North American isolates of P. destructans fall into the same clonal lineage, descendent from the same isolate responsible for the first recorded WNS outbreak in NY 152. P. destructans has also

been isolated from multiple locations across Europe where it appears to be non-lethal to

bats, indicative of a longer period of co-adaptation with its host (as discussed above).

European isolates of P. destructans also possess a level of genetic diversity that is consistent with a substantial evolutionary history in this location. The North American lineage of P. destructans clusters among European isolates in phylogenomic analyses indicating these isolates are all closely related 153. An extensive study of 71 American and

28 European isolates based on 8 unique genetic loci identified several haplotypes among

European strains, one of which was identical to all American strains tested 154. This

provides strong evidence that the isolate responsible for WNS in N. America originated

from Europe. Importantly, experimental infection with a European isolate of P. destructans

was able to cause disease identical to WNS in a North American bat species confirming the plausibility of the “novel pathogen” theory 155.

Morphology, growth and niche-range of P. destructans

P. destructans is a filamentous Ascomycete fungus of the order Leotiomycetes which contains fungi of diverse morphologies and lifestyles. The growth morphology of P. destructans exhibits branching septate hyphae with characteristically elongated, curved conidia. P. destructans is haploid and its clonal expansion in North America indicates

asexual reproduction is its primary mode of propagation. However, sequencing of the

putative mating type locus demonstrated the presence of a heterothallic mating system

156. While all tested North American isolates of P. destructans share the same mating type,

multiple isolates from Europe have been found to possess the opposing mating type,

raising the possibility of sexual reproduction. However, while European isolates also show

significant genetic variation, studies thus far have found little evidence of recombination,

suggesting mating is a rare occurrence 150,154. P. destructans is a psychrophilic fungus and has optimal growth between ~12 and 16 °C with an upper limit of ~ 19 °C 157. This growth

range is well-aligned with bat hibernation temperatures which for most species range from

-10 to ~20 °C. M. lucifugus, a species highly susceptible to WNS, hibernates between -4

and 13 °C 158. The wide host range of P. destructans consisting of at least 25 bat species

suggests it is a generalist pathogen of bats (Whitenosesyndrome.org and 159). However,

P. destructans also remains capable of saprophytic growth and can utilize a range of

energy sources and organic substrates 160,161. This ability enables P. destructans to persist

and even proliferate in cave sediments which likely act as an environmental reservoir in

the transmission of WNS 162,163. The ability for environmental growth has also likely

contributed to the establishment of P. destructans across a wide geographic range.

Though the isolate responsible for WNS in N. America likely originated in Europe, P.

destructans has also been found throughout Asia and even from soil in Antarctica 164.

Phylogenic history of P. destructans and cross-species comparisons

Except for P. destructans, and isolated examples of superficial infections by P.

pannorum165, all identified Pseudogymnoascus species are non-pathogenic and exist

primarily as saprophytes 153. Though P. destructans is foreign to N. America, several of its

closest known relatives are native to the region (Figure 3). Most Pseudogymnoascus

isolates even come from the same cave environments inhabited by P. destructans. The

common ancestor of all Pseudogymnoascus species is estimated to have diverged from

other Leotiomycetes about 300 million years ago (mya), however the closest relatives of

P. destructans in N. America likely diverged ~20 mya 166. All characterized

Pseudogymnoascus isolates also exhibit similar cellular morphology with filamentous growth and formation of conidia. Like P. destructans, characterized Pseudogymnoascus species are haploid. All known species are also heterothallic, but genomic analyses demonstrate little evidence of recombination, again matching findings in P. destructans 3.

However, a recent comparative genomic study of P. destructans and six of its closest

known relatives by Palmer et al.166 did reveal some striking differences. The genome of P. destructans contained about 2 to 10 times the amount of repetitive DNA compared to its relatives, with repeat sequences making up roughly 38% of its genome. Interestingly, the

N. American lineage of P. destructans possesses at least one active retrovirus, suggesting this expansion of repetitive DNA is an ongoing process.167 The increase in repetitive sequences also correlated with a 10% reduction of protein-coding genes in P. destructans.

Much of the gene reduction is explained by a loss of secreted proteins with roughly 50% fewer genes containing a secretion signal. Extracellular carbohydrate-active enzymes

(CAZymes) were particularly reduced in P. destructans with about one-third of the average number of its relatives. Palmer et al did not find evidence of expansions in any specific gene families within P. destructans, though it did possess the highest number of lineage- specific genes. These findings are in contrast to pathogenic fungi such as B. dendrobatidis

and B. salamandrivorans where expansions in subtilisin-like peptidases and other

virulence-associated gene families are present 31. The number of secreted peptidases in

P. destructans is also reduced compared to its relatives, though its genome still contains over 200 putative peptidases, including multiple conserved subtilisin-like peptidases.

While the reduced secretome possessed by P. destructans contrasts with B. dendrobatidis, it is consistent with a larger trend as fungi that function as animal pathogens have smaller secretomes than saprophytic fungi on average. It has been proposed that smaller secretomes could even be an immune-evasive adaptation as many secreted fungal proteins can be immunogenic 63.

In addition to genomic differences, P. destructans possesses notable phenotypic

differences compared to its relatives. Alongside a dramatic loss in CAZyme-encoding

genes, P. destructans secretes lower levels of chitinase and glucanase activity and has a

more limited ability to use carbohydrate carbon sources than related species 161,168. P.

destructans is also much more sensitive to osmotic, pH-induced and chemical stress169

and grows more slowly in vitro, even at temperatures close to its own optimum 168. Though

P. destructans clearly retains the ability for saprophytic growth these differences suggest this ability may be reduced in comparison to related fungi. This hypothesis is supported by experiments demonstrating that several Pseudogymnoascus species can outcompete

P. destructans in vitro. Interestingly P. destructans also secretes fewer secondary metabolites and several of its relatives secrete compounds that can inhibit its growth, suggesting that these compounds may contribute to the competitive fitness of some related Pseudogymnoascus species 170. Together, these phenotypic and genomic

differences may reflect adaptation of P. destructans towards a more specialized,

pathogenic lifestyle.

Putative virulence factors in P. destructans

As P. destructans was completely uncharacterized and largely unknown prior to

the discovery of WNS, little is known regarding fungal factors that contribute to its

virulence. To date no single factor has been shown to be required or even directly

contribute to its ability to infect bats. However, progress is currently being made on this front and several studies have identified putative virulence factors that may be important to the pathogenesis of WNS. The P. destructans genome contains a variety of secreted enzymes including peptidases and lipases 166. Both classes of enzymes are known to serve as virulence factors in other fungi causing cutaneous infections, including the prominent invasive animal pathogen B. dendrobatidis. Transcriptomic analysis of P. destructans indicates that multiple peptidases and at least one lipase are also upregulated during infection relative to in vitro growth, making these interesting candidates for study

171. A variety of other peptidases including multiple subtilisin-like serine peptidases, associated with virulence in B dendrobatidis, appear to be constitutively expressed indicating that these may also be active during host infection. Many genes involved in the fungal heat-shock response are upregulated and many cell-wall remodeling enzymes are differentially expressed during infection as well, suggesting that these processes may be important during host colonization 172. The genome of P. destructans also contains multiple

biosynthetic gene clusters (BSGs) that are predicted to produce secondary metabolites

166. In other pathogenic fungi, BSGs are known to produce toxins, siderophores and other

effectors that can function as virulence factors. Riboflavin is one secondary metabolite

that is produced in large quantities by P. destructans when growing on bat wing tissue. At

quantities believed to be present in bats with WNS, Riboflavin can cause necrotic death

in cultures of primary bat fibroblasts, implicating this molecule in tissue damage and lesion

formation 173. P. destructans has also been shown to produce triacetylfusaranine C and

desferrichrome siderophores during growth on live bat tissue 174. These iron-scavenging

molecules are important for metabolic requirements in other fungal pathogens as iron can

often be limited in the host environment 175.

Existing tools to study virulence in P. destructans (this will be quick)

The relatively early stage of WNS research also means that many of the tools used to study virulence in other fungal pathogens have yet to be fully developed. Furthermore, the psychrophilic nature of P. destructans makes the use of typical mammalian infection models impossible due to host body temperatures. Experimental infection models of M. lucifugus and other captive bat species have been established to study disease pathology and gene expression 114,132. However, bat infection studies require a high level of expertise

and resources and are not amenable for the high-throughput studies needed to examine

multiple putative virulence factors. A few studies have also developed ex-vivo176 and cell-

based173 methods to study the interaction of P. destructans with bat tissue. However, these methods still require access to live bats, are technically complex, and also not ideal for high-throughput studies. A further challenge to WNS research is a lack of a robust and efficient gene-targeting method. Agrobacterium-mediated transformation has been successfully applied to delete a single gene (URE1) in P. destructans, however this system demonstrates only a ~2% homologous recombination rate and has yet to be successfully applied to target additional genes 177.

Overview

Prior to the discovery of WNS in 2006, P. destructans was unstudied,

uncharacterized and its existence virtually unknown. Accordingly, very little was

understood regarding virulence in this pathogenic fungus at the onset of the work presented here, and we faced a broad and largely imprecise set of open research

27 questions (in addition to a lack of tools described above). We therefore took a similarly broad approach in our investigations, with the basic goal of gaining a better understanding of virulence traits in P. destructans. With little existing knowledge on which to build our studies, we focused on factors tied to virulence in other well-studied fungi, and on cross- species comparisons to identify differences associated with virulence. In Chapter 2, the analysis of secreted proteins produced by P. destructans and its closest known relatives is described. This analysis identifies multiple enzymes unique to the P. destructans secretome including a serine carboxypeptidase, PdCP1, which we subsequently clone, purify and characterize. As secreted enzymes and peptidases especially contribute to pathogenicity in many other fungi, the enzymes identified here including PdCP1 may represent virulence factors in P. destructans. In Chapter 3, Galleria mellonella larvae are used to establish a novel infection model for P. destructans, as these are widely used to study virulence in other fungal pathogens. Here, I show that larval killing by P. destructans depends on a live infection and that fungal spores become significantly more virulent upon germination. I also demonstrate the utility of this model for screening chemical inhibitors against P. destructans. Lastly, in Chapter 4, a cross-species comparison of carbon metabolism in P. destructans and closely related non-pathogens is reported, revealing a strikingly limited set of utilizable carbon sources in P. destructans and evidence that it may be more dependent on mitochondrial respiration. Interestingly, limited carbon metabolism and increased mitochondrial activity are features present in other pathogenic fungi, suggesting these adaptations may be broadly conducive to host colonization. Together this work represents some of the first efforts to identify and characterize molecular factors that may contribute to virulence in P. destructans and establishes a novel method for studying its virulence. While much remains to be learned about this deadly pathogen, these findings shed light on the development of virulence in P. destructans and may open new avenues of research going forward.

Figures

A Extinctions due to B Infectious Disease

26% 74%

Fungi

Other infectious agents

Figure 1. The growing threat of invasive fungi. (A) A survey of all published or recorded extinction events reveals a recent and drastic increase in the number of fungal-driven extinctions. (B) Fungi are responsible for 74% of all recorded extinctions caused by infectious agents. Adapted from Fisher et al (2012).

Figure 2. The geographic range of WNS. The latest spreadmap from www.whitenosesyndrome.org demonstrates the continued spread of WNS in N. America.

Figure 3. Schematic tree showing relative relationships of different Pseudogymnoascus species. Based on Palmer et al (2018).

References

1 O'Brien, H. E., Parrent, J. L., Jackson, J. A., Moncalvo, J. M. & Vilgalys, R. Fungal community analysis by large-scale sequencing of environmental samples. Appl Environ Microbiol 71, 5544-5550, doi:10.1128/AEM.71.9.5544-5550.2005 (2005).

2 Blackwell, M. The fungi: 1, 2, 3 ... 5.1 million species? Am J Bot 98, 426-438, doi:10.3732/ajb.1000298 (2011).

3 Leushkin, E. V. et al. Comparative genome analysis of Pseudogymnoascus spp. reveals primarily clonal evolution with small genome fragments exchanged between lineages. BMC Genomics 16, 400, doi:10.1186/s12864-015-1570-9 (2015).

4 Staats, C. C. et al. Comparative genome analysis of entomopathogenic fungi reveals a complex set of secreted proteins. BMC Genomics 15, 822, doi:10.1186/1471-2164-15-822 (2014).

5 Sana, S. et al. Origin and invasion of the emerging infectious pathogen Sphaerothecum destruens. Emerg Microbes Infect 6, e76, doi:10.1038/emi.2017.64 (2017).

6 Allender, M. C., Raudabaugh, D. B., Gleason, F. H. & Miller, A. N. The natural history, ecology, and epidemiology of Ophidiomyces ophiodiicola and its potential impact on free-ranging snake populations. Fungal Ecology 17, 187-196, doi:10.1016/j.funeco.2015.05.003 (2015).

7 Blehert, D. S. et al. Bat white-nose syndrome: an emerging fungal pathogen? Science 323, 227, doi:10.1126/science.1163874 (2009).

8 Fisher, M. C. et al. Emerging fungal threats to animal, plant and ecosystem health. Nature 484, 186-194, doi:10.1038/nature10947 (2012).

9 Hubbes, M. The American elm and Dutch elm disease. The Forestry Chronicle 75, 265-273, doi:10.5558/tfc75265-2 (1999).

10 Davelos, A. L. & Jarosz, A. M. Demography of American chestnut populations: effects of a pathogen and a hyperparasite. Journal of Ecology 92, 675-685, doi:10.1111/j.0022-0477.2004.00907.x (2004).

11 Pennisi, E. Armed and dangerous. Science 327, 804-805, doi:10.1126/science.327.5967.804 (2010).

12 Tatum, L. A. The southern corn leaf blight epidemic. Science 171, 1113-1116, doi:10.1126/science.171.3976.1113 (1971).

13 Fisher, M. C., Gow, N. A. & Gurr, S. J. Tackling emerging fungal threats to animal health, food security and ecosystem resilience. Philos Trans R Soc Lond B Biol Sci 371, doi:10.1098/rstb.2016.0332 (2016).

14 Lorch, J. M. et al. Snake fungal disease: an emerging threat to wild snakes. Philos Trans R Soc Lond B Biol Sci 371, doi:10.1098/rstb.2015.0457 (2016).

15 Collins, J. P. History, novelty, and emergence of an infectious amphibian disease. Proc Natl Acad Sci U S A 110, 9193-9194, doi:10.1073/pnas.1305730110 (2013).

16 Tekaia, F. & Latge, J. P. Aspergillus fumigatus: saprophyte or pathogen? Curr Opin Microbiol 8, 385-392, doi:10.1016/j.mib.2005.06.017 (2005).

17 Casadevall, A., Steenbergen, J. N. & Nosanchuk, J. D. ‘Ready made’ virulence and ‘dual use’ virulence factors in pathogenic environmental fungi — the Cryptococcus neoformans paradigm. Current Opinion in Microbiology 6, 332-337, doi:10.1016/s1369-5274(03)00082-1 (2003).

18 Goodman, N. L. & Larsh, H. W. Environmental factors and growth of Histoplasma capsulatum in soil. Mycopathol Mycol Appl 33, 145-156 (1967).

19 Lips, K. R. Overview of chytrid emergence and impacts on amphibians. Philos Trans R Soc Lond B Biol Sci 371, doi:10.1098/rstb.2015.0465 (2016).

20 Askew, D. S. Aspergillus fumigatus: virulence genes in a street-smart mold. Curr Opin Microbiol 11, 331-337, doi:10.1016/j.mib.2008.05.009 (2008).

21 Steenbergen, J. N., Shuman, H. A. & Casadevall, A. Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Proc Natl Acad Sci U S A 98, 15245-15250, doi:10.1073/pnas.261418798 (2001).

22 Monod, M. et al. Secreted proteases from pathogenic fungi. Int J Med Microbiol 292, 405-419, doi:10.1078/1438-4221-00223 (2002).

23 Lotters, S. et al. The link between rapid enigmatic amphibian decline and the globally emerging chytrid fungus. Ecohealth 6, 358-372, doi:10.1007/s10393- 010-0281-6 (2009).

24 Skerratt, L. F. et al. Spread of Chytridiomycosis Has Caused the Rapid Global Decline and Extinction of Frogs. EcoHealth 4, 125-134, doi:10.1007/s10393-007- 0093-5 (2007).

25 Berger, L. et al. Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proc Natl Acad Sci U S A 95, 9031-9036 (1998).

26 Stuart, S. N. et al. Status and trends of amphibian declines and extinctions worldwide. Science 306, 1783-1786, doi:10.1126/science.1103538 (2004).

27 McCallum, M. L. Amphibian Decline or Extinction? Current Declines Dwarf Background Extinction Rate. Journal of Herpetology 41, 483-491, doi:10.1670/0022-1511(2007)41[483:Adoecd]2.0.Co;2 (2007).

28 Rosenblum, E. B., Voyles, J., Poorten, T. J. & Stajich, J. E. The deadly chytrid fungus: a story of an emerging pathogen. PLoS Pathog 6, e1000550, doi:10.1371/journal.ppat.1000550 (2010).

29 Rosenblum, E. B. et al. Complex history of the amphibian-killing chytrid fungus revealed with genome resequencing data. Proc Natl Acad Sci U S A 110, 9385- 9390, doi:10.1073/pnas.1300130110 (2013).

30 Olson, D. H. et al. Mapping the global emergence of Batrachochytrium dendrobatidis, the amphibian chytrid fungus. PLoS One 8, e56802, doi:10.1371/journal.pone.0056802 (2013).

31 Joneson, S., Stajich, J. E., Shiu, S. H. & Rosenblum, E. B. Genomic transition to pathogenicity in chytrid fungi. PLoS Pathog 7, e1002338, doi:10.1371/journal.ppat.1002338 (2011).

32 Farrer, R. A. et al. Genomic innovations linked to infection strategies across emerging pathogenic chytrid fungi. Nat Commun 8, 14742, doi:10.1038/ncomms14742 (2017).

33 Mitchell, K. M., Churcher, T. S., Garner, T. W. & Fisher, M. C. Persistence of the emerging pathogen Batrachochytrium dendrobatidis outside the amphibian host greatly increases the probability of host extinction. Proc Biol Sci 275, 329-334, doi:10.1098/rspb.2007.1356 (2008).

34 Voyles, J. et al. Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian declines. Science 326, 582-585, doi:10.1126/science.1176765 (2009).

35 Brutyn, M. et al. Batrachochytrium dendrobatidis zoospore secretions rapidly disturb intercellular junctions in frog skin. Fungal Genet Biol 49, 830-837, doi:10.1016/j.fgb.2012.07.002 (2012).

36 Fites, J. S. et al. The invasive chytrid fungus of amphibians paralyzes lymphocyte responses. Science 342, 366-369, doi:10.1126/science.1243316 (2013).

37 Pessier, A. P., Nichols, D. K., Longcore, J. E. & Fuller, M. S. Cutaneous chytridiomycosis in poison dart frogs (Dendrobates spp.) and White's tree frogs (Litoria caerulea). J Vet Diagn Invest 11, 194-199, doi:10.1177/104063879901100219 (1999).

38 Clark, R. W., Marchand, M. N., Clifford, B. J., Stechert, R. & Stephens, S. Decline of an isolated timber rattlesnake (Crotalus horridus) population: Interactions between climate change, disease, and loss of genetic diversity. Biological Conservation 144, 886-891, doi:10.1016/j.biocon.2010.12.001 (2011).

39 Allender, M. C., Baker, S., Britton, M. & Kent, A. D. Snake fungal disease alters skin bacterial and fungal diversity in an endangered rattlesnake. Sci Rep 8, 12147, doi:10.1038/s41598-018-30709-x (2018).

40 Brown, G. D. et al. Hidden killers: human fungal infections. Sci Transl Med 4, 165rv113, doi:10.1126/scitranslmed.3004404 (2012).

41 Kim, J. Y. Human fungal pathogens: Why should we learn? J Microbiol 54, 145- 148, doi:10.1007/s12275-016-0647-8 (2016).

42 Dagenais, T. R. & Keller, N. P. Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis. Clin Microbiol Rev 22, 447-465, doi:10.1128/CMR.00055-08 (2009).

43 Almeida, F., Wolf, J. M. & Casadevall, A. Virulence-Associated Enzymes of Cryptococcus neoformans. Eukaryot Cell 14, 1173-1185, doi:10.1128/EC.00103- 15 (2015).

44 Garcia-Vidal, C., Viasus, D. & Carratala, J. Pathogenesis of invasive fungal infections. Curr Opin Infect Dis 26, 270-276, doi:10.1097/QCO.0b013e32835fb920 (2013).

45 Garfoot, A. L. & Rappleye, C. A. Histoplasma capsulatum surmounts obstacles to intracellular pathogenesis. FEBS J 283, 619-633, doi:10.1111/febs.13389 (2016).

46 Vivek-Ananth, R. P. et al. Comparative systems analysis of the secretome of the opportunistic pathogen Aspergillus fumigatus and other Aspergillus species. Sci Rep 8, 6617, doi:10.1038/s41598-018-25016-4 (2018).

47 Munoz, J. F. et al. The Dynamic Genome and Transcriptome of the Human Fungal Pathogen Blastomyces and Close Relative Emmonsia. PLoS Genet 11, e1005493, doi:10.1371/journal.pgen.1005493 (2015).

48 Holbrook, E. D., Edwards, J. A., Youseff, B. H. & Rappleye, C. A. Definition of the extracellular proteome of pathogenic-phase Histoplasma capsulatum. J Proteome Res 10, 1929-1943, doi:10.1021/pr1011697 (2011).

49 Weber, S. S. et al. Analysis of the secretomes of Paracoccidioides mycelia and yeast cells. PLoS One 7, e52470, doi:10.1371/journal.pone.0052470 (2012).

50 Liu, O. W. et al. Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans. Cell 135, 174-188, doi:10.1016/j.cell.2008.07.046 (2008).

51 Campbell, L. T. et al. Cryptococcus strains with different pathogenic potentials have diverse protein secretomes. Eukaryot Cell 14, 554-563, doi:10.1128/EC.00052-15 (2015).

52 Olszewski, M. A. et al. Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous

system invasion. Am J Pathol 164, 1761-1771, doi:10.1016/S0002- 9440(10)63734-0 (2004).

53 Mirbod-Donovan, F. et al. Urease produced by Coccidioides posadasii contributes to the virulence of this respiratory pathogen. Infect Immun 74, 504- 515, doi:10.1128/IAI.74.1.504-515.2006 (2006).

54 Rutherford, J. C. The emerging role of urease as a general microbial virulence factor. PLoS Pathog 10, e1004062, doi:10.1371/journal.ppat.1004062 (2014).

55 Branzk, N. et al. Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol 15, 1017-1025, doi:10.1038/ni.2987 (2014).

56 Portela, M. B. et al. Ectophosphatase activity in Candida albicans influences fungal adhesion: study between HIV-positive and HIV-negative isolates. Oral Dis 16, 431-437, doi:10.1111/j.1601-0825.2009.01644.x (2010).

57 Kiffer-Moreira, T. et al. An ectophosphatase activity in Candida parapsilosis influences the interaction of fungi with epithelial cells. FEMS Yeast Res 7, 621- 628, doi:10.1111/j.1567-1364.2007.00223.x (2007).

58 Bernard, M. et al. Characterization of a cell-wall acid phosphatase (PhoAp) in Aspergillus fumigatus. Microbiology 148, 2819-2829, doi:10.1099/00221287-148- 9-2819 (2002).

59 Cox, G. M. et al. Superoxide Dismutase Influences the Virulence of Cryptococcus neoformans by Affecting Growth within Macrophages. Infection and Immunity 71, 173-180, doi:10.1128/iai.71.1.173-180.2003 (2003).

60 Frohner, I. E., Bourgeois, C., Yatsyk, K., Majer, O. & Kuchler, K. Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance. Mol Microbiol 71, 240- 252, doi:10.1111/j.1365-2958.2008.06528.x (2009).

61 Youseff, B. H., Holbrook, E. D., Smolnycki, K. A. & Rappleye, C. A. Extracellular superoxide dismutase protects Histoplasma yeast cells from host-derived oxidative stress. PLoS Pathog 8, e1002713, doi:10.1371/journal.ppat.1002713 (2012).

62 McDonagh, A. et al. Sub-telomere directed gene expression during initiation of invasive aspergillosis. PLoS Pathog 4, e1000154, doi:10.1371/journal.ppat.1000154 (2008).

63 Krijger, J. J., Thon, M. R., Deising, H. B. & Wirsel, S. G. Compositions of fungal secretomes indicate a greater impact of phylogenetic history than lifestyle adaptation. BMC Genomics 15, 722, doi:10.1186/1471-2164-15-722 (2014).

64 da Silva Dantas, A. et al. Thioredoxin regulates multiple hydrogen peroxide- induced signaling pathways in Candida albicans. Mol Cell Biol 30, 4550-4563, doi:10.1128/MCB.00313-10 (2010).

65 Missall, T. A. & Lodge, J. K. Thioredoxin reductase is essential for viability in the fungal pathogen Cryptococcus neoformans. Eukaryot Cell 4, 487-489, doi:10.1128/EC.4.2.487-489.2005 (2005).

66 Bernhard Hube, F. S., Michael Bossenz, Anna Mazur, Marianne Kretschmar, Wilhelm Schäfer. Secreted lipases of Candida albicans: cloning, characterisation and expression analysis of a new gene family with at least ten members. Arch Microbiol 174, 362-374, doi:10.1007/ (2000).

67 Mayer, F. L., Wilson, D. & Hube, B. Candida albicans pathogenicity mechanisms. Virulence 4, 119-128, doi:10.4161/viru.22913 (2013).

68 Gacser, A. et al. Lipase 8 affects the pathogenesis of Candida albicans. Infect Immun 75, 4710-4718, doi:10.1128/IAI.00372-07 (2007).

69 A. Ibrahim, F. M., S. Filler, Y. Banno, G. Cole, Y. Kitajima, J. Edwards, Jr, Y. Nozawa, M. Ghannoum. Evidence implicating phospholipase as a virulence factor of Candida albicans. Infection and Immunity 63 1993-1998, doi:https://iai.asm.org/content/iai/63/5/1993.full.pdf (1995).

70 Hube, B. et al. The role and relevance of phospholipase D1 during growth and dimorphism of Candida albicans. Microbiology 147, 879-889, doi:10.1099/00221287-147-4-879 (2001).

71 Leidich, S. D. et al. Cloning and disruption of caPLB1, a phospholipase B gene involved in the pathogenicity of Candida albicans. J Biol Chem 273, 26078-26086 (1998).

72 Wright, L. C. et al. Cryptococcal lipid metabolism: phospholipase B1 is implicated in transcellular metabolism of macrophage-derived lipids. Eukaryot Cell 6, 37-47, doi:10.1128/EC.00262-06 (2007).

73 Santangelo, R. et al. Role of Extracellular Phospholipases and Mononuclear Phagocytes in Dissemination of Cryptococcosis in a Murine Model. Infection and Immunity 72, 2229-2239, doi:10.1128/iai.72.4.2229-2239.2004 (2004).

74 Li, X. et al. Disruption of the phospholipase D gene attenuates the virulence of Aspergillus fumigatus. Infect Immun 80, 429-440, doi:10.1128/IAI.05830-11 (2012).

75 Wu, G. et al. Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny, Physiology, and Niche Adaptation on Human Skin. PLoS Genet 11, e1005614, doi:10.1371/journal.pgen.1005614 (2015).

76 White, T. C. et al. Fungi on the skin: dermatophytes and Malassezia. Cold Spring Harb Perspect Med 4, doi:10.1101/cshperspect.a019802 (2014).

77 Monod, M. Secreted proteases from dermatophytes. Mycopathologia 166, 285- 294, doi:10.1007/s11046-008-9105-4 (2008).

78 Yike, I. Fungal proteases and their pathophysiological effects. Mycopathologia 171, 299-323, doi:10.1007/s11046-010-9386-2 (2011).

79 Rawlings, N. D., Waller, M., Barrett, A. J. & Bateman, A. MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 42, D503-509, doi:10.1093/nar/gkt953 (2014).

80 [2] Families of serine peptidases. doi:10.1016/0076-6879(94)44004-2.

81 Hedstrom, L. Serine protease mechanism and specificity. Chem Rev 102, 4501- 4524 (2002).

82 Dunn, B. M. Structure and mechanism of the pepsin-like family of aspartic peptidases. Chem Rev 102, 4431-4458 (2002).

83 Cerdà-Costa, N. & Xavier Gomis-Rüth, F. Architecture and function of metallopeptidase catalytic domains. Protein Science 23, 123-144, doi:10.1002/pro.2400 (2014).

84 Perona, J. J. & Craik, C. S. Structural basis of substrate specificity in the serine proteases. Protein Sci 4, 337-360, doi:10.1002/pro.5560040301 (1995).

85 Morschhäuser, J., Virkola, R., Korhonen, T. K. & Hacker, J. Degradation of human subendothelial extracellular matrix by proteinase-secreting Candida albicans. FEMS Microbiology Letters 153, 349-355, doi:10.1111/j.1574- 6968.1997.tb12595.x (2006).

86 Schaller, M. et al. Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis. Mol Microbiol 34, 169-180 (1999).

87 Villar, C. C., Kashleva, H., Nobile, C. J., Mitchell, A. P. & Dongari-Bagtzoglou, A. Mucosal tissue invasion by Candida albicans is associated with E-cadherin degradation, mediated by transcription factor Rim101p and protease Sap5p. Infect Immun 75, 2126-2135, doi:10.1128/IAI.00054-07 (2007).

88 Ollert, M. W. et al. Mechanisms of adherence of Candida albicans to cultured human epidermal keratinocytes. Infect Immun 61, 4560-4568 (1993).

89 Meiller, T. F. et al. A novel immune evasion strategy of candida albicans: proteolytic cleavage of a salivary antimicrobial peptide. PLoS One 4, e5039, doi:10.1371/journal.pone.0005039 (2009).

90 Kaminishi, H. et al. Degradation of humoral host defense by Candida albicans proteinase. Infect Immun 63, 984-988, doi:0019-9567 (1995).

91 Gropp, K. et al. The yeast Candida albicans evades human complement attack by secretion of aspartic proteases. Mol Immunol 47, 465-475, doi:10.1016/j.molimm.2009.08.019 (2009).

92 B. Hube, D. S., F. Odds, D. Hess, M. Monod, W. Schäfer, A. Brown, N. Gow. Disruption of Each of the Secreted Aspartyl Proteinase Genes SAP1, SAP2, and

SAP3 of Candida albicans Attenuates Virulence. Infection and Immunity 65, 3529-3538 (1997).

93 Sanglard, D., Hube, B., Monod, M., Odds, F. C. & Gow, N. A. A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infect Immun 65, 3539-3546 (1997).

94 Lermann, U. & Morschhauser, J. Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans. Microbiology 154, 3281-3295, doi:10.1099/mic.0.2008/022525-0 (2008).

95 Correia, A. et al. Limited role of secreted aspartyl proteinases Sap1 to Sap6 in Candida albicans virulence and host immune response in murine hematogenously disseminated candidiasis. Infect Immun 78, 4839-4849, doi:10.1128/IAI.00248-10 (2010).

96 Cooper, K. G., Zarnowski, R. & Woods, J. P. Histoplasma capsulatum encodes a dipeptidyl peptidase active against the mammalian immunoregulatory peptide, substance P. PLoS One 4, e5281, doi:10.1371/journal.pone.0005281 (2009).

97 Chen, L. C., Blank, E. S., & Casadevall, A. . Extracellular proteinase activity of Cryptococcus neoformans. Clinical and diagnostic laboratory immunology 3 570- 574 (1996 Clinical and diagnostic laboratory immunology).

98 Yoo, J. i., Lee, Y. S., Song, C. Y. & Kim, B. S. Purification and Characterization of a 43-Kilodalton Extracellular Serine Proteinase from Cryptococcus neoformans. Journal of Clinical Microbiology 42, 722-726, doi:10.1128/jcm.42.2.722-726.2004 (2004).

99 Clarke, S. C. et al. Integrated Activity and Genetic Profiling of Secreted Peptidases in Cryptococcus neoformans Reveals an Aspartyl Peptidase Required for Low pH Survival and Virulence. PLoS Pathog 12, e1006051, doi:10.1371/journal.ppat.1006051 (2016).

100 Pinti, M. et al. Identification and characterization of an aspartyl protease from Cryptococcus neoformans. FEBS Lett 581, 3882-3886, doi:10.1016/j.febslet.2007.07.006 (2007).

101 Na Pombejra, S., Salemi, M., Phinney, B. S. & Gelli, A. The Metalloprotease, Mpr1, Engages AnnexinA2 to Promote the Transcytosis of Fungal Cells across the Blood-Brain Barrier. Front Cell Infect Microbiol 7, 296, doi:10.3389/fcimb.2017.00296 (2017).

102 Vu, K. et al. Invasion of the central nervous system by Cryptococcus neoformans requires a secreted fungal metalloprotease. MBio 5, e01101-01114, doi:10.1128/mBio.01101-14 (2014).

103 Farnell, E., Rousseau, K., Thornton, D. J., Bowyer, P. & Herrick, S. E. Expression and secretion of Aspergillus fumigatus proteases are regulated in response to different protein substrates. Fungal Biol 116, 1003-1012, doi:10.1016/j.funbio.2012.07.004 (2012).

104 Reichard, U. et al. Sedolisins, a new class of secreted proteases from Aspergillus fumigatus with endoprotease or tripeptidyl-peptidase activity at acidic pHs. Appl Environ Microbiol 72, 1739-1748, doi:10.1128/AEM.72.3.1739-1748.2006 (2006).

105 Kauffman, H. F., Tomee, J. F. C., van de Riet, M. A., Timmerman, A. J. B. & Borger, P. Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production. Journal of Allergy and Clinical Immunology 105, 1185-1193, doi:10.1067/mai.2000.106210 (2000).

106 Behnsen, J. et al. Secreted Aspergillus fumigatus protease Alp1 degrades human complement proteins C3, C4, and C5. Infect Immun 78, 3585-3594, doi:10.1128/IAI.01353-09 (2010).

107 Nierman, W. C. et al. Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438, 1151-1156, doi:10.1038/nature04332 (2005).

108 Kolattukudy, P. E. et al. Evidence for possible involvement of an elastolytic serine protease in aspergillosis. Infect Immun 61, 2357-2368 (1993).

109 Sriranganadane, D. et al. Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH. Proteomics 11, 4422- 4433, doi:10.1002/pmic.201100234 (2011).

110 Jousson, O. et al. Secreted subtilisin gene family in Trichophyton rubrum. Gene 339, 79-88, doi:10.1016/j.gene.2004.06.024 (2004).

111 R. Tsuboi, I. K., K. Takamori, and H. Ogawa. Isolation of a Keratinolytic Proteinase from Trichophyton mentagrophytes with Enzymatic Activity at Acidic pH. Infection and Immunity 57, 3479-3483, doi:0019-9567/89/113479-05 (1989).

112 Baldo, A. et al. Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes. J Med Microbiol 57, 1152-1156, doi:10.1099/jmm.0.47827-0 (2008).

113 Baldo, A. et al. Mechanisms of skin adherence and invasion by dermatophytes. Mycoses 55, 218-223, doi:10.1111/j.1439-0507.2011.02081.x (2012).

114 Lorch, J. M. et al. Experimental infection of bats with Geomyces destructans causes white-nose syndrome. Nature 480, 376-378, doi:10.1038/nature10590 (2011).

115 Lorch, J. M. et al. First Detection of Bat White-Nose Syndrome in Western North America. mSphere 1, doi:10.1128/mSphere.00148-16 (2016).

116 .

117 Frick, W. F. et al. An emerging disease causes regional population collapse of a common North American bat species. Science 329, 679-682, doi:10.1126/science.1188594 (2010).

118 Kalka, M. B., Smith, A. R. & Kalko, E. K. Bats limit arthropods and herbivory in a tropical forest. Science 320, 71, doi:10.1126/science.1153352 (2008).

119 Williams-Guillen, K., Perfecto, I. & Vandermeer, J. Bats limit insects in a neotropical agroforestry system. Science 320, 70, doi:10.1126/science.1152944 (2008).

120 Maine, J. J. & Boyles, J. G. Bats initiate vital agroecological interactions in corn. Proc Natl Acad Sci U S A 112, 12438-12443, doi:10.1073/pnas.1505413112 (2015).

121 Boyles, J. G., Cryan, P. M., McCracken, G. F. & Kunz, T. H. Conservation. Economic importance of bats in agriculture. Science 332, 41-42, doi:10.1126/science.1201366 (2011).

122 Courtin, F., Stone, W. B., Risatti, G., Gilbert, K. & Van Kruiningen, H. J. Pathologic findings and liver elements in hibernating bats with white-nose syndrome. Vet Pathol 47, 214-219, doi:10.1177/0300985809358614 (2010).

123 Meteyer, C. U. et al. Histopathologic criteria to confirm white-nose syndrome in bats. J Vet Diagn Invest 21, 411-414, doi:10.1177/104063870902100401 (2009).

124 Makanya, A. N. & Mortola, J. P. The structural design of the bat wing web and its possible role in gas exchange. J Anat 211, 687-697, doi:10.1111/j.1469- 7580.2007.00817.x (2007).

125 Warnecke, L. et al. Pathophysiology of white-nose syndrome in bats: a mechanistic model linking wing damage to mortality. Biol Lett 9, 20130177, doi:10.1098/rsbl.2013.0177 (2013).

126 Verant, M. L. et al. White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating bat host. BMC Physiol 14, 10, doi:10.1186/s12899-014-0010-4 (2014).

127 McGuire, L. P., Mayberry, H. W. & Willis, C. K. R. White-nose syndrome increases torpid metabolic rate and evaporative water loss in hibernating bats. Am J Physiol Regul Integr Comp Physiol 313, R680-R686, doi:10.1152/ajpregu.00058.2017 (2017).

128 Reeder, D. M. et al. Frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome. PLoS One 7, e38920, doi:10.1371/journal.pone.0038920 (2012).

129 Bernard, R. F. & McCracken, G. F. Winter behavior of bats and the progression of white-nose syndrome in the southeastern United States. Ecol Evol 7, 1487- 1496, doi:10.1002/ece3.2772 (2017).

130 Bouma, H. R., Carey, H. V. & Kroese, F. G. Hibernation: the immune system at rest? J Leukoc Biol 88, 619-624, doi:10.1189/jlb.0310174 (2010).

131 Sahdo, B. et al. Body temperature during hibernation is highly correlated with a decrease in circulating innate immune cells in the brown bear (Ursus arctos): a common feature among hibernators? Int J Med Sci 10, 508-514, doi:10.7150/ijms.4476 (2013).

132 Field, K. A. et al. The White-Nose Syndrome Transcriptome: Activation of Anti- fungal Host Responses in Wing Tissue of Hibernating Little Brown Myotis. PLoS Pathog 11, e1005168, doi:10.1371/journal.ppat.1005168 (2015).

133 Lilley, T. M. et al. Immune responses in hibernating little brown myotis (Myotis lucifugus) with white-nose syndrome. Proc Biol Sci 284, doi:10.1098/rspb.2016.2232 (2017).

134 Rapin, N. et al. Activation of innate immune-response genes in little brown bats (Myotis lucifugus) infected with the fungus Pseudogymnoascus destructans. PLoS One 9, e112285, doi:10.1371/journal.pone.0112285 (2014).

135 Moore, M. S. et al. Hibernating little brown myotis (Myotis lucifugus) show variable immunological responses to white-nose syndrome. PLoS One 8, e58976, doi:10.1371/journal.pone.0058976 (2013).

136 Prendergast, B. J., Freeman, D. A., Zucker, I. & Nelson, R. J. Periodic arousal from hibernation is necessary for initiation of immune responses in ground squirrels. Am J Physiol Regul Integr Comp Physiol 282, R1054-1062, doi:10.1152/ajpregu.00562.2001 (2002).

137 Moore, M. S. et al. Energy conserving thermoregulatory patterns and lower disease severity in a bat resistant to the impacts of white-nose syndrome. J Comp Physiol B 188, 163-176, doi:10.1007/s00360-017-1109-2 (2018).

138 Meteyer, C. U., Barber, D. & Mandl, J. N. Pathology in euthermic bats with white nose syndrome suggests a natural manifestation of immune reconstitution inflammatory syndrome. Virulence 3, 583-588, doi:10.4161/viru.22330 (2012).

139 Langwig, K. E. et al. Sociality, density-dependence and microclimates determine the persistence of populations suffering from a novel fungal disease, white-nose syndrome. Ecol Lett 15, 1050-1057, doi:10.1111/j.1461-0248.2012.01829.x (2012).

140 Frank, C. L. et al. The resistance of a North American bat species (Eptesicus fuscus) to White-nose Syndrome (WNS). PLoS One 9, e113958, doi:10.1371/journal.pone.0113958 (2014).

141 Frank, C. L. et al. The Effects of Cutaneous Fatty Acids on the Growth of Pseudogymnoascus destructans, the Etiological Agent of White-Nose Syndrome (WNS). PLoS One 11, e0153535, doi:10.1371/journal.pone.0153535 (2016).

142 Ingala, M. R., Ravenelle, R. E., Monro, J. J. & Frank, C. L. The effects of epidermal fatty acid profiles, 1-oleoglycerol, and triacylglycerols on the susceptibility of hibernating bats to Pseudogymnoascus destructans. PLoS One 12, e0187195, doi:10.1371/journal.pone.0187195 (2017).

143 Pannkuk, E. L. et al. Fatty acid methyl ester profiles of bat wing surface lipids. Lipids 49, 1143-1150, doi:10.1007/s11745-014-3951-2 (2014).

144 Hayman, D. T., Pulliam, J. R., Marshall, J. C., Cryan, P. M. & Webb, C. T. Environment, host, and fungal traits predict continental-scale white-nose syndrome in bats. Sci Adv 2, e1500831, doi:10.1126/sciadv.1500831 (2016).

145 Grieneisen, L. E., Brownlee-Bouboulis, S. A., Johnson, J. S. & Reeder, D. M. Sex and hibernaculum temperature predict survivorship in white-nose syndrome affected little brown myotis (Myotis lucifugus). R Soc Open Sci 2, 140470, doi:10.1098/rsos.140470 (2015).

146 Puechmaille, S. J. et al. Pan-European distribution of white-nose syndrome fungus (Geomyces destructans) not associated with mass mortality. PLoS One 6, e19167, doi:10.1371/journal.pone.0019167 (2011).

147 Bandouchova, H. et al. Pseudogymnoascus destructans: evidence of virulent skin invasion for bats under natural conditions, Europe. Transbound Emerg Dis 62, 1-5, doi:10.1111/tbed.12282 (2015).

148 Kovacova, V. et al. White-nose syndrome detected in bats over an extensive area of Russia. BMC Vet Res 14, 192, doi:10.1186/s12917-018-1521-1 (2018).

149 Pikula, J. et al. White-nose syndrome pathology grading in Nearctic and Palearctic bats. PLoS One 12, e0180435, doi:10.1371/journal.pone.0180435 (2017).

150 Zukal, J. et al. White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America. Sci Rep 6, 19829, doi:10.1038/srep19829 (2016).

151 Bandouchova, H. et al. Alterations in the health of hibernating bats under pathogen pressure. Sci Rep 8, 6067, doi:10.1038/s41598-018-24461-5 (2018).

152 Khankhet, J. et al. Clonal expansion of the Pseudogymnoascus destructans genotype in North America is accompanied by significant variation in phenotypic expression. PLoS One 9, e104684, doi:10.1371/journal.pone.0104684 (2014).

153 Drees, K. P. et al. Phylogenetics of a Fungal Invasion: Origins and Widespread Dispersal of White-Nose Syndrome. MBio 8, doi:10.1128/mBio.01941-17 (2017).

154 Leopardi, S., Blake, D. & Puechmaille, S. J. White-Nose Syndrome fungus introduced from Europe to North America. Curr Biol 25, R217-R219, doi:10.1016/j.cub.2015.01.047 (2015).

155 Warnecke, L. et al. Inoculation of bats with European Geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome. Proc Natl Acad Sci U S A 109, 6999-7003, doi:10.1073/pnas.1200374109 (2012).

156 Palmer, J. M. et al. Molecular characterization of a heterothallic mating system in Pseudogymnoascus destructans, the fungus causing white-nose syndrome of bats. G3 (Bethesda) 4, 1755-1763, doi:10.1534/g3.114.012641 (2014).

157 Verant, M. L., Boyles, J. G., Waldrep, W., Jr., Wibbelt, G. & Blehert, D. S. Temperature-dependent growth of Geomyces destructans, the fungus that causes bat white-nose syndrome. PLoS One 7, e46280, doi:10.1371/journal.pone.0046280 (2012).

158 Webb, P. I., Speakman, J. R. & Racey, P. A. How hot is a hibernaculum? A review of the temperatures at which bats hibernate. Canadian Journal of Zoology 74, 761-765, doi:10.1139/z96-087 (1996).

159 Zukal, J. et al. White-nose syndrome fungus: a generalist pathogen of hibernating bats. PLoS One 9, e97224, doi:10.1371/journal.pone.0097224 (2014).

160 Raudabaugh, D. B. & Miller, A. N. Nutritional capability of and substrate suitability for Pseudogymnoascus destructans, the causal agent of bat white-nose syndrome. PLoS One 8, e78300, doi:10.1371/journal.pone.0078300 (2013).

161 Wilson, M. B., Held, B. W., Freiborg, A. H., Blanchette, R. A. & Salomon, C. E. Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans, the cause of white-nose syndrome in bats. PLoS One 12, e0178968, doi:10.1371/journal.pone.0178968 (2017).

162 Reynolds, H. T., Ingersoll, T. & Barton, H. A. Modeling the environmental growth of Pseudogymnoascus destructans and its impact on the white-nose syndrome epidemic. J Wildl Dis 51, 318-331, doi:10.7589/2014-06-157 (2015).

163 Verant, M. L. et al. Determinants of Pseudogymnoascus destructans within bat hibernacula: implications for surveillance and management of white-nose syndrome. J Appl Ecol 55, 820-829, doi:10.1111/1365-2664.13070 (2018).

164 Gomes, E. C. Q. et al. Cultivable fungi present in Antarctic soils: taxonomy, phylogeny, diversity, and bioprospecting of antiparasitic and herbicidal metabolites. Extremophiles 22, 381-393, doi:10.1007/s00792-018-1003-1 (2018).

165 Gianni, C., Caretta, G. & Romano, C. Skin infection due to Geomyces pannorum var. pannorum. Fallbericht. Hautinfektion durch Geomyces pannorum var. pannorum. Mycoses 46, 430-432, doi:10.1046/j.1439-0507.2003.00897.x (2003).

166 Palmer, J. M., Drees, K. P., Foster, J. T. & Lindner, D. L. Extreme sensitivity to ultraviolet light in the fungal pathogen causing white-nose syndrome of bats. Nat Commun 9, 35, doi:10.1038/s41467-017-02441-z (2018).

167 Thapa, V. et al. Using a Novel Partitivirus in Pseudogymnoascus destructans to Understand the Epidemiology of White-Nose Syndrome. PLoS Pathog 12, e1006076, doi:10.1371/journal.ppat.1006076 (2016).

168 Reynolds, H. T. & Barton, H. A. Comparison of the white-nose syndrome agent Pseudogymnoascus destructans to cave-dwelling relatives suggests reduced saprotrophic enzyme activity. PLoS One 9, e86437, doi:10.1371/journal.pone.0086437 (2014).

169 Chaturvedi, V., DeFiglio, H. & Chaturvedi, S. Phenotype profiling of white-nose syndrome pathogen Pseudogymnoascus destructans and closely-related Pseudogymnoascus pannorum reveals metabolic differences underlying fungal lifestyles. F1000Res 7, 665, doi:10.12688/f1000research.15067.2 (2018).

170 Lilley, T. M. et al. White-nose syndrome survivors do not exhibit frequent arousals associated with Pseudogymnoascus destructans infection. Front Zool 13, 12, doi:10.1186/s12983-016-0143-3 (2016).

171 Donaldson, M. E. et al. Growth medium and incubation temperature alter the Pseudogymnoascus destructans transcriptome: implications in identifying virulence factors. Mycologia 110, 300-315, doi:10.1080/00275514.2018.1438223 (2018).

172 Reeder, S. M. et al. Pseudogymnoascus destructans transcriptome changes during white-nose syndrome infections. Virulence 8, 1695-1707, doi:10.1080/21505594.2017.1342910 (2017).

173 Flieger, M. et al. Vitamin B2 as a virulence factor in Pseudogymnoascus destructans skin infection. Sci Rep 6, 33200, doi:10.1038/srep33200 (2016).

174 Mascuch, S. J. et al. Direct detection of fungal siderophores on bats with white- nose syndrome via fluorescence microscopy-guided ambient ionization mass spectrometry. PLoS One 10, e0119668, doi:10.1371/journal.pone.0119668 (2015).

175 Hissen, A. H., Wan, A. N., Warwas, M. L., Pinto, L. J. & Moore, M. M. The Aspergillus fumigatus siderophore biosynthetic gene sidA, encoding L-ornithine N5-oxygenase, is required for virulence. Infect Immun 73, 5493-5503, doi:10.1128/IAI.73.9.5493-5503.2005 (2005).

176 Cornelison, C. T. et al. A preliminary report on the contact-independent antagonism of Pseudogymnoascus destructans by Rhodococcus rhodochrous strain DAP96253. BMC Microbiol 14, 246, doi:10.1186/s12866-014-0246-y (2014).

177 Zhang, T., Ren, P., Chaturvedi, V. & Chaturvedi, S. Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum. Fungal Genet Biol 81, 73-81, doi:10.1016/j.fgb.2015.05.009 (2015).

CHAPTER 2: Characterization of PdCP1, a Serine Carboxypeptidase from Pseudogymnoascus destructans, the Causal Agent of White-nose Syndrome

This chapter was published in Biological Chemistry (v.399, issue 12, online October 2018). I performed all experiments with the following exceptions: Giselle Knudsen performed Mass spectrometry on fungal secretomes and SDS-Page gel bands in Figures 1 and 2C, Anthony O’Donoghue and Zhenze Jiang performed MSP-MS experiments in Figure 4, and Supplementary Figures 3 and 4 and Zhenze performed peptide cleavage mapping shown in Figure 3D. I performed all data analysis except for initial analysis of MSP-MS data which was performed by Zhenze Jiang. I also wrote the original manuscript with edits from Richard Bennett, Giselle Knudsen and Anthony O’Donoghue and designed all figures

Characterization of PdCP1, a Serine Carboxypeptidase from Pseudogymnoascus destructans, the Causal Agent of White-nose Syndrome

Chapman Beekman1, Zhenze Jiang2, Brian M. Suzuki2, Jonathan M. Palmer4,

Daniel L. Lindner4, Anthony J. O’Donoghue2, Giselle M. Knudsen3

and Richard J. Bennett1*

1Department of Molecular Microbiology and Immunology, Brown University, Providence, RI, USA. 2Skaggs School of Pharmacy and Pharmaceutical Sciences, UC San Diego, La Jolla, USA; Center for Discovery and Innovation in Parasitic Diseases, UC San Diego, La Jolla, USA 3Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA 4Center for Forest Mycology Research, Northern Research Station, USDA Forest Service, Madison, WI, USA * correspondence: Richard Bennett, Box G-B6, 171 Meeting Street, Providence, RI 02912. Tel (401) 863-6341. Email: [email protected]

Short title: Analysis of P. destructans carboxypeptidase 1

Abstract

Pseudogymnoascus destructans is a pathogenic fungus responsible for White- nose Syndrome, a disease afflicting multiple species of North American bats. P. destructans infects susceptible bats during hibernation, invading dermal tissue and causing extensive tissue damage. In contrast, other Pseudogymnoascus species are non- pathogenic and cross-species comparisons may therefore reveal factors that contribute to virulence. In this study, we compared the secretome of P. destructans with that from several closely related Pseudogymnoascus species. A diverse set of hydrolytic enzymes were identified, including a putative serine peptidase, PdCP1, that was unique to the P. destructans secretome. A recombinant form of PdCP1 was purified and substrate preference determined using a multiplexed-substrate profiling method based on enzymatic degradation of a synthetic peptide library and analysis by mass spectrometry.

Most peptide substrates were sequentially truncated from the carboxyl terminus revealing that this enzyme is a bona fide carboxypeptidase. Peptides with arginine located close to the carboxy terminus were rapidly cleaved, and a fluorescent substrate containing arginine was therefore used to characterize PdCP1 activity and to screen a selection of peptidase inhibitors. Antipain and leupeptin were found to be the most potent inhibitors of PdCP1 activity.

Keywords: Bat infection; Peptidase; Secretome; Virulence; Proteomics

Introduction

Pseudogymnoascus destructans is a fungal pathogen responsible for White-nose

Syndrome (WNS), a disease affecting multiple bat species across North America. First discovered in 2006, this disease has spread rapidly across 33 U.S. states and 7 Canadian provinces, killing approximately 6 million bats in the process (U.S. Fish and Wildlife

Service 2018). The mortality rate for WNS is over 90% for some species, including the

once-common Little Brown bat (Myotis lucifugus) which is now threatened with extinction.

Losses to bat populations caused by WNS have substantial impacts not only on their

native ecosystems but also on human agriculture where bats play an important role in

limiting insect pests (Boyles 2011; Maine and Boyles 2015).

P. destructans is an invasive species originating from Europe where it has co-

evolved with bats and colonizes native species in a largely non-lethal manner (Leopardi

et al. 2015; Puechmaille et al. 2011; Zukal et al. 2016). P. destructans is not related to

any known animal pathogens and other Pseudogymnoascus species exist as non-

pathogenic saprophytes living off of dead organic matter in cave environments (Reynolds

and Barton 2014; Palmer et al. 2018). Cross-species comparisons may therefore be an

effective strategy for identifying factors contributing to virulence. P. destructans is a cold-

loving, psychrophilic fungus that targets its host during hibernation, when body

temperatures are close to that of the surrounding cave (Meteyer et al. 2009). In species

that are susceptible to WNS P. destructans exhibits highly invasive growth, penetrating

sub-dermal connective tissue and causing deep-seated lesions (Pikula et al. 2017).

Infection disrupts hibernation and aroused bats deplete their energy stores, ultimately

resulting in death (Warnecke et al. 2013). However, despite the prevalence of WNS in

North America, the molecular factors that may enable host and environmental colonization

by P. destructans remain poorly defined.

Fungi rely on secreted hydrolytic enzymes such as glycosidases, lipases and

peptidases to breakdown larger polymers into diffusible energy sources. Saprophytic

fungi generally secrete a larger number of hydrolytic enzymes than pathogenic fungi due

to the wide range of nutrient sources available (Krijger et al. 2014; Lowe and Howlett

2012). However, secreted enzymes can also play important roles during pathogenesis

(Campbell et al. 2015). For fungal species that exist both as saprophytes and

opportunistic pathogens, extracellular enzymes may serve a dual purpose promoting both

pathogenic and non-pathogenic lifestyles (Casadevall et al. 2003; Krappmann 2016).

Secreted peptidases have received particular attention as fungal virulence factors.

Peptidases are classified by their catalytic residue (e.g., serine, cysteine, etc.) and by

whether they cleave near the amino- or carboxyl-terminal sites (exopeptidases) or at

peptide bonds distal from the termini (endopeptidases) (Rawlings et al. 2014). A variety

of secreted peptidase activities have been identified from fungal pathogens, with serine,

aspartyl and metallo endopeptidases, metallo aminopeptidases and serine

carboxypeptidases being the most common (Monod et al. 2002; Yike 2011). These enzymes can promote pathogenesis through multiple mechanisms. For example, degradation of host structural proteins by fungal peptidases can contribute to pathogenicity by enabling invasion and/or adherence to host tissue (Sanglard et al. 1997;

Morschhauser et al. 1997; Ollert et al. 1993). Additionally, degradation of host immune components may allow immune evasion and promote survival in the host (Behnsen et al.

2010; Kaminishi et al. 1995; Thekkiniath et al. 2013). Secreted peptidases are especially important to dermatophytes, a specialized group of fungi that, like P. destructans, colonize mammalian skin, where these enzymes serve roles in both nutrient acquisition and tissue invasion (Monod 2008; Baldo et al. 2012).

P. destructans has been shown to secrete multiple peptidases including a serine

endopeptidase, Destructin-1, which is capable of degrading extracellular-matrix (ECM)

proteins that may facilitate tissue invasion (O’Donoghue et al. 2015). P. destructans- conditioned media also contained peptidase activity that had a preference for cleavage at the carboxy termini of peptides, and a proteomic analysis identified two putative serine carboxypeptidases in the secretome (O’Donoghue et al. 2015). Serine carboxypeptidases are common to many fungi and are important for the final stages of protein catabolism, releasing single amino acids that can serve as diffusible carbon/nitrogen sources

(Sriranganadane et al. 2011; Monod et al. 2002; Zaugg et al. 2008). Characterization of serine carboxypeptidases produced by P. destructans may therefore shed light on the metabolic capacity of this fungus and how it can colonize both its mammalian host and the environment.

In this study, we compared the secretome of P. destructans with related non-

pathogenic species during growth in a defined medium and found that PdCP1, a putative

serine carboxypeptidase, was uniquely detected in the P. destructans secretome. A recombinant form of this enzyme was overexpressed, purified and its activity analyzed using synthetic substrates and an unbiased multiplex substrate profiling by mass spectrometry (MSP-MS) approach (O'Donoghue et al. 2012). These analyses established that PdCP1 acts as a carboxypeptidase with broad specificity, indicating that it can degrade a wide range of protein substrates. The optimal conditions for PdCP1 activity are defined and effective inhibitors identified, which will allow for further investigation of the role of this peptidase in P. destructans biology.

Results

Secretome analysis in P. destructans and related species

The secretomes of P. destructans and four of its closest known non-pathogenic

relatives, P. verrucosus, P. sp. 03VT05, P. sp. 05NY08, and P. sp. WSF3629 (Palmer et

al., 2018), were analyzed by LC-MS/MS (liquid chromatography-tandem mass

spectrometry) following growth in RPMI medium. The number of identified proteins in

each secretome ranged from 92 in P. destructans to 178 in P. sp. 05NY08 (Supp. Tables

1-5). Functional annotations were assigned using the PANNZER annotation tool

(Koskinen et al. 2015) and indicated that most secreted proteins possessed putative enzymatic activity, ranging from 63% in P. sp. WSF3629 to 76% in P. destructans (Supp.

Tables 6-10). Most secreted enzymes fit into one of three broad activity/specificity categories based on functional annotations: glycosidases, reductive/oxidative (redox) enzymes, and peptidases. The number and proportion of putative redox enzymes and glycosidases in the P. destructans secretome was generally lower than that found in the non-pathogenic secretomes (Fig. 1). In contrast, the P. destructans secretome possessed the second-highest number of peptidases and a higher proportion of these enzymes

(9.3%) than its relatives (4.0% to 9.0%). Most of the secreted peptidases from P. destructans were identified in previous studies (O’Donoghue et al. 2015; Pannkuk et al.

2015). However, additional peptidases were also detected for the first time including an ortholog of tripeptidyl aminopeptidase SED2, a putative virulence factor from Aspergillus fumigatus (Reichard et al. 2006), as well as aspartyl- and metallo-endopeptidases (Table

1).

To compare secreted peptidases in more detail, each peptidase in the P. destructans secretome was searched for orthologs against each non-pathogenic proteome using Blastp (Altschul et al. 1990). All secreted peptidases from P. destructans had orthologs in each of the related genomes (using a threshold of >80% coverage and

>60% identity) and most of these orthologs were also detected in the LC-MS/MS secretome analysis (Table 1). However, the orthologs of two peptidases from P. destructans were not detected by LC-MS/MS in any of the four non-pathogenic secretomes: a putative metallopeptidase (GMDG_07199) and PdCP1 (GMDG_05452), a putative serine carboxypeptidase. PdCP1 showed substantial homology to members of the S10 peptidase family, comprising all known serine carboxypeptidase enzymes, and this included conservation of the key catalytic residues (Supp. Fig. 1). Within this group it shared greatest homology with members of the carboxypeptidase O (S10.014) subfamily that are present in a wide range of fungal species, but few of which have been characterized.

Recombinant expression and purification of PdCP1

For this study, we focused our efforts on characterizing PdCP1 and expressed the recombinant protein in the yeast Pichia pastoris. To achieve this, the PdCP1 ORF was placed under the control of a methanol-inducible P. pastoris promoter and expressed with an N-terminal S. cerevisiae secretion signal peptide and C-terminal 6x His epitope tag.

Multiple independent PdCP1-transformed P. pastoris clones were isolated and induced, and peptidase activity in the media examined using an internally-quenched (IQ) fluorescent peptide substrate with the sequence Mca-PKRLSALLK-DNP. Peptidase activity was detected in the media of transformed, but not untransformed, P. pastoris cells using this substrate.

We attempted to purify recombinant PdCP1 from P. pastoris-conditioned medium using a nickel-agarose column but were unable to recover sufficient activity for biochemical studies. However, the recombinant peptidase was successfully isolated by cation-exchange chromatography yielding a diffuse protein band of ~99 kDa by SDS-

PAGE analysis (Fig. 2A). Treatment of this sample with PNGase F to remove potential N-

linked glycosylation reduced the size of the purified protein band to ~57 kDa, consistent

with the predicted mass of recombinant PdCP1 (59 kDa, Fig. 2B). The protein band was

excised, digested with trypsin and the peptides sequenced by LC-MS/MS (Fig. 2C). These

data confirmed that the isolated protein was PdCP1.

Peptidase activity in the purified PdCP1 sample was evaluated using the IQ

substrate from pH 2.2 to 8.6 to determine the optimal pH for subsequent assays (Fig. 3A).

Maximum activity was observed at pH 4.6 with greater than 50% of maximal activity

maintained between pH 2.6 and 6.5. Enzyme assays at the optimal pH showed a ~3.4-

fold increase in specific activity in the purified sample over that in the P. pastoris-

conditioned medium (Fig. 3B). To establish that PdCP1 is responsible for cleavage of the

IQ substrate, a catalytically-inactivated PdCP1 mutant (S207A) that lacks the predicted

catalytic serine residue (see protein alignment, Supp. Fig. 1) was also purified using the

same protocol. The purified PdCP1-S207A protein formed a similar band on SDS-PAGE

(Supp. Fig. 2) but no peptidase activity was detected (Fig. 3C). Mass spectrometry

analysis of the IQ substrate after incubation with active PdCP1 identified multiple

cleavages in the C-terminal region (Fig. 3D) consistent with PdCP1 possessing a bona

fide carboxypeptidase activity.

Analysis of PdCP1 substrate specificity

To determine the substrate specificity preference of PdCP1, we incubated the enzyme with an equimolar mixture of 228 unique tetradecapeptide substrates at pH 4.6.

This same set of peptides was previously used to define the substrate preference of the serine peptidase Destructin-1 secreted by P. destructans (O’Donoghue et al. 2015).

Cleavage of any of the 2,964 unique peptide bonds within this library can be detected by

LC-MS/MS sequencing and the method is therefore referred to as multiplex substrate profiling by mass spectrometry (MSP-MS) (O'Donoghue et al. 2012). After 5 minutes incubation at pH 4.6, PdCP1 had removed single amino acids from the carboxyl terminus of 76 of the 14-mer peptides. Many of the new 13-mer products were progressively degraded to 12-mer, 11-mer and 10 mer peptides. Additional data was generated by incubating PdCP1 with the peptide mixture for 15, 60 and 240 minutes and, in all cases, there was a clear time-dependent increase in cleaved peptides that were truncated at the carboxyl terminus (Fig. 4A).

The highly processive degradation of peptides from the carboxyl terminus is illustrated in Figure 4B which depicts the cleavage of a single substrate from the peptide library. The peak intensity of the LGWHAnFRKYPInA peptide decreased by >99% during the 4-hour assay with eight unique cleavage products detected (note that lower case n corresponds to norleucine, which is a Met mimic). The abundance of the 13-mer cleavage product increased after 15 minutes and then subsequently decreased between the 15 and

60-minute time points. A 12-mer peptide subsequently formed which showed maximal intensity at 60 minutes before it too was degraded. Only the intensities of the two smallest cleavage products, LGWHAnF and LGWHAn, continued to increase throughout the time course.

To further define the substrate specificity of PdCP1, a frequency plot was

generated for all cleavage sites that occur within 15 minutes at the carboxyl terminus of

the peptide library. Although the enzyme has broad specificity, it exhibits a clear

preference for substrates containing Arg, norleucine and Leu residues when compared to

Gly, Asp or Ala residues (Fig. 4C). Cleavage takes place between the P1 and P1ʹ residues

and therefore the carboxyl terminal residues are situated in the P1ʹ position. In the P1

position, Arg, Nle, Gln, Leu and Ala were favored while Asp, Pro and His were disfavored.

Arg and Pro were significantly enriched in the P2 position, while positively charged

residues were most commonly seen at P3.

Taken together, unbiased peptide cleavage assays reveal that PdCP1 is a carboxypeptidase with a broad specificity indicating it is likely capable of digesting the carboxy-termini of a wide range of protein and peptide substrates, although it also exhibits a preference for particular amino acids at the P2, P1 and P1’ positions.

Characterization of PdCP1 activity using AMC substrates

To continue our biochemical analysis, we sought to identify a fluorescent reporter substrate that mimicked the substrate preference found in the peptide digestion assay.

Peptide substrates with a C-terminal 7-Amino-4-Methylcoumarin (AMC) group are generally used to assay aminopeptidases and endopeptidases, but if the AMC group can be accommodated in the S1ʹ pocket then these substrates can also be cleaved by a carboxypeptidase (Takeuchi et al. 2014; Kunugi et al. 1985). PdCP1 has broad specificity in the P1ʹ position and a preference for Arg in the P1 position. We therefore predicted that this enzyme would hydrolyze a peptide substrate containing an Arg-AMC bond. We evaluated a small collection of fluorescent substrates that all contain an Arg-AMC bond

(Fig. 5A) and found that PdCP1 was unable to cleave Arg-AMC and Gly-Arg-AMC, and

had weak activity against dipeptides capped on the amino terminus with a

benzyloxycarbonyl group. However, when tripeptide substrates were evaluated, PdCP1

rapidly hydrolyzed Boc-Leu-Arg-Arg-AMC but not N-Benzoyl-Phe-Val-Arg-AMC. These data indicate that a preferred P2 amino acid such as Arg is important for efficient hydrolysis of peptide-AMC substrates by PdCP1. We subsequently tested a tetrapeptide substrate containing another preferred P2 amino acid and found that the substrate Boc-Ala-Gly-Pro-

Arg-AMC was hydrolyzed faster than all other substrates tested.

Using the Boc-Ala-Gly-Pro-Arg-AMC as a reporter substrate we assayed PdCP1 activity under a variety of pH conditions and were surprised to find that the optimal activity of PdCP1 on this substrate was pH 6.2 (Fig. 5B). At this pH, hydrolysis of Boc-Ala-Gly-

Pro-Arg-AMC is ~6.5-times faster than at pH 4.6, whereas the IQ substrate was cleaved

~1.5-times faster at pH 4.6 than pH 6.2. We therefore repeated the MSP-MS assay with

PdCP1 at pH 6.2 and compared the rate of product formation to that at pH 4.6. At each timepoint, PdCP1 generated a greater number of cleavages at pH 4.6 than at pH 6.2.

Additionally, less than 5 cleavages were unique to the pH 6.2 condition at any timepoint

(Supp. Fig. 4A), establishing that PdCP1 activity is greater at pH 4.6 than 6.2 on unmodified peptides, and that its substrate specificity is not significantly altered by changes in pH. While it remains unclear why PdCP1 cleaves Boc-Ala-Gly-Pro-Arg-AMC faster at pH 6.2 than at pH 4.6, the lower pH may be due to AMC (pKa = 7.8) being partially protonated below pH 5 which negatively affects fluorescent intensity (R. Srinivasan 1974).

Using the Boc-Ala-Gly-Pro-Arg-AMC at pH 6.2, PdCP1 activity was found to decrease in the presence of increasing salt concentrations (Fig. 5C) with 50% and 25% of maximal activity present at 100 mM and 500 mM NaCl, respectively. In addition, PdCP1 exhibited optimal activity at 20°C (Fig. 5D). Therefore, PdCP1 appears to be cold adapted

to function optimally near the maximum temperature supporting growth of P. destructans

(Chaturvedi et al. 2010).

PdCP1 activity was also tested against a panel of peptidase inhibitors (Fig. 6A).

Serine peptidase inhibitors 6-aminohexanoic acid (6-AHA) and aprotinin, as well as

serine/cysteine peptidase inhibitors antipain, chymostatin and leupeptin inhibited PdCP1

activity by 50% or more, consistent with the classification of PdCP1 as a serine-

carboxypeptidase. Under these conditions, leupeptin and antipain completely inhibited

activity and an IC50 was calculated with these compounds. Leupeptin and antipain each have a P1-Arg residue adjacent to a reactive aldehyde group and had IC50 values of 104 to 129 nM and 472 to 559 nM (95% confidence intervals) respectively (Fig. 6B). In addition, we assayed PdCP1 with chymostatin which contains a disfavored P1-Phe residue adjacent to a reactive aldehyde group. As expected, this compound only weakly inhibited PdCP1 with an IC50 between 17 and 35µM (95% confidence interval). These studies establish leupeptin as a potent inhibitor of PdCP1 activity and provide new chemical tools with which the function of PdCP1 in P. destructans can be assessed in

future experiments.

Discussion

P. destructans is responsible for White-nose Syndrome, a disease that continues to afflict numerous bat species in North America. However, little is known about how this filamentous fungus causes disease in the mammalian host, particularly given that closely related species are harmless saprophytes. Saprophytic fungi generally possess a greater number of secreted enzymes than animal pathogens, which may reflect their need for greater metabolic flexibility (Krijger et al. 2014). It has also been suggested that smaller secretomes are an adaptation by animal pathogens to evade host immune responses as

many secreted proteins likely have immunogenic potential (Campbell et al. 2015; Krijger

et al. 2014).

Here, we reveal that the P. destructans secretome is more limited in scope than that of four related saprophytic species. Reduced numbers of secreted redox enzymes and glycosidases from P. destructans compared to relatives contributed to this difference.

This is consistent with a recent analysis that showed a marked loss of genes encoding

putative secreted proteins in the P. destructans genome, especially those for

carbohydrate-active enzymes (179 encoding genes vs. 463-544 in related species, Palmer

et al. 2018). The same study also found that the P. destructans genome contains fewer peptidase-encoding genes than its non-pathogenic relatives. In contrast to the genomic analysis, however, we found that the P. destructans secretome contained the second highest number of peptidases (and the highest proportion of peptidases overall). Secreted peptidases may be important for pathogenicity as these enzymes are widely implicated as virulence factors in a range of disease-causing fungi (Monod 2008; Jousson et al. 2004;

Martinez-Rossi et al. 2017; Monod et al. 2002; Yike 2011). Together, these data suggest that P. destructans has streamlined its secretome allowing it to evade host immunity while potentially maintaining factors important for a pathogenic lifestyle.

We chose to examine one peptidase, PdCP1, in more detail given that it was uniquely detected in the P. destructans secretome and that carboxypeptidase activity had previously been observed in this secretome (O’Donoghue et al. 2015). Biochemical characterization of carboxypeptidases is often challenging due to a lack of effective substrates for kinetic assays. However, PdCP1 was able to hydrolyze an internally- quenched (IQ) fluorescent substrate between Leu and Lys(DNP) indicating that it had unusual substrate specificity. To our knowledge, this is the first example of a mono- carboxypeptidase being able to efficiently process an IQ substrate, although this ability

has previously been described for a human di-carboxypeptidase, cathepsin B (Cotrin et

al. 2004). The ability of PdCP1 to process an IQ substrate facilitated the isolation and

subsequent characterization of this enzyme.

PdCP1 specificity was analyzed in detail using an MSP-MS assay containing a

diverse library of tetradecapeptide substrates (O'Donoghue et al. 2012). This analysis established PdCP1 as a strict mono-carboxypeptidase as processive cleavage of single

C-terminal residues was observed on multiple target peptides. PdCP1 continued to hydrolyze the C-terminus of each peptide until unfavored amino acids were encountered.

In this manner, PdCP1 hydrolyzed at least the first C-terminal residue from 175 of the 228 peptides following 1 h incubation, indicating it possesses a relatively broad substrate specificity. PdCP1was also able to cleave all possible amino acids at the P1ʹ position in this assay, albeit at different rates (see Supp. Fig. 3), thereby explaining its ability to hydrolyze an IQ substrate with a bulky non-natural amino acid such as Lys(DNP).

PdCP1 also demonstrated a requirement for substrates of a certain length, as it was able to cleave a tripeptide substrate (Boc-Leu-Arg-Arg-AMC) at a much higher rate than mono- or dipeptide substrates of a similar composition (z-Arg-Arg-AMC and Arg-

AMC, see Fig. 5A). This indicates that residues distal to the scissile bond (P2 and further) are important for substrate recognition. Biochemical analysis of PdCP1 using the AMC reporter substrate also revealed a temperature optimum of 20ºC and inhibition by high salt concentrations. The low temperature optimum of PdCP1 is consistent with P. destructans being unable to grow above ~18ºC. While the sensitivity of PdCP1 to NaCl is unusual, it is not immediately clear how this might impact its activity during growth of P. destructans on its host or in the environment.

Although it possessed a broad substrate specificity, PdCP1 also demonstrated a notable preference for basic residues such as Arg. This preference is consistent with

other serine carboxypeptidases, all of which belong to the S10 peptidase family (Rawlings

et al. 2014). The acidic pH optimum of PdCP1 (pH 4.6) also matches that of other serine

carboxypeptidases which are typically most active at pH 3-5 (Breddam 1986; Remington

1993). PdCP1 activity was inhibited by multiple serine/cysteine peptidase inhibitors

including leupeptin and antipain, two peptide-aldehyde-based inhibitors. Both leupeptin

and antipain contain Arg residues that correspond to the P1 substrate position, and the

sensitivity of PdCP1 to these compounds is therefore consistent with its preference for

basic residues.

The broad specificity of PdCP1 suggests it could serve a general metabolic

function in P. destructans by generating free amino acids. Recent studies indicate that P.

destructans possesses a more limited carbon metabolism than non-pathogenic relatives but has retained the ability to use single amino acids as sole carbon sources (Wilson et al. 2017; Palmer et al. 2018). Amino acids released by PdCP1 could therefore provide an important carbon and nitrogen source in addition to supporting de novo protein synthesis.

As P. destructans is not an obligate pathogen but retains the ability to grow environmentally as a saprophyte (Reynolds et al. 2015), PdCP1 could function during both lifestyles. Peptidases may be particularly important during infection, however, as mammalian dermal tissue is highly abundant in a variety of potential protein substrates

(Mikesh et al. 2013). Additionally, the pH range of PdCP1 suggests it could be active on mammalian skin which is slightly acidic (Matousek and Campbell 2002; Blank 1939).

Dermatophytes similarly colonize mammalian skin and use secreted peptidases to break down keratin and other dermal proteins into amino acids. These fungi use a synergistic combination of peptidases, with endopeptidases generating new peptide termini for subsequent digestion by exopeptidases (Monod 2008; Baldo et al. 2012). The breakdown of host proteins by dermatophytes and other fungal pathogens is also known to contribute

to pathogenesis by promoting adherence and invasion of host tissues (Baldo et al. 2012;

Baldo et al. 2008). By analogy, P. destructans similarly secretes a variety of endo and exopeptidases and the combination of these peptidases may contribute to host colonization and tissue invasion by this species. Further studies of secreted peptidases from P. destructans and their roles during infection may therefore lead to a better understanding of WNS pathogenesis and could reveal important avenues of intervention against this deadly fungus.

Materials and Methods

Strains and culture media

P. destructans strain 20631-21 (purchased from ATCC), P. verrucosus, P. sp.

05NY8, P. sp. 03VT05, and P. sp. WSF3629 (CFMR culture collection) and P. pastoris

PichiaPink™ Strain-1 (ade2, Invitrogen) were maintained on Yeast Extract Peptone

Dextrose (YPD) agar. P. pastoris PdCP1 and PdCP1-S207A expression strains were

maintained on Synthetic Complete Dextrose agar lacking adenine (SCD-Ade). Low pH

BMGY and BMMY were made according to PichiaPink™ (Invitrogen) manual recipes with

the exception that potassium-phosphate buffer, pH 6, was replaced with phosphate-citrate

buffer, pH 3, as preliminary screening of media conditions revealed the highest level of

peptidase activity from PdCP1-expressing cells when grown at this pH.

Secretome generation

For generation of fungal secretome samples, spores were collected from YPD plates using a solution of 0.05% Tween 20 (Sigma-Aldrich) and rubbing the surface of the agar. Spores were passed through sterile Miracloth (Millipore, # 475855) to remove larger hyphal material and adjusted to 4 x 105/mL in RPMI medium (Thermo-Fisher). Inoculated

RPMI was divided into multiple 5 mL cultures in glass culture tubes and incubated at 14°C

on a rotator drum. After 1 week of growth, the conditioned medium containing secreted

proteins (secretome) was harvested and separated from mycelia by passing through

Miracloth. Secretomes were buffer exchanged into phosphate-buffered saline (PBS) +

20% glycerol and concentrated 100-fold using 0.5 mL 10 kDa MWCO centrifugal filters

(Amicon, # UFC501024), flash frozen in liquid nitrogen and stored at -80°C. Three

biological replicates were generated and analyzed for each species.

Generation of recombinant PdCP1 expression constructs

To generate the PdCP1 expression construct, a P. pastoris codon-optimized

version of the PdCP1 (GMDG_05452) open reading frame was generated (GeneArt®

Strings™, Invitrogen). This construct lacks the endogenous secretion signal sequence

and contains a C-terminal 6x His epitope tag. The gene was inserted into the pPinkα-HC plasmid (PichiaPink™ expression system, Invitrogen) between the StuI and KpnI restriction sites in frame with the N-terminal α secretion signal present in the pPinkα-HC plasmid and transformed into E. coli JM109 cells (Promega). Successful generation of the desired plasmid was confirmed by restriction fragment analysis and Sanger sequencing. The PdCP1-S207A expression construct was generated by site-directed mutagenesis of the existing PdCP1 expression plasmid. Mutagenesis was conducted by

PCR amplification of the PdCP1-expression plasmid with Phusion polymerase (Thermo-

Fisher) in HF-buffer for 18 cycles, with a 60°C annealing step and an 8-minute elongation

step. PCR used primers containing the desired mutation (primer 1-

CATTACTGGTGAGGCTTATGCTGGTCAGTATATCC, primer 2-

GGATATACTGACCAGCATAAGCCTCACCAGTAATG, mutated codon underlined). The resulting PCR product was treated with DpnI (New England Biolabs) to digest template

DNA and re-transformed into JM109 cells. Successful mutagenesis was confirmed by

Sanger DNA sequencing.

Transformation of P. pastoris

PdCP1 and PdCP1-S207A expression plasmids were linearized within the pPinkα-

HC TRP2 locus using EcoNI (New England Biolabs) and transformed into competent P.

pastoris PPink Strain-1 cells by electroporation using established protocols (PichiaPink™

manual, Invitrogen). Briefly, 7-10 µg of linearized DNA was mixed with 80 µl of freshly

prepared competent P. pastoris cells in a 0.2 cm electroporation cuvette and pulsed once

at 2 kV in a MicroPulser™ electroporator (BioRad). Electroporated cells were immediately

diluted with 1 mL YPD + 1 M sorbitol, recovered at 30°C for 2.5 h, and plated on SCD-Ade agar to select for the expression construct. Presence of the expression construct was confirmed by PCR.

PdCP1 expression and purification

To generate recombinant proteins, PdCP1 or PdCP1-S207A expressing P.

pastoris strains were grown for ~24 h in 25 mL low-pH BMGY at 25°C with shaking (220

rpm). This starter culture was used to inoculate 1 L low-pH BMGY cultures which were

grown under the same conditions to an optical density (OD600) of 2-3. Cultures were

centrifuged (5 min, 1500 g), and the cells resuspended in 200 mL low-pH BMMY induction

medium and cultured under the same conditions for 48 h. During culture in BMMY 500 µl

of MeOH was added every 12 hours to maintain induction of the expression construct.

BMMY culture supernatant containing secreted recombinant protein was harvested via

centrifugation (15 min, 3500 g), diluted 1:1 in Binding buffer (10 mM phosphate-citrate, pH

5, 10 mM NaCl) and concentrated to ~ 50 mL using a stir-cell concentrator (Amicon model

8400) at 4°C. The concentrated supernatant was diluted again to 400 mL in Binding buffer and re-concentrated to ~40 mL. The concentrated, buffer-exchanged supernatant was

then loaded onto a gravity-flow chromatography column (BioRad 731-1550) packed with

0.5 mL of sulphopropyl (SP) Sepharose FastFlowTm (GE healthcare) beads pre-washed

in Binding buffer. The column was washed with 10 mL Binding buffer and elutions (2 mL

each) conducted with increasing concentrations of NaCl (25, 50, 100, 150, 200, 250, and

500 mM). Fractions containing the recombinant PdCP1/PdCP1-S207A protein were

determined by activity assays and/or SDS-PAGE and Coomassie/silver staining with

subsequent LC-MS/MS analysis of putative PdCP1/PdCP1-S207A gel bands.

SDS-PAGE

For SDS-PAGE analysis of PdCP1/PdCP1-S207A proteins and SP

chromatography fractions, samples were mixed with 1/5 volume of 6x Laemmli buffer and

heated at 85°C for 5 min prior to loading. 5-20 µL of sample was loaded per well of a Mini-

PROTEAN® TGX Stain-Free™ gel (BioRad, #456-8086). Resulting gel bands were

visualized by Coomassie blue (Thermo-Fisher) or silver staining. Precision Plus Protein™

standards (BioRad, #1610373) were used to estimate the molecular weights of sample

gel bands. For mass spectrometry, gel bands of interest were cut out on a methanol-

cleaned surface using a scalpel and placed in a microcentrifuge tube with an equal volume

of water. For SDS-PAGE analysis of PNGase F-treated PdCP1, purified PdCP1 was

denatured and incubated for 1 h with PNGase F following the manufacturer’s protocol

(New England Biolabs) and 25µL was loaded alongside equal volumes of mock-treated

PdCP1 protein and PNGase F alone.

Fluorescent peptidase assays

Fluorescent peptidase activity assays were conducted with the internally-

quenched (IQ) peptide substrate (7-methoxycoumarin-4-yl)acetyl-PKRLSALL-K(2-4-

Dinitrophenyl) or peptide substrates modified with a C-terminal 7-amino-4-methylcoumarin

(AMC) group. AMC substrate sequences are provided in figures legends. All fluorescent

peptidase assays were conducted in black 96-well assay plates with a final reaction

volume of 50-100 µl and 100-600 ng of PdCP1/PdCP1-S207A protein per reaction. The

IQ substrate was used at a final concentration of 40 µM and AMC substrates were used

at a final concentration of 10 µM for all assays. AMC substrate assays were conducted in

25 mM phosphate citrate buffer (pH 4.6 or 6.2) with 0.05% Tween 20. IQ substrate assays

were conducted in 25 mM phosphate citrate buffer pH 4.6. For pH analysis, pH 2.2-7.8 reactions were performed in 25 mM phosphate citrate, whereas pH 7.4-8.6 reactions were

performed in 25 mM Tris-HCL. Overlapping pH reactions in both buffers were used to

normalize activity between buffers. All assays were read immediately after combining

substrate and enzyme in a Synergy HT plate reader (AMC substrates: 360/40 nm and

460/40 nm, IQ substrate: 320/20 nm and 400/30 nm).

MSP-MS assay

5 nM of PdCP1 was incubated in triplicate with a mixture of 228 synthetic

tetradecapeptides (0.5 µM each) in 10 mM sodium citrate, pH 4.5, and 10 mM sodium

citrate, pH 6.2. 10% of the reaction mixture was removed after 5, 15, 60 and 240 min of

incubation and the enzyme activity quenched with 6.4 M GuHCl and stored immediately

at -80°C. Prior to MS/MS analysis, samples were thawed, acidified to

trifluoroacetic acid and desalted using C18 LTS tips (Rainin).

Mass Spectrometry

For mass spectrometry analysis of fungal secretions, 5-20 µg of each secretome was prepared by in solution digestion with trypsin. Samples were denatured and reduced

with solid urea (8 M final) and DTT (10 mM), and incubated at 56°C for 20 min.

Iodoacetamide was added to each sample (15 mM final) using a freshly-made stock solution, and samples were incubated in the dark at 22°C for 1 h. Reactions were quenched with an additional 5 mM DTT, then diluted with 50 mM ammonium bicarbonate to 1 M urea final concentration prior to addition of sequencing-grade trypsin (Sigma-

Aldrich, or Promega) at 37 °C for 4 h (1:20 wt:wt trypsin to total protein). Digestions were

stopped with 5 µl 10% formic acid, desalted with C18 Zip-tips (Millipore) and dried in a

Speedvac concentrator to fully remove solvent. Peptides were separated by liquid

chromatography (LC) with nanoACUITY (Waters) ultra-performance instrument for

reverse-phase chromatography with a C18 column at 10,000 psi (BEH130, 1.7-μm bead size, 100 µm × 100 mm) and sequenced using an LTQ-Orbitrap Velos mass spectrometer.

A 300 nl/min flow rate with a linear gradient over 42 min from 2% to 30% (vol/vol)

acetonitrile in 0.1% formic acid was used for LC, as previously reported (O’Donoghue et

al. 2015).

Survey scans were recorded over 350-1,400 m/z, and MS/MS was performed

using higher-energy collisional dissociation (HCD) activation on the 10 most-intense

precursor ions, with minimum signal of 2,000 counts, using an isolation width of 2.5 amu,

and a normalized collision energy of 30 over a mass range of 350–1500 m/z. Internal

recalibration of both MS and MS/MS scans was performed using a

polydimethylcyclosiloxane ion with m/z = 445.120025 (Olsen et al. 2005). Data were

analyzed using in-house PAVA software to generate peak lists and Protein Prospector

software v.5.20 was used for searching peptide hits against whole proteome databases

generated for the corresponding species (Guan et al. 2011). Each species database was concatenated with an equal number of randomized (decoy) protein sequences for determination of false discovery rate (FDR)(Elias and Gygi 2007). Parent and fragment ion error tolerances of 20 ppm and 30 ppm, respectively, were used and up to one missed trypsin cleavage was allowed during protein identification, and carbamidomethylated cysteines were set as a fixed modification. N-terminal acetylation, acetylation, oxidation and removal of start methionine, N-terminal pyroglutamate from glutamine and oxidation of methionine were included as variable modifications. Proteins are reported at <1% FDR cutoff in the provided secretome lists (Supp. Tables 1-5) and were required to have a best expectation value less than 1x10-5 in at least 2 of 3 replicates for additional confidence.

Proteins in each list were functionally annotated using PANNZER software based on descriptions (DEs) assigned to each amino acid sequence (webserver) (Koskinen et al.

2015). Proteins were manually categorized by putative enzymatic activity based on the assigned functional annotations. Peptidase orthologues were assigned by reciprocal

Blastp searches of P. destructans amino acid sequences vs. each related species using

cutoffs of at least 80% coverage and 60% sequence identity to define orthologues.

Protein bands from SDS-PAGE were digested with trypsin according to the UCSF

in gel digestion protocol (http://msf.ucsf.edu/protocols.html). For protein and peptide

identifications, mass spectrometric data was processed and searched with Protein

Prospector as described above.

For identification of peptide cleavage sites in MSP-MS substrates, 0.22 ng of the

peptide mixture dissolved in 0.1% formic acid, was injected into a Q Exactive Mass

Spectrometer (Thermo) equipped with an Ultimate 3000 HPLC. Peptides were separated

by reverse phase chromatography on a C18 column (1.7 um bead size, 75 um x 20 cm,

heated to 65°C) at a flow rate of 400 nl min-1 using a 55 min linear gradient from 5% B to

30% B, with solvent A being 0.1% formic acid in HPLC grade water and solvent B being

0.1% formic acid in acetonitrile. Survey scans were recorded over a 150–2000 m/z range

at 70000 resolution at 200 m/z (AGC target 1×106, 75 ms maximum injection time).

MS/MS was performed in data-dependent acquisition mode with HCD fragmentation (30 normalized collision energy) on the 10 most intense precursor ions at 17500 resolution at

200 m/z (AGC target 5×104, 120 ms maximum injection time, dynamic exclusion 15s).

The cleavages within the IQ fluorescent substrate were determined by mass spectrometry using the same method as described above except using a 20-min linear gradient from

5% B to 50% B and MS/MS acquisition was performed on top 5 most intense precursor ions.

Peak integration and peptide identification were performed using Peaks software

(Bioinformatics Solutions Inc.). Quantification data are normalized by LOWESS and filtered by 0.3 peptide quality. Missing and zero values are imputed with random numbers in the range of the average of smallest 5% of the data ± sd. Differences between each

69 time point and no-enzyme control were analyzed for statistical significance by multiple t- test. When compared to the control reaction, peptide cleavage products with >10-fold change in peak area intensity and p-value <0.05 were identified and the peptide sequence corresponding to the P4 to P4' subsite positions were used to make IceLogo frequency plots (Colaert et al. 2009). Raw mass spectrometry data files and peak list files have been deposited at ProteoSAFE (http://massive.ucsd.edu) with accession number

MSV000082319. (During review, data may be accessed at: ftp://[email protected] with the password "a").

Acknowledgements

We thank Matt Ravalain for his advice on inhibitors of PdCP1. Funding for this project was provided by a National Science Foundation grant (NSF-1456787) to RJB and Skaggs

School of Pharmacy and Pharmacutical Sciences to AJO. Funding for JMP and DLL was provided by the Northern Research Station, USDA Forest Service. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Figures

Figure 1. LC-MS/MS analysis of secretomes from P. destructans and four non- pathogenic relatives reveals a wide range of secreted enzymes. Proteins detected by LC- MS/MS were functionally annotated using PANNZER (Koskinen et al. 2015) and proteins with putative enzymatic activity were categorized by enzyme class.

Figure 2: Recombinant expression and purification of PdCP1, a putative serine carboxypeptidase. (A) SDS-PAGE analysis of the supernatant from P. pastoris cells engineered to overexpress PdCP1 (1) and PdCP1 protein purified by SP chromatography (2). (B) SDS-PAGE analysis of purified PdCP1 before (1) and after (2) PNGase F treatment to remove N-linked glycosyl groups (lane 3 contains PNGase F enzyme alone). (C) Amino acid sequence of the recombinant PdCP1 protein. Highlighted residues indicate peptide fragments detected by LC-MS/MS from an excised putative PdCP1 gel band.

Figure 3: PdCP1 activity vs IQ substrate. (A) PdCP1 activity was assayed across a range of pH conditions from pH 2.2 to 8.6 using the IQ substrate. Error bars = SD using 6 - replicates from 2 independent experiments. (B) Relative peptidase activity vs. IQ substrate in conditioned medium (CM) from PdCP1-expressing P. pastoris cells and the purified PdCP1 sample (error bars = SD of 6 replicates). (C) Kinetic activity plot of IQ substrate cleavage by PdCP1 and S207A mutant measured by fluorescence (each data point is average of 3 replicates). (D) Liquid chromatography trace of IQ substrate cleavage products after incubation with PdCP1. Individual peaks were analyzed by MS/MS and the corresponding cleavage products identified (labeled 1-7)). The T on each x axis indicates incubation time of substrate with PdCP1 prior to analysis (0 and 4 hours).

Figure 4: Characterization of PdCP1 substrate specificity by MSP-MS assay. (A) Histogram indicating total number and bond position of all PdCP1-generated cleavages detected at each time point. (B) Bar charts indicating measured intensities of fragments generated by PdCP1-cleavage of a representative MSP-MS substrate across each time point. (Error bars = SD of at least 3 replicates per condition). (C) Substrate specificity signature based on C-terminal PdCP1-generated cleavages detected after 15 minutes using IceLogo. (Colaert et al. 2009). Favored residues are indicated above the horizontal line and disfavored residues below. Horizontal positions of residues indicate their distance from the cleaved peptide bond between P1 and P1’ (only cleavages with P < 0.05 and 10-

fold increase in intensity over no-enzyme condition were used in figure, assay conducted at pH 4.6).

Figure 5: Screening of AMC substrates and characterization of PdCP1 activity. (A) Bar chart indicating cleavage rates for PdCP1 on select AMC substrates. (B-D) Relative PdCP1 activity on AGPR-AMC substrate across a range of pH values (B), NaCl concentrations (C) and temperatures (D). Error bars = SD of at least 3 replicates per condition.

Figure 6: Screening peptidase inhibitors against PdCP1. (A) Relative PdCP1 activity on AGPR-AMC substrate upon application of individual peptidase inhibitors. (B) Inhibition curve of antipain, chymostatin and leupeptin against PdCP1 (AGPR-AMC substrate). Error bars = SD of 3 replicates per condition.

Table 1: A list of putative peptidases detected in the P. destructans secretome, gene ID’s and species where orthologues were detected in the corresponding secretome. Peptidases uniquely detected in the P. destructans secretome are in bold.

Supplementary Figures

Supplementary Figure 1. SDS-PAGE analysis of PdCP1 (1) and PdCP1-S207A purified by ion exchange chromatography

Carboxypeptidase_O_A.parasiticus MRAATAIASLFL--VGSVVGLENPHRKAKAVQRAHQHKTVLPRAVPVARDDDYKYLTNKT Carboxypeptidase_4_A.fumigatus] MHFAVIVITLLV--ASSAVALENPHRKAVRPIK-LDHGHLKPRAVTVADDGGYKYLNKQT Carboxypeptidase_O_S.sclerotiorum MRGLNSLALLLAASATSVVAILPHHEAHAKYAKRSLPKQILARQPVVRSRSTSKYLTNST Carboxypeptidase_O_B.fuckeliana MRGLNSLAVLLAASVTSVVAVLPHHEAHAKYAKRAQPKHVLPRQPALKPRSISKYLTNST Carboxypeptidase_O_P.pannorum MKTIRALLLLVAAS--AVTAAQNPHEKAAKYVQKKAPEPVLSKH-ASRAVNTAQFLNKHT PdCP1 MQTIRALLLLLAAS--VVTAAQNPHEKAAKYVQKRSAEPVVSKH-ASRAANTAHFLNNKT *: : *. ... *. : : : . ::*.: *

Carboxypeptidase_O_A.parasiticus ERFLVNGTGIPEVDFDVGETYAGLLPNSPA--GNSSLFFWFFPSQNPKAQDEITIWLNGG Carboxypeptidase_4_A.fumigatus] QRFLVNGTGIPEVDFDVGESYAGTLPNTPA--GNSSLFFWFFPSQNPKAHDEITIWLNGG Carboxypeptidase_O_S.sclerotiorum ASFAVNGTALPEVDFDIGESYAGTLPVSTNASDTNRLWFWFFPTDNPAAEKEITIWLNGG Carboxypeptidase_O_B.fuckeliana ASFAVNGTALPEIDFDIGESYAGTLPISTNASDTNRLWFWFFPSENPLAEKEITIWLNGG Carboxypeptidase_O_P.pannorum AKFAVNGSELPEVDFNIGESYAGTLPISSKQDDENRLWFWFFPSSNPAAKKEITIWLNGG PdCP1 AKFAINGSALPEVDFNIGESYAGTLPISSKKDDENRLWFWFFPSSNPAAKKEITIWLNGG * :**: :**:**::**:*** ** : . . *:*****:.** *..*********

Carboxypeptidase_O_A.parasiticus PGCSSLDGLLQENGPFLWLPGTYKPARNPYSWTNLTNVVYIDQPAGTGFSPGPSTVDDEE Carboxypeptidase_4_A.fumigatus] PGCSSLDGLLQENGPFLWQSGTYKPIRNPYSWTNLTNMVYVDQPAGTGFSPGPSTVNNEE Carboxypeptidase_O_S.sclerotiorum PGCSSLDGFFQENGPLSWQSGTYAPILNPYSWTNLTNMIWIDQPVSTGFSPGDILVDDEI Carboxypeptidase_O_B.fuckeliana PGCSSLDGLFQENGPFSWQSGTYAPIPNPYSWTNLTNMIWIDQPVSTGYSPGDILVDDEI Carboxypeptidase_O_P.pannorum PGCSSLNGLFQENGPFLWQPGTYEPFANPYSWVNLTNMIYIDQPVSTGFSPGTILVDDEG PdCP1 PGCSSLNGLFQENGPFLWQPGTYAPFANPYSWVNLTNMIYIDQPVSTGFSPGTVLVDDEN ******:*::*****: * *** * *****.****::::***..**:*** *::*

Carboxypeptidase_O_A.parasiticus GVAAQFKSWFKHFVDTFGLHGHKVYLTGESYAGQYIPYIASAMLDEEDEKYFNVKGIQIN Carboxypeptidase_4_A.fumigatus] DVARQFKSWFKHFVDTFNLHGRKVYITGESYAGQYIPYIASAMLDEKDKKYFNVKGIQIN Carboxypeptidase_O_S.sclerotiorum DVGNQFAAFWKNFIDTFSMQGYKVYITGESYAGQYIPYIASNFLDRNDTIYYNLKGIQIN Carboxypeptidase_O_B.fuckeliana DVGNQFAAFWKNFIDTFSMQGYKIYITGESYAGQYIPYIASNFLDRNDTTYYNLKGIQIN Carboxypeptidase_O_P.pannorum DVAEQFMGFWKNFIDTFSLQGYKIYITGESYAGQYIPYIASGMLDTEDKKYFNVKGVQIN PdCP1 DVAEQFMGFWKNFIDTFSMQGYKIYITGESYAGQYIPYIASGMLDTKDNKYFNVKGVQIN .*. ** .::*:*:***.::* *:*:*************** :** :* *:*:**:***

Carboxypeptidase_O_A.parasiticus DPSINDDSVMIYAPAVRHLNHYTNVFALNDTFLADVNSRADKCGFNKFLDE-ALTYPPPK Carboxypeptidase_4_A.fumigatus] DPSINDDSVMIYAPAVSHLNQYLNVFSLNDTFVKHINKRAEECGYNKFLDE-AITYPPPK Carboxypeptidase_O_S.sclerotiorum DPSINEFDTMGSAPVTAAALYYQNILNLNDTYIANITARAKSCGYTEFL-EYATVFPPAG Carboxypeptidase_O_B.fuckeliana DPSINDFDTMGSAPVTAAALYYQNVLNLNDTYIANITARAKSCGYTDFL-EYASVFPPAG Carboxypeptidase_O_P.pannorum DPSINSDDVLLHAPVVPALNYFNNVINLNESFIANITARADSCGYTDFFNKYTTEFPPSG PdCP1 DPSINTDDVLLHTPIVPALNYFNNVINLNESFIANITARADSCGYTDFFNKWTTEFPPSS ***** ..: :* . : *:: **:::: .:. **..**:..*: : : :**

Carboxypeptidase_O_A.parasiticus DFPVLPEI-NSECAIWDDIVAAAYDVNPCFNYYHLTDYCPYLWSEMGFPSLAGGPNNYFN Carboxypeptidase_4_A.fumigatus] EFPVAPDPSKNNCALWDDIVEAAYYVNPCFNFYHLTDFCPYLWDEMGFPSLAGGPNNYFN Carboxypeptidase_O_S.sclerotiorum PIPTAPSSEEYGCDLYDDIYNAAYYVNPCFNIYHLTDYCPYLWDELGFPSLGGGPNNYFN Carboxypeptidase_O_B.fuckeliana PIPTAPSSSEYGCDLYDDIYNAVYYVNPCFNIYHLTDFCPFLWDELGFPSLAGGPNNYFN Carboxypeptidase_O_P.pannorum KLPPAPNYKKPGCDIYDEVYNAAYYENPCFNIYHLTDYCPYQWNELGFPSLAGGPNNYFN PdCP1 KIPTAPSWKEPGCDIFDEVYNAAYYENPCFNIYHLTDYCPYQWNLLGFPSMAGGPNNYFN :* *. : * ::*:: *.* ***** *****:**: *. :****:.********

Carboxypeptidase_O_A.parasiticus RTDVQKALHVPR-TDYSVCG-ETTIFKNGDQSPPSALGPLPSVIERTNNTIIGHGWLDYL Carboxypeptidase_4_A.fumigatus] RSDVQKALHVPP-TDYSVCG-ETTIFAKGDQSVPSALGPLPSVIERTNNVLIGHGWLDYL Carboxypeptidase_O_S.sclerotiorum RTDVKEVIHAPVDTDYFVCTGGPNLFPNGDKSIDSALGPLPSVIERTNNVIIGHGLLDFL Carboxypeptidase_O_B.fuckeliana RTDVKEVLHAPVDVDYFVCTGGPNLFPEGDKSIDSALGPLPSVIERTNNVIIGHGLLDFL Carboxypeptidase_O_P.pannorum RTDVKKAIHAPPDANYMVCGGGDNLFPHGDKSIESGLGPLPSVIERTNNTIIGHGLLDFL PdCP1 RTDVQKAINAPP-TNYMVCGGGHNLFPNGDKSIESSLGPLPSVIERTNNTIIGHGLLDFL *:**::.::.* .:* ** .:* .**:* *.*************.:**** **:*

Carboxypeptidase_O_A.parasiticus LFLNGSLVTIQNMTWNGAQGFQKPPV--EPLYVPYHYGLAELAN---GNA--PEPFTLIA Carboxypeptidase_4_A.fumigatus] LFVNGSLATIQNMTWNGAQGFQHPPV--EPLYVPYHYGLAELVT---STA--PNPYTLNA Carboxypeptidase_O_S.sclerotiorum LFANGSLITIQNMTWNGLQGFQEAPSSTQNLYVPYHQSLGTILTIANAAIPNTPPQNDVA Carboxypeptidase_O_B.fuckeliana LFANGSLITIQNMTWHGLQGFQEAPSSTQNFYVPYHQSIGEILSIVNDAIPNSPPQYDTA Carboxypeptidase_O_P.pannorum LFANGSLITIQNMTWNGAQGFQTSPFKEQNFYVPYHQTNGDILQVVNGIEPHV--FTDTA PdCP1 LFANGSLISIQNMTWNGAQGFQTSPFKKQNFYVPYHQTNGEILQYANGINTHV--FTDTA ** **** :******:* **** * : :***** . : *

Carboxypeptidase_O_A.parasiticus GAGLLGTAHTERGLTFSSVYLAGHEIPQYVPGAAYRQLEFLLGRIENLQQRGGYTA---- Carboxypeptidase_4_A.fumigatus] GAGYLGTAHTERGLTFSTVYMAGHEIPQYTPGAAYRQLEFLLGRIDNLSSPGSYTA---- Carboxypeptidase_O_S.sclerotiorum GAGFQGTWHTERGLTFATVNLAGHEIPQYTPGVGYRQLEFLLGRIANLSVVGDYTT-QTG Carboxypeptidase_O_B.fuckeliana GAGYQGTWHTERGLTFSTVNLAGHEIPQYTPGAGYRHLEFLLGRIANLSVVGDYTT-QTG Carboxypeptidase_O_P.pannorum GGGFQGVTHTERGLTFVTVNLAGHMIPQYVPGAAYRQLEFLLGRVSSLEQRGDFTTGPQG PdCP1 GGGFQGVTHTERGLTFVTVNLAGHMIPQYVPGAAYRQLEFLLGRVSSLEQRGDFTTGPQG *.* *. ******** :* :*** ****.**..**:*******: .*. *.:*:

Carboxypeptidase_O_A.parasiticus ------Carboxypeptidase_4_A.fumigatus] ------Carboxypeptidase_O_S.sclerotiorum NFTGVSAPLKMRGLEY Carboxypeptidase_O_B.fuckeliana NFTGVSAPLKMRGL-- Carboxypeptidase_O_P.pannorum NYTGVSPPLK------PdCP1 NYTGGTW------

Supplementary Figure 2. Amino acid alignment of PdCP1 and protein members of Carboxypeptidase O family (MEROPS S10.014).

Tyr Trp Val Thr Ser Arg Gln

Pro Asn Met

Leu Lys Amino acid acid Amino in P1' Ile

His Gly Phe

Glu Asp Ala

0 2 4 6 8

# of detected cleavages at 60 minutes

Supplementary Figure 3. Bar chart indicating the number of detected (MSP-MS) PdCP1-generated cleavages containing the indicated Amino acids at the P1’ position (C- terminal Amino acid that is freed upon cleavage). Data represents all cleavages with P < .05 and 10-fold increase over no-enzyme condition after 60 minutes at pH 4.6

Supplementary Figure 4. MSP-MS analysis of PdCP1 at pH 6.2 A .Venn diagrams representing total number of PdCP1-dependent cleavages detected in MSP library at each time point for pH4.6 (green) and 6.2 (purple) conditions. B. Histogram indicating total number and bond position of all PdCP1 generated cleavages detected at each time point. C. Substrate specificity signature, based on C-terminal PdCP1-generated cleavages detected after 60 minutes at pH 6.2 using IceLogo 2. Favored residues are indicated above the horizontal line and disfavored residues below. Horizontal positions of residues indicate their distance from the cleaved peptide bond (between P1 and P1’) D. bar charts indicating measured intensities of fragments generated by PdCP1-cleavage of 2 representative MSP-MS substrates across each time point

References

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology 215; 8.

Baldo, A., Monod, M., Mathy, A., Cambier, L., Bagut, E. T., Defaweux, V., Symoens, F., Antoine, N., and Mignon, B. (2012). Mechanisms of skin adherence and invasion by dermatophytes. Mycoses 55; 218-23.

Baldo, A., Tabart, J., Vermout, S., Mathy, A., Collard, A., Losson, B., and Mignon, B. (2008). Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes. J Med Microbiol 57; 1152-6.

Behnsen, J., Lessing, F., Schindler, S., Wartenberg, D., Jacobsen, I. D., Thoen, M., Zipfel, P. F., and Brakhage, A. A. (2010). Secreted Aspergillus fumigatus protease Alp1 degrades human complement proteins C3, C4, and C5. Infect Immun 78; 3585- 94.

Blank, I. H. (1939). Measurement of pH of the skin surface. Journal of Investigative Dermatology 2; 231-34.

Boyles, J. G., Cryan, P. M., Mccracken, G. F., Kunz, T. H. (2011). Economic importance of bats in agriculture. Science 332; 2.

Breddam, K. (1986). Serine Carboxypeptidases; a Review. Carlsberg Research Communications 51; 45.

Campbell, L. T., Simonin, A. R., Chen, C., Ferdous, J., Padula, M. P., Harry, E., Hofer, M., Campbell, I. L., and Carter, D. A. (2015). Cryptococcus strains with different pathogenic potentials have diverse protein secretomes. Eukaryot Cell 14; 554-63.

Casadevall, A., Steenbergen, J. N., and Nosanchuk, J. D. (2003). ‘Ready made’ virulence and ‘dual use’ virulence factors in pathogenic environmental fungi — the Cryptococcus neoformans paradigm. Current Opinion in Microbiology 6; 332-37.

Chaturvedi, V., Springer, D. J., Behr, M. J., Ramani, R., Li, X., Peck, M. K., Ren, P., Bopp, D. J., Wood, B., Samsonoff, W. A., Butchkoski, C. M., Hicks, A. C., Stone, W. B., Rudd, R. J., and Chaturvedi, S. (2010). Morphological and molecular characterizations of psychrophilic fungus Geomyces destructans from New York bats with White Nose Syndrome (WNS). PLoS One 5; e10783.

Colaert, N., Helsens, K., Martens, L., Vandekerckhove, J., and Gevaert, K. (2009). Improved visualization of protein consensus sequences by iceLogo. Nat Methods 6; 786-7.

Cotrin, S. S., Puzer, L., De Souza Judice, W. A., Juliano, L., Carmona, A. K., and Juliano, M. A. (2004). Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides to define substrate specificity of carboxydipeptidases; assays with human cathepsin B. Anal Biochem 335; 244-52.

Elias, J. E., and Gygi, S. P. (2007). Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat Methods 4; 207- 14.

Guan, S., Price, J. C., Prusiner, S. B., Ghaemmaghami, S., and Burlingame, A. L. (2011). A data processing pipeline for mammalian proteome dynamics studies using stable isotope metabolic labeling. Mol Cell Proteomics 10; M111 010728.

Jousson, O., Lechenne, B., Bontems, O., Mignon, B., Reichard, U., Barblan, J., Quadroni, M., and Monod, M. (2004). Secreted subtilisin gene family in Trichophyton rubrum. Gene 339; 79-88.

Kaminishi, H., Miyaguchi, H., Tamaki, T., Suenaga, N., Hisamatsu, M., Mihashi, I., Matsumoto, H., Maeda, H., and Hagihara, Y. (1995). Degradation of humoral host defense by Candida albicans proteinase. Infection and Immunity 63; 984-88.

Koskinen, P., Toronen, P., Nokso-Koivisto, J., and Holm, L. (2015). PANNZER; high- throughput functional annotation of uncharacterized proteins in an error-prone environment. Bioinformatics 31; 1544-52.

Krappmann, S. (2016). How to invade a susceptible host: cellular aspects of aspergillosis. Curr Opin Microbiol 34; 136-46.

Krijger, J. J., Thon, M. R., Deising, H. B., and Wirsel, S. G. (2014). Compositions of fungal secretomes indicate a greater impact of phylogenetic history than lifestyle adaptation. BMC Genomics 15; 722.

Kunugi, S., Fukuda, M., and Hayashi, R. (1985). Action of serine carboxypeptidases on endopeptidase substrates, peptide-4-methyl-coumaryl-7-amides. European Journal of Biochemistry 153; 4.

Leopardi, S., Blake, D., and Puechmaille, S. J. (2015). White-Nose Syndrome fungus introduced from Europe to North America. Curr Biol 25; R217-9.

Lowe, R. G. T., and Howlett, B. J. (2012). Indifferent, affectionate, or deceitful: lifestyles and secretomes of fungi. PLoS Pathog 8; e1002515.

Maine, J. J., and Boyles, J. G. (2015). Bats initiate vital agroecological interactions in corn. Proc Natl Acad Sci U S A 112; 12438-43.

Martinez-Rossi, N. M., Peres, N. T., and Rossi, A. (2017). Pathogenesis of dermatophytosis: sensing the host tissue. Mycopathologia 182; 215-27.

Matousek, J. L., and Campbell, K. L. (2002). A comparative review of cutaneous pH. Veterinary Dermatology 13; 293-300.

Meteyer, C. U., Buckles, E. L., Blehert, D. S., Hicks, A. C., Green, D. E., Shearn-Bochsler, V., Thomas, N. J., Gargas, A., and Behr, M. J. (2009). Histopathologic criteria to confirm white-nose syndrome in bats. Journal of Veterinary Diagnostic Investigation 21; 411-14.

Mikesh, L. M., Aramadhaka, L. R., Moskaluk, C., Zigrino, P., Mauch, C., and Fox, J. W. (2013). Proteomic anatomy of human skin. J Proteomics 84; 190-200.

Monod, M. (2008). Secreted proteases from dermatophytes. Mycopathologia 166; 285- 94.

Monod, M., Capoccia, S., Lechenne, B., Zaugg, C., Holdom, M., and Jousson, O. (2002). Secreted proteases from pathogenic fungi. Int J Med Microbiol 292; 405-19.

Morschhauser, J., Virkola, R., Korhonen, T. K., and Hacker, J. (1997). Degradation of human subendothelial extracellular matrix by proteinase-secreting Candida albicans. FEMS Microbiol Lett 153; 349-55.

O'Donoghue, A. J., Eroy-Reveles, A. A., Knudsen, G. M., Ingram, J., Zhou, M., Statnekov, J. B., Greninger, A. L., Hostetter, D. R., Qu, G., Maltby, D. A., Anderson, M. O., Derisi, J. L., Mckerrow, J. H., Burlingame, A. L., and Craik, C. S. (2012). Global identification of peptidase specificity by multiplex substrate profiling. Nat Methods 9; 1095-100.

O’Donoghue, A. J., Knudsen, G. M., Beekman, C., Perry, J. A., Johnson, A. D., Derisi, J. L., Craik, C. S., and Bennett, R. J. (2015). Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome. Proc Natl Acad Sci U S A 112; E3152.

Ollert, M. W., Sohnchen, R., Korting, H. C., Ollert, U., Brautigam, S., and Brautigam, W. (1993). Mechanisms of adherence of Candida albicans to cultured human epidermal-keratinocytes. Infection and Immunity 61; 4560-68.

Olsen, J. V., De Godoy, L. M., Li, G., Macek, B., Mortensen, P., Pesch, R., Makarov, A., Lange, O., Horning, S., and Mann, M. (2005). Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4; 2010-21.

Palmer, J. M., Drees, K. P., Foster, J. T., and Lindner, D. L. (2018). Extreme sensitivity to ultraviolet light in the fungal pathogen causing white-nose syndrome of bats. Nat Commun 9; 35.

Pannkuk, E. L., Risch, T. S., and Savary, B. J. (2015). Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans. PLoS One 10; e0120508.

Pikula, J., Amelon, S. K., Bandouchova, H., Bartonicka, T., Berkova, H., Brichta, J., Hooper, S., Kokurewicz, T., Kolarik, M., Kollner, B., Kovacova, V., Linhart, P., Piacek, V., Turner, G. G., Zukal, J., and Martinkova, N. (2017). White-nose syndrome pathology grading in Nearctic and Palearctic bats. PLoS One 12; e0180435.

Puechmaille, S. J., Wibbelt, G., Korn, V., Fuller, H., Forget, F., Muhldorfer, K., Kurth, A., Bogdanowicz, W., Borel, C., Bosch, T., Cherezy, T., Drebet, M., Gorfol, T., Haarsma, A. J., Herhaus, F., Hallart, G., Hammer, M., Jungmann, C., Le Bris, Y., Lutsar, L., Masing, M., Mulkens, B., Passior, K., Starrach, M., Wojtaszewski, A.,

Zophel, U., and Teeling, E. C. (2011). Pan-European distribution of white-nose syndrome fungus (Geomyces destructans) not associated with mass mortality. PLoS One 6; e19167.

R. Srinivasan, R. J. V. G., C.S. Angadiyavar and R.W. Dreyfus. (1974). Anomalous fluorescence and laser emission from 7-alkylaminocoumarins in acid solutions. Chemical Physics Letters 25; 537-40. Rawlings, N. D., Waller, M., Barrett, A. J., and Bateman, A. (2014). MEROPS: the database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 42; D503-9.

Reichard, U., Lechenne, B., Asif, A. R., Streit, F., Grouzmann, E., Jousson, O., and Monod, M. (2006). Sedolisins, a new class of secreted proteases from Aspergillus fumigatus with endoprotease or tripeptidyl-peptidase activity at acidic pHs. Appl Environ Microbiol 72; 1739-48.

Remington, S. R. (1993). Serine carboxypeptidases: a new and versatile family of enzymes. Current Opinion in Biotechnology 4; 7.

Reynolds, H. T., and Barton, H. A. (2014). Comparison of the white-nose syndrome agent Pseudogymnoascus destructans to cave-dwelling relatives suggests reduced saprotrophic enzyme activity. PLoS One 9; e86437.

Reynolds, H. T., Ingersoll, T., and Barton, H. A. (2015). Modeling the environmental growth of Pseudogymnoascus destructans and its impact on the white-nose syndrome epidemic. J Wildl Dis 51; 318-31.

Sanglard, D., Hube, B., Monod, M., Odds, F. C., and Gow, N. A. R. (1997). A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infection and Immunity 65; 3539-46.

Sriranganadane, D., Waridel, P., Salamin, K., Feuermann, M., Mignon, B., Staib, P., Neuhaus, J. M., Quadroni, M., and Monod, M. (2011). Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH. Proteomics 11; 4422-33.

Takeuchi, M., Takagi, Y., Ebisui, R., Toyama, T., and Ichishima, E. (2014). Mode of action on fluorogenic substrates of acid carboxy-peptidases from Aspergillus. Agricultural and Biological Chemistry 53; 1177-78. Thekkiniath, J. C., Zabet-Moghaddam, M., San Francisco, S. K., and San Francisco, M. J. (2013). A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis is induced by thyroid hormone and degrades antimicrobial peptides. Fungal Biol 117; 451-61.

U.S. Fish and Wildlife Service. (2018). White-Nose Syndrome Fact Sheet: The devastating disease of hibernating bats in North America. Updated 7/2/2018.

Warnecke, L., Turner, J. M., Bollinger, T. K., Misra, V., Cryan, P. M., Blehert, D. S., Wibbelt, G., and Willis, C. K. (2013). Pathophysiology of white-nose syndrome in bats: a mechanistic model linking wing damage to mortality. Biol Lett 9; 20130177.

Wilson, M. B., Held, B. W., Freiborg, A. H., Blanchette, R. A., and Salomon, C. E. (2017). Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans, the cause of white-nose syndrome in bats. PLoS One 12; e0178968.

Yike, I. (2011). Fungal proteases and their pathophysiological effects. Mycopathologia 171; 299-323.

Zaugg, C., Jousson, O., Lechenne, B., Staib, P., and Monod, M. (2008). Trichophyton rubrum secreted and membrane-associated carboxypeptidases. Int J Med Microbiol 298; 669-82.

Zukal, J., Bandouchova, H., Brichta, J., Cmokova, A., Jaron, K. S., Kolarik, M., Kovacova, V., Kubatova, A., Novakova, A., Orlov, O., Pikula, J., Presetnik, P., Suba, J., Zahradnikova, A., Jr., and Martinkova, N. (2016). White-nose syndrome without borders: Pseudogymnoascus destructans infection tolerated in Europe and Palearctic Asia but not in North America. Sci Rep 6; 19829.

CHAPTER 3: Galleria mellonella as an insect model for P. destructans, the cause of White-nose Syndrome in bats

This chapter was published in Plos One (2018 Sep 5;13(9)). I performed all experiments with the following exceptions: Lauren Meckler performed drug susceptibility experiments in Figure 4D. Eleonor Kim assisted with larval injections for experiments shown in Figure 3. I performed all data analysis, wrote the original manuscript with edits from Richard Bennett, and designed all figures.

Galleria mellonella as an insect model for P. destructans, the cause of White-nose Syndrome in bats

Chapman Beekman, Lauren Meckler, Eleanor Kim, and Richard J. Bennett*

Department of Molecular Microbiology and Immunology, Brown University, Providence, RI USA.

* correspondence: [email protected]

Abstract

Pseudogymnoascus destructans is the fungal pathogen responsible for White- nose Syndrome (WNS), a disease that has killed millions of bats in North America over the last decade. A major obstacle to research on P. destructans has been the lack of a tractable infection model for monitoring virulence. Here, we establish a high-throughput model of infection using larvae of Galleria mellonella, an invertebrate used to study host- pathogen interactions for a wide range of microbial species. We demonstrate that P. destructans can kill G. mellonella larvae in an inoculum-dependent manner when infected larvae are housed at 13°C or 18°C. Larval killing is an active process, as heat-killed P. destructans spores caused significantly decreased levels of larval death compared to live spores. We also show that fungal spores that were germinated prior to inoculation were able to kill larvae 3-4 times faster than non-germinated spores. Lastly, we identified chemical inhibitors of P. destructans and used G. mellonella to evaluate these inhibitors for their ability to reduce virulence. We demonstrate that amphotericin B can effectively block larval killing by P. destructans and thereby establish that this infection model can be used to screen biocontrol agents against this fungal pathogen.

Introduction

Pseudogymnoascus destructans is the fungal species responsible for White-nose

Syndrome (WNS), a disease currently devastating bat populations across North America.

P. destructans is a psychrophilic fungus and colonizes susceptible bat species during hibernation, causing depletion of energy stores and death of the host. Since it was first discovered in New York State in 2006, WNS has spread to 32 US states and 5 Canadian provinces [1]. This rapid spread, combined with a mortality rate approaching 100% for several species, has led to an estimated 6 million bats being killed by WNS [1]. As a result, one of the most common bat species in the North-East US, the little brown bat

(Myotis lucifugus), is now threatened with regional extinction. The loss of bats can harm both local ecosystems and agriculture as they play a crucial role in controlling insect pest populations, and it has been estimated that bat populations lost to WNS could cost the agricultural industry as much as $23 billion per year [2].

Despite the impact of WNS, a clear understanding of the factors that allow P. destructans to infect its host remain elusive. Studies have suggested several attributes may be important for fungal virulence including the production of small molecule effectors

[3], protease secretion [4, 5], lipid utilization [6], as well as the fungal heat shock response, cell wall remodeling, and micronutrient acquisition [7]. A significant obstacle to evaluation of these hypotheses has been the lack of a tractable infection model. The psychrophilic nature of P. destructans (maximum growth temperature of ~18 °C) has made standard mammalian infection models unfeasible. Laboratory-based WNS models using live bats have been utilized [8, 9], but require specialized equipment and long infection timelines, and are impractical for high-throughput studies.

The lack of an accessible infection model for P. destructans has also limited the testing of therapeutic agents to treat WNS. Studies have identified several agents that

can inhibit P. destructans growth on laboratory media [10-12], yet these are difficult to test in a natural setting. Treating a live infection entails additional complications that are not present during growth on laboratory media, such as host drug toxicity or drug degradation

by the host. Additionally, available carbon sources and growth conditions can affect fungal

drug susceptibility [13, 14]. Treatment of WNS also poses unique challenges given that

infection occurs only in hibernating bat populations, often located in remote habitats.

Development of a simple host model of P. destructans infection would therefore be of

considerable value and could accelerate the identification of an effective treatment for

WNS.

For several fungal pathogens, larvae of the greater wax moth, Galleria mellonella,

have provided a simple yet effective alternative to mammalian infection models. G.

mellonella has been employed to study virulence in human fungal pathogens including

Candida [15-17], Aspergillus [18, 19] and Fusarium [20, 21] species. Importantly, many results obtained using G. mellonella reproduce findings from mammalian infection studies

[15, 19, 21, 22], indicating that larval infection shows parallels with that in higher eukaryotes. Though insects lack an adaptive immune system, their immune response closely resembles the mammalian innate immune response at both a structural and functional level [23]. G. mellonella hemocytes are functionally analogous to mammalian phagocytes and generate reactive oxygen species (ROS) for microbial killing [24, 25].

These features make G. mellonella a relevant model for WNS, as evidence suggests hibernating bats can mount an innate immune response to P. destructans but are unable to activate adaptive immunity [26-28]. To our knowledge, no previous studies using G. mellonella have been conducted at temperatures below 20°C, yet larvae can be maintained at such temperatures making their use as a P. destructans infection model an attractive possibility.

Here, we examine the feasibility of using G. mellonella larvae as an invertebrate

model for infection with P. destructans. Our results indicate that live P. destructans

spores, but not heat-killed spores, are lethal to G. mellonella larvae, and that killing is

augmented if spores are induced to germinate prior to inoculation. We also perform a

screen to identify chemical inhibitors of P. destructans growth and evaluate their efficacy

during infection. These experiments establish that insect larvae can be used as a high-

throughput model for P. destructans enabling the screening of potential treatments for

WNS.

Results

Evaluation of G. mellonella as a suitable host for P. destructans

To test if P. destructans can establish an infection in G. mellonella, larvae were injected with 104, 105, or 106 fungal spores in phosphate-buffered saline (PBS) or mock

infected with PBS alone. Injected larvae were incubated at either 13°C or 18°C as

appropriate temperatures for growth of P. destructans. At 18°C, the upper limit for growth

of P. destructans, increased inoculum size correlated with increased killing of Galleria

larvae (Fig 1A). In contrast, at 13°C, only the highest inoculum of 106 spores per larva

demonstrated increased lethality above the PBS-injected control group (p = 0.0167, log-

rank test, Fig 1B). At the highest inoculum, the rate of killing was similar at both 13°C and

18°C, with 50% mortality reached ~20-25 days after infection. Larvae injected with the

highest fungal inoculum also showed an increase in pigmentation within 2 weeks of

injection at both temperatures (Fig 1C). Increased pigmentation in Galleria is likely due to

melanization of host tissues, and is indicative of an immune response to infectious agents

[33]. Less pigmentation was observed in larvae that received smaller inoculums (104 or

105 spores) and was completely absent in PBS-injected controls. Thus, pigmentation was a specific response to fungal spores and not due to physical injury caused by the injection.

At 18°C, several larvae appeared to form pupae while no pupae were observed at 13°C for the duration of the experiment. To avoid potential impacts of pupation on larval survival, all subsequent experiments were conducted at 13°C.

Lethal infections require live spores

The killing of G. mellonella by P. destructans spores could be due to active

proliferation of fungal cells in the larvae, or due to the host response to spores regardless

of whether the spores were viable. To distinguish between these possibilities, heat-killed

spores were prepared by treatment at 65°C for 30 minutes. Plating of heat-treated spores

onto YPD medium established that less than 0.1% of spores were viable (Fig 2A, inset).

Heat-killed and live spores were used to infect G. mellonella and larval death was

monitored daily. While some mortality was observed in larvae infected with heat-killed

spores, live P. destructans spores caused significantly greater killing of the larvae (P <

0.0001) (Fig 2A). Histology demonstrated a substantial fungal burden within larval tissue

after infection (Fig 2B), with many hyphal forms of P. destructans observed (Fig 2C).

Counts of colony-forming units (CFUs) were also conducted and showed that CFUs

(normalized by larval weight) increased during the first 2 weeks of infection with an

average of 29 CFUs/mg, 71 CFUs/mg and 64 CFUs/mg detected after 1,2, or 3 weeks,

respectively. Together, these results indicate the proliferation of P. destructans cells and

the formation of hyphal filaments within larval tissue, and are consistent with active fungal

growth leading to larval killing.

Virulence of P. destructans is increased by pre-germinating fungal spores

We examined whether spore germination could have an impact on the survival times of larvae. P. destructans spores were harvested and incubated at 13°C in liquid

YPD medium for varying amounts of time to allow germination prior to infection of G. mellonella. Spores were cultured for 0, 6, 12 or 24 hours in YPD and microscopic examination revealed that germ tube formation was clearly visible in those grown for 12-

24 hours (Fig 3A). The extent of germination in each of the inoculums was calculated by counting spores with visible germ tubes versus non-germinated spores. We found that

~50% of spores incubated for 24 h in YPD had formed germ tubes and many cells had already formed hyphae (Fig 3A and 3B). This contrasts with ~35% of spores having germinated after 12 h in YPD, while at 0 h or 6 h less than 10% of spores had visible germ tubes.

Germinated P. destructans spores were found to kill larvae much more rapidly than non-germinated spores. Live spores that had been pre-incubated for 24 hours at 13°C killed 50% of infected larvae within ~10 days, whereas larvae injected with spores germinated for 0, 6 or 12 h reached 50% mortality at approximately 45 days, 20 days or

15 days, respectively (Fig 3C). Increased melanization was also observed in larvae infected with spores that were pre-germinated (both live and heat-killed), indicating that germinated spores may elicit a stronger immune response from the host (Fig 3D).

However, the increased melanization did not directly impact mortality rates as germinated heat-killed spores still produced little death in the host.

Antifungal drug screen with Phenotype Microarray (PM) plates

We next examined the feasibility of using G. mellonella larvae for evaluating antifungal agents against P. destructans. An in vitro screen was conducted to identify

chemical inhibitors of P. destructans using the Phenotype Microarray (PM) system (Biolog) which consists of 96 well plates (PM21-25) coated with a panel of 120 chemical compounds at 4 different concentrations (for a full list of compounds see S1 Table)[29].

P. destructans spores suspended in YPD were used to inoculate each PM well and growth was monitored over the course of 11 days by measuring optical density (OD595). The resulting growth curves were analyzed using DuctApe software to generate an “activity index” representing relative fungal growth in the presence of a given compound (0 = no growth to 9 = maximal growth)[30]. Of the 120 compounds tested, approximately one third

(42/120) were able to fully inhibit growth of P. destructans (activity index = 0) under at least one of the concentrations tested (Fig 4A). The compounds that were able to fully inhibit growth are predicted to act on a wide range of cellular targets including the cell membrane, cell wall, protein synthesis and cellular respiration (Fig 4B, S1 Table).

However, two prominent classes of inhibitory compounds included those targeting the cell membrane (p < 0.0001, Fisher’s exact test) and antipsychotics/efflux pump inhibitors (p = 0.0136), suggesting that these may represent aspects of P. destructans

biology

that are particularly sensitive to perturbation.

The sensitivity of P. destructans to chemical inhibitors was compared to that of the

human fungal pathogen, Candida albicans, which was previously evaluated in PM plate

assays (Fig 4B, orange bars [31]). C. albicans cells similarly showed a significant

enrichment for inhibitory compounds targeting the cell membrane (p = 0.0260).

Additionally, all four of the compounds annotated as efflux pump inhibitors within the PM

panel (trifluoperazine, thioridazine, promethazine and chlorpromazine) were able to fully

inhibit growth of both P. destructans and C. albicans. This indicates that the sensitivity of

P. destructans to compounds targeting the cellular membrane and efflux pumps is not

unique among fungal pathogens. This comparison also revealed that P. destructans was

sensitive to a smaller number of PM compounds than C. albicans (42 and 68 compounds,

respectively, Fig 4C). Only 5 inhibitory compounds were unique to P. destructans and

these were benzamidine, cadmium chloride, ceftriaxone, sodium azide and sodium

thiosulfate. This may indicate that P. destructans is generally more resistant to chemical

inhibitors than C. albicans, although the growth media differed between these two studies

which limits direct comparisons between the two species.

Evaluation of PM drug screen hits

A subset of the 42 inhibitory compounds identified from the PM screen were

selected for further evaluation. Compounds known to possess high toxicity toward animal species were avoided. For each test compound, the minimum inhibitory concentration

(MIC) was determined by testing a range of drug concentrations against P. destructans grown in YPD using the broth dilution method. The MIC of each compound was defined as the minimum concentration at which at least 80% of fungal growth was inhibited. The majority of compounds tested showed MICs within the millimolar range, however two

compounds, trifluoperazine and sodium thiosulfate, had MICs in the micromolar range

(130 µM or 53 µg/mL and 12.5 µM or 2 µg/mL, respectively) indicating they are relatively potent inhibitors of P. destructans growth (Table 1).

Each test compound was also evaluated to determine if it was fungistatic or

fungicidal. To determine fungicidal activity, P. destructans spores were exposed to test

compounds at a concentration 10-fold higher than the determined MIC for 24 h and plated

onto YPD to assess their viability (Fig 4D). A compound was considered fungicidal if P.

destructans was unable to grow on YPD plates after exposure. Of the compounds tested,

only trifluoperazine showed fungicidal activity whereas all other compounds appeared

fungistatic.

Inhibitory compounds can block killing of G. mellonella larvae by P. destructans

Trifluoperazine and sodium thiosulfate, the most potent inhibitors of P. destructans identified in vitro, were tested for their ability to block killing of G. mellonella larvae. Two widely used antifungal drugs, amphotericin B and fluconazole [34], were also tested in these experiments. We note that P. destructans has previously been shown to be susceptible to fluconazole in a dose-dependent manner [10], while amphotericin B fully inhibited P. destructans growth under 3 of 4 concentrations tested in the Biolog screen.

For each assay, P. destructans spores were harvested, pre-germinated, resuspended in

PBS containing the test compound and injected into Galleria larvae alongside controls

(spores in PBS alone). Treatment of spores with sodium thiosulfate or fluconazole failed to reduce larval killing compared to infections with untreated spores (Fig 5A and 5B).

However, both trifluoperazine and amphotericin B blocked larval killing by P. destructans, reducing the rate of mortality to that produced by heat-killed spores (p < 0.0001, log-rank test, Fig 5C and 5D). In these experiments, untreated spores killed 50% of larvae after 20 days and 100% of larvae by 42 days, whereas >90% of larvae inoculated with trifluoperazine-treated spores and 65% of larvae inoculated with amphotericin B-treated spores were still viable 60 days post-infection.

Amphotericin B and trifluoperazine were also tested for their efficacy as treatments post infection. However, when each compound was injected 2 hours after infection only amphotericin B significantly reduced larval killing (Fig 6A), while trifluoperazine showed no protection against P. destructans (Fig 6B). Thus, whereas 50% of larvae infected with live spores were killed within 22 days (and with less than 10% of larvae surviving to 60 days), over 50% of larvae treated with amphotericin B survived for 60 days or more.

Overall, these experiments demonstrate the utility of the Galleria model for the

evaluation of P. destructans inhibitors and provide a proof-of-principle for screening of

potential WNS treatments through the validation of amphotericin B as an in vivo inhibitor

of pathogenesis.

Discussion

In this work, we establish the invertebrate Galleria mellonella as a suitable model

for examining the virulence of P. destructans, the pathogen responsible for WNS. Our

experiments demonstrate that G. mellonella larvae are susceptible to P. destructans at

temperatures compatible with the growth of this fungus (13°C or 18°C). To our knowledge,

this is the first evidence that the virulence of cold-loving, psychrophilic species can be

studied in G. mellonella. Infection with P. destructans proceeds slowly, with larvae typically succumbing to infection over the course of 1-2 months. This time-frame is similar to infections of hibernating bats, where WNS gradually leads to progressive tissue damage and depletion of host reserves [8, 9].

P. destructans caused larval killing in an inoculum-dependent manner, with the most effective inoculum being 106 spores per larva. These inoculums are similar to those

described for other fungal pathogens in G. mellonella [20], although the length of the

infection required for killing by P. destructans is considerably longer. Experiments using

heat-killed P. destructans spores, histology of larval tissue and recovery of fungal cells

from infected larvae reveal that larval killing requires active fungal proliferation and is not

simply a consequence of collateral damage from the host response to the inoculum. This

is relevant as dead spores from some fungal species can kill larvae even in the absence

of a live infection [35]. Together, these findings support the use of G. mellonella larvae as

an accessible system for studying pathogenicity during P. destructans infection.

We found that pre-germination of P. destructans spores enhanced virulence levels, as spores germinated for 24 hours prior to infection were able to kill larvae ~3-4 times faster than non-germinated spores. There may be several non-mutually exclusive factors contributing to this phenomenon. One possibility is that spore germination does not readily

occur within the larvae , so that pre-germinated spores exhibit an advantage as they have

progressed past this rate-limiting step. Alternatively, germinated spores could be more

resistant to phagocytosis or other immune defenses than non-germinated spores. In line

with this, cell shape and/or size has been shown to determine interactions between some

fungal species and immune cells, with elongated hyphal cells being more resistant to

phagocytosis than smaller yeast cells [36]. P. destructans spores that have initiated hyphae formation may therefore present a greater challenge to clearance by larval hemocytes. Remodeling of the fungal cell wall during spore germination may also impact immune responses. C. albicans hyphal cells, but not yeast cells, can block phagosome maturation and induce macrophage lysis due to expression of hyphal-specific cell wall components [37, 38]. It is possible that a similar mechanism allows germinated P. destructans spores to resist G. mellonella hemocytes, thereby accelerating infection. On the other hand, conidia from Aspergillus fumigatus possess hydrophobins [39] and melanins [40] that mask immunogenic components of the cell wall. Upon germination, A. fumigatus cells shed these components exposing surfaces that stimulate immune cell activation. The increased melanization of larvae injected with germinated spores suggests that P. destructans germ tubes may similarly stimulate a stronger immune response than non-germinated spores. However, given that germinated P. destructans spores are more virulent than non-germinated spores, this increased response is not protective and could even be detrimental to long-term survival in this model.

In addition to establishing conditions for infection of G. mellonella with P. destructans, we also used this model to test whether inhibitors of fungal growth could

improve infection outcomes. A screen using Biolog PM plates identified 42 compounds

that inhibited P. destructans in vitro, with effective inhibitors significantly enriched in

compounds targeting the cell membrane and efflux pumps. A comparison with C. albicans

[31] indicated that this species is similarly sensitive to these classes of inhibitors, although

differences in growth conditions make it difficult to compare inhibitor responses between

fungal species. We note that Chaturvedi et al. previously performed a more extensive

screen of 1,920 compounds of which 1.4% were highly effective at inhibiting P.

destructans growth, including several azole drugs, a fungicide (phenylmercuric acetate), as well as several biocides [10]. The fraction of effective compounds identified was similar to that of high-throughput screens for compounds against other fungal species, whereas the high hit rate in our more limited screen (35%) is presumably due to Biolog compounds having been pre-selected for those with potential antifungal activity.

We subsequently focused our analysis on trifluoperazine and sodium thiosulfate from the Biolog screen. Trifluoperazine is an antipsychotic drug that has been shown to have antimicrobial properties against a wide range of fungal and bacterial species [41,

42], whereas sodium thiosulfate has been used as a topical treatment for fungal infections

[43]. We compared these inhibitors with two well-established antifungal drugs, fluconazole

and amphotericin B, the latter of which was also an effective inhibitor of P. destructans

growth in our screen. Of these four compounds, trifluoperazine and amphotericin B

effectively blocked larval killing by P. destructans when applied prior to infection, yet only

amphotericin B was effective when used post infection. The ineffectiveness of

trifluoperazine post infection could be due to metabolism of the compound by G. mellonella

or an inability of the drug to access sites of P. destructans infection. However, this compound could still have value as a preventative disinfectant as spores exposed to trifluoperazine for 2 h were rendered completely avirulent. Both trifluoperazine and amphotericin B are fungicidal (our data and [44]), suggesting this mode of action may be

100

most effective for treatment of this fungus. Curiously, trifluoperazine was also tested as

part of the Chaturvedi et al. screen but was not found to inhibit P. destructans growth [10], possibly due to differences in drug concentrations or culture conditions. The use of fungicidal drugs would also be beneficial for treatment of WNS in nature, as delivery of a single application to hibernating bat populations would be considerably more practical than multiple applications. The G. mellonella model can therefore enable screening of anti-P. destructans compounds and may help accelerate the development of an effective WNS treatment.

Finally, we note that the G. mellonella model also has limitations that should be considered. First, WNS is a mammalian disease in which infection follows the invasion of external dermal tissues, whereas Galleria is an invertebrate species where fungal spores

are directly introduced into the insect hemolymph. The larval model therefore lacks

important features that occur during fungal colonization and invasion of dermal tissues.

Second, G. mellonella, like all insects, lacks an adaptive immune system. However, this

difference may not be critical for modeling P. destructans infections, as overall immune

function is down-regulated in hibernating animals [45], and adaptive immune responses

are particularly lacking in hibernating bats, whereas innate pro-inflammatory signaling is

activated in response to P. destructans [26-28]. Neutrophils are also present in

hibernating bats [27, 28, 46], although neutrophil recruitment and activation by P.

destructans may primarily occur upon a return to euthermia [47]. A number key aspects

of innate immunity are, in fact, shared between G. mellonella and mammals, including

pathogen-associated molecular pattern (PAMP) receptors, anti-microbial peptides and

phagocytic cells [20]. Galleria hemocytes share similarities with mammalian neutrophils

and can phagocytose microbes, generate reactive oxygen species (ROS) and produce

extracellular net-like structures for microbial killing [23-25]. The G. mellonella immune

101 system, while lacking adaptive immunity, therefore shows parallels to that of the natural host for P. destructans.

In conclusion, we demonstrate that G. mellonella represents a highly accessible model for the analysis of P. destructans, the primary cause of WNS. While there are limitations to this model its simplicity, ease of use, and affordability make it an attractive system for high-throughput screening of antifungal agents, as well as for the analysis of fungal mutants that may be defective in virulence. We suggest its inclusion will add to the growing list of tools available for the study of this emerging mammalian pathogen.

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Materials and Methods

Strains and culture conditions

Pseudogymnoascus destructans strain 20631-21 (ATCC stock: MYA-4855) was

used for all experiments. P. destructans cultures were cultured on yeast extract-peptone-

dextrose (YPD) medium at 13°C prior to spore collection.

G. mellonella virulence assays

G. mellonella larvae were obtained from Vanderhorst Wholesale (St. Marys, OH).

Prior to infection, larvae were stored at 13°C and used within 1 week of delivery. For all

experiments, inoculums were prepared by harvesting P. destructans spores from 2-3 week-old cultures grown on YPD plates using a solution of 0.05% Tween-20 (Sigma) and rubbing the surface of the plates with a glass spreader to release spores. Spores were isolated from larger hyphae by filtering through a layer of sterile miracloth, centrifuged and resuspended in 5 mL sterile phosphate-buffered saline, pH 7.4 (PBS). Spores were counted on a light microscope (Leica DM750) using a hemocytometer and adjusted to the desired concentration. Heat-killed spores were prepared by incubating inoculums for at least 30 min at 65°C and re-cooling briefly on ice prior to injection. Efficacy of heat killing

was confirmed by plating 1000 spores/plate on YPD and checking for viable colonies. For

pre-germination experiments, P. destructans spores were adjusted to 1x107 cells/mL in

liquid YPD and incubated at 13°C in a shaking incubator (200 rpm) for the specified time

period. Spore germination was assessed by microscopic analysis of germ tubes versus

un-germinated spores. Approximately 400 spores were counted in two independent

experiments. Inoculums were then prepared by adjusting spores to 1x108 cells/mL in

sterile PBS. Larvae of similar size were randomly selected for each experiment and those

showing discoloration were discarded. Each worm was injected with 10 µL of inoculating

103 solution just below the second to last left proleg using a 10 µL glass syringe with a 26S gauge needle (Hamilton, 80300). Infected larvae and controls were maintained at 13°C and checked daily. Larvae were recorded as dead if no movement was observed upon contact. For antifungal experiments, P. destructans spores were germinated for 6 h in

YPD then either (1) resuspended in each compound in PBS for 2 hours or less prior to injection, or (2) compounds were applied to larvae 2 hours post-infection by a second 10

µL injection at the last right proleg. Control larvae for these latter experiments received a second injection containing only PBS.

Histology

Larvae were injected with 12 h-germinated P. destructans spores or an equal volume of PBS as described above and incubated at 13°C. At 1, 2 and 3 weeks post- infection, larvae were removed and injected with 100 uL of 4 % paraformaldehyde using an insulin syringe, submerged in 2 mL of the fixative solution and incubated for 3 days at

4°C to allow complete fixation. Fixed larvae were then cut into 4-5 segments using a scalpel, embedded in paraffin and sectioned at 5 µm using a Leica EG1150C microtome.

Sections were stained using Modified Grocott’s Methanamine Silver (GMS) Stain

(Thermo, #87008) and imaged by light microscopy (Leica DM750).

Colony Forming Units (CFUs) in Infected Larvae

Larvae were injected with 12 h-germinated live or heat-killed P. destructans spores and incubated at 13°C. At 1, 2 and 3 weeks, infected larvae were removed, weighed and placed on ice for 5-10 minutes to immobilize. Larvae were then homogenized in PBS containing kanamycin (110 µg/mL), doxycycline (250 µg/mL), penicillin (1 mg/mL) and streptomycin (500 µg/ml) by slicing into small segments with a scalpel and grinding with a

104

plastic syringe plunger against a cell strainer (70 µm, Fisher) contained within a 6-well

tissue culture plate. Each larval homogenate was plated neat and as 1:100 dilutions on

YPD plates and plates were counted for P. destructans colonies after 8-10 days at 13°C.

Phenotype Microarray (PM) screen

P. destructans spores were harvested, washed in PBS, counted, and adjusted to

1x106 cells/mL in YPD. Next, 100 µL of the spore solution was used to inoculate each

well of PM plates 21-25 (Biolog, Hayward, CA). Each well contains one of 120 different

chemical compounds at 4 different concentrations [29]. Plates were incubated at 13°C

and growth in each well was monitored daily by measuring optical density (OD595 nm) in

a Synergy HT plate reader (Biotek, Winooski, VT) for 11 days. OD readings were compiled

to construct growth curves which were analyzed using DuctApeTM software [30] to convert each growth curve into an “activity index” (0-9) representing relative growth of P. destructans in response to each chemical. Data shown represents the average activity indices from 2 independent experiments.

Comparison of P. destructans and C. albicans PM data

Data from the PM drug screen on P. destructans was compared with published

data obtained for C. albicans using the same PM drug panel and DuctApe analysis

software [31]. Inhibitory compounds were defined as those producing an activity index of

0 at any concentration. Individual compounds were compared using the BioVenn [32]

categorized by “mode of action” and each category was evaluated for enrichment of

inhibitory compounds (Fishers exact test, Graphpad Prism v.5).

105

Determination of minimum inhibitory concentrations (MICs)

P. destructans spores were harvested and adjusted to 1x106 spores/mL in YPD

solutions containing test compounds. These solutions were then used to inoculate

individual wells of clear plastic 96-well tissue culture plates (100 µL/well, 6 replicate

wells/condition). Plates were incubated at 13°C and growth (OD595) measured after 240 h using a Synergy HT plate reader (Biotek). OD values from each of the 6 replicate wells were averaged and then divided by the average growth in control wells containing spores in YPD to determine the fraction of growth. The MIC was defined as the lowest concentration of each compound able to reduce P. destructans growth by at least 80%.

Evaluation of compounds for fungicidal activity

Harvested P. destructans spores were adjusted to 1x106 spores/mL in YPD containing the test compound and incubated at 13°C for 24 h. Next, serial dilutions of the spore solutions were made in YPD and 2 µL of each dilution spotted onto YPD plates, incubated at 13°C for 1 week and imaged using a Chemidoc imaging system (BioRad).

Acknowledgements

We would like to thank Iuliana Ene for assistance with PM data analysis, Corey

Frazer and Iuliana Ene for comments on the paper, and Matthew Anderson and Matthew

Hirakawa for help with larval injections.

Funding

Funding for this project was provided by a National Science Foundation grant

(NSF-1456787) to RJB. The funders had no role in study design, data collection and

analysis, decision to publish, or preparation of the manuscript.

106

Figures and Tables

Figure 1. Inoculation of G. mellonella larvae with P. destructans spores leads to larval killing in an inoculum- and temperature-dependent manner. (A,B) 10 larvae each were injected with 104, 105 or 106 spores or an equal volume of PBS (control) and kept at 13°C (A) or 18°C (B). Larvae were monitored daily and deaths recorded. p-values represent Log-rank test in infections with 106 spores v. PBS control. (C) Images of infected larvae 16 days post-inoculation. Melanization can be seen in infected larvae and increases with inoculum size.

107

Figure 2. Effective killing of larvae requires live P. destructans spores. (A) Larvae were injected with 106 live spores or an equal number of heat-killed (HK) spores, kept at 13°C and monitored daily. 20 larvae were used per condition in two independent experiments, p-value represents result of Log-rank test on Live vs. HK. Right inset: representative YPD agar plates on which 1000 spores from live and heat-killed inoculums were plated to confirm efficacy of heat-killing. (B) histology of larval tissues injected with live 12 h-germinated P. destructans spores (red bar) or mock-infected (green bar) and fixed 1, 2 or 3 weeks post-infection. Fungal elements were visualized using modified Grocott’s Silver Stain (GMS) and light microscopy (10x magnification, scale bar = 200 µm). (C) Histology of infected larval tissue 2 weeks after infection with live 12 h-germinated P. destructans spores, stained with modified GMS and imaged by light microscopy (40x, scale bar = 50 µm). (D) CFUs indicating P. destructans colonies recovered from larvae infected with 12 h-germinated live (red dots) or heat-killed (HK, black squares) spores. Data points represent individual larvae.

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Figure 3. Pre-germinated P. destructans spores kill larvae more effectively than non-germinated spores. (A) Representative images of inoculums demonstrate formation of germ tubes by fungal spores after 12 and 24 h in YPD. (B) Quantification of germ tube formation at each time point. Data represents analysis of >= 400 spores from two independent experiments for each time-point. (C) Larvae were each injected with either 106 live or heat-killed (HK) spores which had been allowed to pre-germinate for 0, 6, 12 or 24 h. Larval deaths were recorded daily. 20 larvae from two independent experiments were used per condition. p-values represent results of Log-rank test on live v. HK spores. (D) Images of larvae infected with spores allowed to germinate for 0, 6, or 24 h (images taken 2 h post-infection) shows greatest melanization in larvae infected with spores pre-germinated for 24 h.

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Figure 4. Identification and evaluation of anti-P. destructans compounds. (A) Heat map representing relative P. destructans growth on PM plates 21-25 which contain a total of 120 test compounds. (B) Bar graph indicating the % of PM compounds within each mode of action/target category that were able to fully inhibit growth of P. destructans (black) or C. albicans (orange) * P <= 0.05, *** P <= 0.001 (Fisher’s exact test). (C) Venn- diagram indicating compounds able to inhibit P. destructans and C. albicans growth. (D) Images of P. destructans spores spotted onto YPD agar as 10-fold dilutions from left (103) to right (100) and allowed to grow for 1 week after an initial 24 h exposure to the indicated compound at 10 times the MIC concentration.

110

Figure 5. Evaluation of anti-P. destructans compounds using the G. mellonella infection model. Survival curves from Galleria larvae injected with spores (106/larva) resuspended in PBS containing sodium thiosulfate (125 µM) (A), fluconazole (100 µg/ml) (B), trifluoperazine (1.3 mM) (C), or amphotericin B (200 µg/ml) (D). Each curve is plotted against control larvae injected with live or heat-killed (HK) spores germinated for 6 h and resuspended in PBS. p-values represent Log-rank test on live-treated vs. live-untreated spores.

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Figure 6. Evaluation of antifungal treatments administered after infection with P. destructans. Survival curves from Galleria larvae injected with spores (106/larva) and treated 2 hours later with trifluoperazine (1.3 mM) (A) or amphotericin B (200 µg/mL) (B). Each curve is plotted against control larvae injected with live or heat-killed (HK) spores which received a second injection of PBS alone. p-value represent Log-rank test on live + amphotericin B vs. live-untreated spores.

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MIC value (80% Chemical Mode of Action inhibition) Caffeine cyclic AMP phosphodiesterase inhibitor 8 mM Boric acid toxic anion 25 mM Benzamidine peptidase inhibitor, fungicide 25 mM membrane, phenothiazine, efflux pump inhibitor, anti- Trifluoperazine psychotic 130 µM Sodium Thiosulfate toxic anion, reducing agent 12.5 µM Ferulic acid antioxidant 6 mM Sodium Caprylate respiration, ionophore, H+ 50 mM Isoniazid inhibitor of fatty acid synthesis 50 mM

Table 1. Selected anti-P. destructans compounds from PM drug screen.

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References

1. White-Nose Syndrome Fact Sheet. US Fish and Wildlife Service. April 2018. 2. Boyles JG, Cryan PM, McCracken GF, Kunz TH. Economic Importance of Bats in Agriculture. Science. 2011;332:2. 3. Flieger M, Bandouchova H, Cerny J, Chudickova M, Kolarik M, Kovacova V, et al. Vitamin B2 as a virulence factor in Pseudogymnoascus destructans skin infection. Sci Rep. 2016;6:33200. doi: 10.1038/srep33200. PubMed PMID: 27620349; PubMed Central PMCID: PMCPMC5020413. 4. Pannkuk EL, Risch TS, Savary BJ. Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans. PLoS One. 2015;10(3):e0120508. doi: 10.1371/journal.pone.0120508. PubMed PMID: 25785714; PubMed Central PMCID: PMCPMC4364704. 5. O’Donoghue, AJ, Knudsen GM, Beekman C, Perry JA, Johnson AD, DeRisi JL, Craik CS, and Bennett RJ. Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus destructans, the causative agent of white-nose syndrome. Proc Natl Acad Sci U S A. 2015;112(24):E3152. doi: 10.1073/pnas.1509071112. PubMed PMID: 26015578; PubMed Central PMCID: PMCPMC4475950. 6. Frank CL, Ingala MR, Ravenelle RE, Dougherty-Howard K, Wicks SO, Herzog C, et al. The Effects of Cutaneous Fatty Acids on the Growth of Pseudogymnoascus destructans, the Etiological Agent of White-Nose Syndrome (WNS). PLoS One. 2016;11(4):e0153535. doi: 10.1371/journal.pone.0153535. PubMed PMID: 27070905; PubMed Central PMCID: PMC4829186. 7. Reeder SM, Palmer JM, Prokkola JM, Lilley TM, Reeder DM, Field KA. Pseudogymnoascus destructans transcriptome changes during white-nose syndrome infections. Virulence. 2017:1-13. doi: 10.1080/21505594.2017.1342910. PubMed PMID: 28614673. 8. Warnecke L, Turner JM, Bollinger TK, Lorch JM, Misra V, Cryan PM, et al. Inoculation of bats with European Geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome. Proc Natl Acad Sci U S A. 2012;109(18):6999-7003. doi: 10.1073/pnas.1200374109. PubMed PMID: 22493237; PubMed Central PMCID: PMCPMC3344949. 9. Lorch JM, Meteyer CU, Behr MJ, Boyles JG, Cryan PM, Hicks AC, et al. Experimental infection of bats with Geomyces destructans causes white-nose syndrome. Nature. 2011;480(7377):376-8. doi: 10.1038/nature10590. PubMed PMID: 22031324. 10. Chaturvedi S, Rajkumar SS, Li X, Hurteau GJ, Shtutman M, Chaturvedi V. Antifungal testing and high-throughput screening of compound library against Geomyces destructans, the etiologic agent of geomycosis (WNS) in bats. PLoS One. 2011;6(3):e17032. doi: 10.1371/journal.pone.0017032. PubMed PMID: 21399675; PubMed Central PMCID: PMCPMC3047530. 11. Wilson MB, Held BW, Freiborg AH, Blanchette RA, Salomon CE. Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans, the cause of white-nose syndrome in bats. PLoS One. 2017;12(6):e0178968. doi: 10.1371/journal.pone.0178968. PubMed PMID: 28617823; PubMed Central PMCID: PMCPMC5472292.

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12. Cornelison CT, Keel K, Gabriel KT, Barlament CK, Tucker TA, Pierce GE, Crow SA Jr. A preliminary report on the contact-independent antagonism of Pseudogymnoascus destructans by Rhodococcus rhodochrous strain DAP96253. BMC Microbiology. 2014;14(246). PubMed Central PMCID: PMCPMC4181622. 13. Ene IV, Heilmann CJ, Sorgo AG, Walker LA, de Koster CG, Munro CA, et al. Carbon source-induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans. Proteomics. 2012;12(21):3164-79. doi: 10.1002/pmic.201200228. PubMed PMID: 22997008; PubMed Central PMCID: PMC3569869. 14. Ene IV, Adya AK, Wehmeier S, Brand AC, MacCallum DM, Gow NA, et al. Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen. Cell Microbiol. 2012;14(9):1319-35. doi: 10.1111/j.1462- 5822.2012.01813.x. PubMed PMID: 22587014; PubMed Central PMCID: PMC3465787. 15. Brennan M TD, Whiteway M, Kavanagh K,. Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae. FEMS Immunology and Medical Microbiology. 2002;34:5. 16. Li DD, Deng L, Hu GH, Zhao LX, Hu DD, Jiang YY, Wang Y. Using Galleria mellonella–Candida albicans Infection Model to Evaluate Antifungal Agents. Biol Pharm Bull. 2013;36(9):1482–7. PubMed Central PMCID: PMC23995660. 17. Mesa-Arango AC, Forastiero A, Bernal-Martinez L, Cuenca-Estrella M, Mellado E, Zaragoza O. The non-mammalian host Galleria mellonella can be used to study the virulence of the fungal pathogen Candida tropicalis and the efficacy of antifungal drugs during infection by this pathogenic yeast. Med Mycol. 2013;51(5):461-72. doi: 10.3109/13693786.2012.737031. PubMed PMID: 23170962. 18. Maurer E, Browne N, Surlis C, Jukic E, Moser P, Kavanagh K, et al. Galleria mellonella as a host model to study Aspergillus terreus virulence and amphotericin B resistance. Virulence. 2015;6(6):591-8. doi: 10.1080/21505594.2015.1045183. PubMed PMID: 26107350; PubMed Central PMCID: PMC4720272. 19. Slater JL, Gregson L, Denning DW, Warn PA. Pathogenicity of Aspergillus fumigatus mutants assessed in Galleria mellonella matches that in mice. Med Mycol. 2011;49 Suppl 1:S107-13. doi: 10.3109/13693786.2010.523852. PubMed PMID: 20950221. 20. Binder U, Maurer E, Lass-Florl C. Galleria mellonella: An invertebrate model to study pathogenicity in correctly defined fungal species. Fungal Biol. 2016;120(2):288-95. doi: 10.1016/j.funbio.2015.06.002. PubMed PMID: 26781383. 21. Coleman JJ, Muhammed M, Kasperkovitz PV, Vyas JM, Mylonakis E. Fusarium pathogenesis investigated using Galleria mellonella as a heterologous host. Fungal Biol. 2011;115(12):1279-89. doi: 10.1016/j.funbio.2011.09.005. PubMed PMID: 22115447; PubMed Central PMCID: PMC3224342. 22. Garcia-Rodas R, Casadevall A, Rodriguez-Tudela JL, Cuenca-Estrella M, Zaragoza O. Cryptococcus neoformans capsular enlargement and cellular gigantism during Galleria mellonella infection. PLoS One. 2011;6(9):e24485. doi: 10.1371/journal.pone.0024485. PubMed PMID: 21915338; PubMed Central PMCID: PMC3168503. 23. Browne N, Heelan M, Kavanagh K. An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes. Virulence.

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2013;4(7):597-603. doi: 10.4161/viru.25906. PubMed PMID: 23921374; PubMed Central PMCID: PMCPMC3906293. 24. Bergin D, Reeves EP, Renwick J, Wientjes FB, Kavanagh K. Superoxide production in Galleria mellonella hemocytes: identification of proteins homologous to the NADPH oxidase complex of human neutrophils. Infect Immun. 2005;73(7):4161-70. doi: 10.1128/IAI.73.7.4161-4170.2005. PubMed PMID: 15972506; PubMed Central PMCID: PMC1168619. 25. Renwick J, Reeves EP, Wientjes FB, Kavanagh K. Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella. Dev Comp Immunol. 2007;31(4):347- 59. doi: 10.1016/j.dci.2006.06.007. PubMed PMID: 16920193. 26. Rapin N, Johns K, Martin L, Warnecke L, Turner JM, Bollinger TK, et al. Activation of innate immune-response genes in little brown bats (Myotis lucifugus) infected with the fungus Pseudogymnoascus destructans. PLoS One. 2014;9(11):e112285. doi: 10.1371/journal.pone.0112285. PubMed PMID: 25391018; PubMed Central PMCID: PMCPMC4229191. 27. Moore MS, Reichard JD, Murtha TD, Nabhan ML, Pian RE, Ferreira JS, et al. Hibernating little brown myotis (Myotis lucifugus) show variable immunological responses to white-nose syndrome. PLoS One. 2013;8(3):e58976. doi: 10.1371/journal.pone.0058976. PubMed PMID: 23527062; PubMed Central PMCID: PMCPMC3604015. 28. Field KA, Johnson JS, Lilley TM, Reeder SM, Rogers EJ, Behr MJ, et al. The White-Nose Syndrome Transcriptome: Activation of Anti-fungal Host Responses in Wing Tissue of Hibernating Little Brown Myotis. PLoS Pathog. 2015;11(10):e1005168. doi: 10.1371/journal.ppat.1005168. PubMed PMID: 26426272; PubMed Central PMCID: PMCPMC4591128. 29. Bochner BR, Gadzinski P, Panomitros E. Phenotype MicroArrays for High- Throughput Phenotypic Testing and Assay of Gene Function. Genome Res. 2001;11:1246–55. doi: 10.1101/. PubMed PMID: 11779830; PubMed Central PMCID: PMC155265. 30. Galardini M, Mengoni A, Biondi EG, Semeraro R, Florio A, Bazzicalupo M, et al. DuctApe: a suite for the analysis and correlation of genomic and OmniLog Phenotype Microarray data. Genomics. 2014;103(1):1-10. doi: 10.1016/j.ygeno.2013.11.005. PubMed PMID: 24316132. 31. Ene IV, Lohse MB, Vladu AV, Morschhauser J, Johnson AD, Bennett RJ. Phenotypic Profiling Reveals that Candida albicans Opaque Cells Represent a Metabolically Specialized Cell State Compared to Default White Cells. MBio. 2016;7(6). doi: 10.1128/mBio.01269-16. PubMed PMID: 27879329; PubMed Central PMCID: PMCPMC5120136. 32. Hulsen T, de Vlieg J, Alkema W. BioVenn - a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. BMC Genomics. 2008;9:488. doi: 10.1186/1471-2164-9-488. PubMed PMID: 18925949; PubMed Central PMCID: PMC2584113. 33. Pye AE. ACTIVATION OF PROPHENOLOXIDASE AND INHIBITION OF MELANIZATION IN THE HAEMOLYMPH OF IMMUNE GALLERIA MELLONELLA LARVAE. Insect Biochemistry. 1977;8:7. 34. Cowen LE. The evolution of fungal drug resistance: modulating the trajectory from genotype to phenotype. Nat Rev Microbiol. 2008;6(3):187-98. Epub 2008/02/05. doi: nrmicro1835 [pii]10.1038/nrmicro1835. PubMed PMID: 18246082.

116

35. Achterman RR, Smith AR, Oliver BG, White TC. Sequenced dermatophyte strains: growth rate, conidiation, drug susceptibilities, and virulence in an invertebrate model. Fungal Genet Biol. 2011;48(3):335-41. doi: 10.1016/j.fgb.2010.11.010. PubMed PMID: 21145410; PubMed Central PMCID: PMC3035951. 36. Erwig LP, Gow NA. Interactions of fungal pathogens with phagocytes. Nat Rev Microbiol. 2016;14(3):163-76. doi: 10.1038/nrmicro.2015.21. PubMed PMID: 26853116. 37. Uwamahoro N, Verma-Gaur J, Shen HH, Qu Y, Lewis R, Lu J, et al. The pathogen Candida albicans hijacks pyroptosis for escape from macrophages. MBio. 2014;5(2):e00003-14. doi: 10.1128/mBio.00003-14. PubMed PMID: 24667705; PubMed Central PMCID: PMCPMC3977349. 38. Bain JM, Louw J, Lewis LE, Okai B, Walls CA, Ballou ER, et al. Candida albicans hypha formation and mannan masking of beta-glucan inhibit macrophage phagosome maturation. MBio. 2014;5(6):e01874. doi: 10.1128/mBio.01874-14. PubMed PMID: 25467440; PubMed Central PMCID: PMC4324242. 39. Aimanianda V, Bayry J, Bozza S, Kniemeyer O, Perruccio K, Elluru SR, et al. Surface hydrophobin prevents immune recognition of airborne fungal spores. Nature. 2009;460(7259):1117-21. doi: 10.1038/nature08264. PubMed PMID: 19713928. 40. Chai LY, Netea MG, Sugui J, Vonk AG, van de Sande WW, Warris A, et al. Aspergillus fumigatus conidial melanin modulates host cytokine response. Immunobiology. 2010;215(11):915-20. doi: 10.1016/j.imbio.2009.10.002. PubMed PMID: 19939494; PubMed Central PMCID: PMC2891869. 41. Eilam Y, Polacheck I, Ben-Gigi G, Chernichovsky D. Activity of phenothiazines against medically important yeasts. Antimicrob Agents Chemother. 1987;31(5):834-6. PubMed PMID: 3300543; PubMed Central PMCID: PMC174848. 42. Vitale RG, Afeltra J, Meis JF, Verweij PE. Activity and post antifungal effect of chlorpromazine and trifluopherazine against Aspergillus, Scedosporium and zygomycetes. Mycoses. 2007;50(4):270-6. doi: 10.1111/j.1439- 0507.2007.01371.x. PubMed PMID: 17576318. 43. Rezabek. Superficial Fungal Infections of the Skin: Diagnosis and Current Treatment Recommendations. Drugs (New York, NY). 1992;43(5):674 - EOA. 44. Anderson TM, Clay MC, Cioffi AG, Diaz KA, Hisao GS, Tuttle MD, et al. Amphotericin forms an extramembranous and fungicidal sterol sponge. Nat Chem Biol. 2014;10(5):400-6. doi: 10.1038/nchembio.1496. PubMed PMID: 24681535; PubMed Central PMCID: PMCPMC3992202. 45. Bouma H. Hibernation: the immune system at rest? Journal of leukocyte biology. 2010;88(4):619. 46. Meteyer CU, Valent M, Kashmer J, Buckles EL, Lorch JM, Blehert DS, et al. Recovery of little brown bats (Myotis lucifugus) from natural infection with Geomyces destructans, white-nose syndrome. J Wildl Dis. 2011;47(3):618-26. doi: 10.7589/0090-3558-47.3.618. PubMed PMID: 21719826. 47. Meteyer CU, Barber D, Mandl JN. Pathology in euthermic bats with white nose syndrome suggests a natural manifestation of immune reconstitution inflammatory syndrome. Virulence. 2012;3(7):583-8. doi: 10.4161/viru.22330. PubMed PMID: 23154286; PubMed Central PMCID: PMC3545935.

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CHAPTER 4: Analysis of Primary Metabolism in P. destructans and Non-Pathogenic Relatives

I performed all experiments in this Chapter with the following exception: Eleanor Kim performed agrobacterium mediated transformations of P. destructans for introduction of the 3VT5-LxrA-consruct1 plasmid shown in Figure 4. I performed all data analysis, wrote this chapter and designed all figures.

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Introduction

The intimate link between primary metabolism and virulence in fungal pathogenesis is well-established.1-5 Like in any other niche, growth in a living host requires the ability utilize available energy sources. However, the host environment presents distinct challenges to nutrient acquisition and carbon metabolism. This is exhibited in mammalian pathogens A. fumigatus6, C. neoformans7 and C. albicans8 where interactions with host cells drive large-scale transcriptional changes in primary carbon metabolism genes. For these fungi, metabolic flexibility and careful regulation of carbon metabolism appear key to pathogenicity. However, metabolic reduction is also observed in some fungal pathogens, where losses of metabolic enzymes is associated with a specialization for growth in the host niche.9-11

The reduced secretome and smaller set of CAZymes in P. destructans observed

here (Chapter 2) and in outside genomic analyses could be suggestive of a more limited

metabolic capability. To test this, we directly evaluated carbon source utilization by P.

destructans using the Biolog Phenotype Microarray (PM) system. We assessed the ability

of P. destructans and related Pseudogymnoascus species to utilize 190 individual carbon

sources. Spores were collected from P. destructans and Pseudogymnoascus sp. 03VT05,

05NY08, P. verrucosus, 24MN13, and WSF3629 and used to inoculate single carbon

source-containing wells of the Biolog PM 1 and 2 assay plates. Growth was then assayed

by optical density (OD600) over the course of 15 days. The resulting data was analyzed

using DuctApe software which constructs fitted growth curves and uses key growth

parameters including lag time, slope and height of each growth curve to generate an

Activity Index (AI) value between 0 (no growth) and 9 (maximal growth). The experiment

was conducted both at 13°C, which is within the optimal growth range for P. destructans

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and at 18°C, close to the maximum growth temperature of P. destructans and nearer the

optimal growth temperature of several non-pathogenic relatives.12,13

Results

P. destructans exhibits limited carbon source utilization

Independent of temperature, P. destructans exhibited substantially reduced AI

values across a wide range of carbon sources suggesting it possesses a much more

limited carbon metabolism compared to non-pathogenic relatives (Figure 1A, B). Even at

a temperature within its optimal growth range (13°C), P. destructans did not produce the highest AI on any individual carbon source. However, P. destructans did exhibit equivalent growth across a limited set of carbon sources and produced the third-highest AI on glucose, indicating that under the conditions tested here, P. destructans has a more limited range of utilizable carbon sources and does not simply exhibit ubiquitously slower growth.

Overall, at 13°C, P. destructans was able to utilize only 59 of the 190 tested carbon sources (AI above that of no carbon source control / AI > 2) whereas each of the non- pathogenic Pseudogymnoascus species were able to utilize between 92 and 164 carbon sources. (For full list of tested carbon sources and resulting AI values see Supplementary

Tables 1 and 2 in Appendix D).

Deficiencies in carbohydrate metabolism

P. destructans showed a particularly limited ability to utilize carbohydrate carbon sources (17 out of 71) and produced the lowest average AI across these substrates

(Figure 1C). The relatively limited carbon metabolism and deficiency of P. destructans to

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utilize carbohydrates here is consistent with the observed genomic14 and proteomic

reduction in secreted CAZymes as well as recent studies which conducted similar PM

analyses14-16. Together these observations suggest a broad shift in the metabolism of P.

destructans away from carbohydrate energy sources. This metabolic shift has implications for saprophytic growth as plant polysaccharides represent one of the most abundant natural sources of organic carbon17,18. The major classes of polysaccharides within the plant cell wall, cellulose, hemi-cellulose and pectin, are heterogeneous polymers largely composed of glucose, galactose, arabinose, xylose, mannose and other monosaccharides.19 The reduction in CAZymes suggests that P. destructans may have

lost some ability to break down these polysaccharides. This is supported in our PM

analysis as P. destructans was unable to utilize polymeric substrates including glycogen, inulin and pectin in contrast to its saprophytic relatives. However, most carbohydrate substrates within the PM panel are mono- or oligo-saccharides and therefore a reduction in polysaccharide digesting enzymes cannot explain the inability of P. destructans to use these substrates.

P. destructans may be missing a key enzyme in Arabinose/Galactose utilization

The inability of P. destructans to utilize monosaccharides suggests that it is also deficient in uptake and/or intracellular carbohydrate catabolism. Interestingly, carbon sources where P. destructans shows the greatest deficiency compared to its relatives

included several sugars abundant in plant polysaccharides such as L-arabinose, D-

galactose and D-xylose. Currently, nothing is known regarding the catabolic pathways for

L-Arabinose, D-Galactose and D-Xylose within the Pseudogymnoascus genus, yet the metabolism of these sugars has been studied in the saprophytic fungi Aspergillus niger/nidulans and Trichoderma reesei. Arabinose catabolism in each of these species

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proceeds via the same pathway with alternating oxidative and reductive reactions

sequentially converting arabinose to L-arabitol, L-xylulose, L-xylitol, D-xylulose and finally

D-xylulose-5-phosphate, enabling entry into the core pentose-phosphate pathway.20-22

The second half of the fungal arabinose pathway is also responsible for the metabolism

of L-xylose which is first converted to L-xylitol in an outside reaction. Genes encoding each

of the enzymes involved in the fungal arabinose pathway have been identified in A.

niger23,24/nidulans25 and T. reesei21. Blastp searches of arabinose pathway enzymes from

Aspergillus/T.reesei within Pseudogymnoascus species revealed putative orthologues with similar levels of homology across all proteomes. However, the closest homolog to the

L-xylulose reductase from Aspergillus species (Blastp 48-52% identity with A. nidulans

LxrA gene) responsible for the 3rd step in the pathway, displayed a large N-terminal truncation in the annotated protein unique to P. destructans. Upon examination of the corresponding genomic locus, P. destructans is missing the putative start codon present in each of the other species (Figure 2). Several alternative start codons are present in the

P. destructans gene; however, a single nucleotide deletion causes a frameshift that covers a conserved catalytic Asn residue26, indicating the function of the encoded protein is likely

disrupted. I used Sanger sequencing to confirm the presence of this frame-shift mutation

in this P. destructans gene (GMDG_07781). If the function of this putative LxrA homolog

in the Pseudogymnoascus species here matches that of A. niger, the observed

polymorphism in P. destructans could explain its inability to utilize arabinose. Interestingly

P. destructans was also unable to utilize other intermediates of the fungal

arabinose/xylose pathway present in the PM panel supporting the idea that this pathway

is non-functional (Figure 3A). In T. reesei, many of the same proteins involved in the

arabinose pathway also function in an alternative galactose pathway (Figure 3B) that

exists in parallel with the widely conserved Leloir pathway for galactose metabolism 27. In

A. niger, paralogs to the arabinose pathway enzymes catalyze various steps in this

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alternative galactose pathway. However, in A. nidulans the alternative galactose pathway

may involve an extra intermediate, L-sorbose and it has been suggested that the

arabinose pathway L-xylulose reductase (LxrA) could catalyze its conversion to D-

sorbitol28 (Figure 3B). P. destructans is also unable to utilize many intermediate

substrates of this fungal-specific pathway, raising the possibility that the disruption of its

putative LxrA ortholog also impacts galactose metabolism.

Is P. destructans more metabolically active than non-pathogenic relatives?

My initial analysis of P. destructans growth on Biolog plates relied on measuring changes in absorbance to examine cellular growth and assess carbon source utilization.

However, the Biolog PM system can also be used in conjunction with a redox-based metabolic dye as an alternative assay method. While the exact chemical composition of this dye is proprietary it is known to be a water-soluble tetrazolium dye similar to other widely used metabolic dyes such as MTT, XTT, MTS, and WST-1. All tetrazolium dyes are based on the same chemical mechanism where reduction of the tetrazole ring converts the molecule to a colored formazan product29. In the case of the Biolog tetrazolium dye, its reduction leads to formation of a dark purple formazan. In mammalian cells, the Biolog dye is reduced by electrons originating from cellular NADH which, depending on the substrate, can be generated by the TCA cycle or directly by catabolic pathways such as glycolysis30. For this reason, the dye is marketed as an assay to measure general metabolic activity and substrate utilization.

We accordingly conducted an analysis of carbon source utilization in

Pseudogymnoascus species using the Biolog dye to measure substrate utilization.

Curiously, the results obtained using the dye were strikingly different from those measuring growth by optical absorbance. Despite exhibiting similar or lower levels of

123 growth on all carbon sources in our initial analysis, P. destructans reduced the Biolog dye to a greater extent than its non-pathogenic relatives across many carbon sources (Figure

4A). This elevated dye reduction by P. destructans can be seen clearly in medium containing glucose, at both 13°C and 18°C (Figure 4B). This result was surprising and suggested that despite lower overall growth, P. destructans may be more metabolically active on many carbon sources.

Is P. destructans more dependent on mitochondrial function than non-pathogenic relatives?

As reduction of tetrazolium-based dyes has been linked to intracellular NADH levels in a wide range of species including bacteria31, mammals29, and fungi32, I measured the levels of NAD+/H within P. destructans and related species to determine whether this could underlie the differences in Biolog dye reduction. Briefly, spores were collected from each species, used to inoculate liquid medium and cultured at 13°C for 3 days. The resulting mycelia were harvested, lysed and separated into equivalent samples for determination of intracellular NAD+ and NADH levels using the NAD+/H glo assay

(Promega). Protein concentration of each mycelial lysate was also determined and used to normalize NAD+/H concentrations across species. However, the measured levels of

NAD+/NADH in in P. destructans were lower compared to non-pathogenic

Pseudogymnoascus species after normalization (Figure 5A). This result was surprising but suggested the increased reduction of the Biolog dye by P. destructans may be due to differences in electron transfer downstream of NADH. In yeast, the mechanism of tetrazolium reduction is dependent on mitochondrial ETC activity as deletion of complex

III and IV subunits eliminates dye reduction32. This suggests that electrons from NADH may pass through the mitochondrial ETC before being transferred to tetrazolium dyes as

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has been observed in bacterial species31. If the metabolism of P. destructans is more dependent on the mitochondrial ETC than its non-pathogenic relatives this may explain its elevated ability to reduce the Biolog dye.

I therefore tested the sensitivity of P. destructans to several ETC inhibitors including rotenone (complex I), antimycin (complex III) and oligomycin (complex IV) during growth in PM culture medium (Figure 5B). Interestingly, P. destructans was more sensitive than related Pseudogymnoascus species to each ETC inhibitor tested. This difference was particularly striking for antimycin and subsequent experiments demonstrated that P. destructans is over 100x more sensitive to this inhibitor than non-

pathogenic Pseudogymnoascus species (Figure 5C). The elevated sensitivity to antimycin and other ETC inhibitors is consistent with P. destructans relying more heavily on mitochondrial ETC and aerobic respiration than other species under the conditions tested here. However, the role of complex III and ETC activity in the reduction of the Biolog dye remains unclear, as color formation was only inhibited by antimycin at concentrations that also inhibited overall fungal growth.

Evidence from various biological systems indicate that the final step in the transfer of electrons to many tetrazolium dyes occurs extracellularly through a process known as plasma membrane electron transport (PMET).29,31-34 The involvement of PMET in tetrazolium reduction has been demonstrated in species ranging from bacteria to mammals and is dependent on the activity of plasma membrane-localized oxidoreductases. However, the specific enzymes responsible for tetrazolium reduction appear to vary greatly between species and growth conditions. In mammalian cells, the

NAD(P)H-dependent quinone oxidoreductase NQO1 is required for reduction of the water- soluble WST-1.33 In the bacterium Lactococcus lactis, quinone oxidoreductases drive the

extracellular reduction of Tetrazolium Violet on solid medium while plasma membrane

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NADH-dehydrogenases NoxA and B are largely responsible for dye reduction in liquid

medium.31 In the fungus C. albicans, membrane-bound ferric reductases are required for reduction of XTT.34 The involvement of membrane-bound or extracellular redox enzymes

in tetrazolium reduction implies that differences in extracellular redox enzymes between

P. destructans and its relatives may also contribute to the difference in Biolog dye

reduction. However, techniques for genetic manipulation of P. destructans will need to be

further developed in order to effectively test this hypothesis.

An additional feature of PMET and associated extracellular tetrazolium reduction

is the involvement of superoxide. A requirement for extracellular superoxide radicals in the

reduction of tetrazolium dyes has been demonstrated in many cases by the inhibitory

effect of exogenous superoxide dismutase enzyme (SOD), a superoxide scavenger

enzyme.33-36 Tetrazolium dyes have even been applied as assays of superoxide

generation during the neutrophil oxidative burst36 and in filamentous fungi to measure redox signaling in the hyphal tip37. This raised the possibility that P. destructans is

generating higher levels of superoxide than its non-pathogenic relatives. As the ability of

reactive oxygen species (ROS) to cause cellular damage is well documented38, if this is the case, it could have clear implications for fungal virulence and WNS pathology.

However, initial experiments in PM medium containing glucose show that the addition of

SOD has no impact on Biolog dye reduction by P. destructans indicating that superoxide is not involved (Figure 5C). Additionally, P. destructans was unable to reduce ferricytochrome C (data not shown), another established assay of superoxide generation39,40, suggesting that the fungus does not produce measurable extracellular superoxide in the conditions tested.

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Discussion

Carbon metabolism and implications for pathogenicity in P. destructans

Here we found that P. destructans is relatively restricted in the range of carbon sources it can utilize, with a particular deficiency in carbohydrate utilization. Interestingly, a recent analysis comprising over 300 yeast genomes revealed widespread and frequent loss of metabolic pathways during species diversification, suggesting a general role for metabolic simplification in fungal evolution 41. Loss of the ability to use various

carbohydrates in P. destructans may therefore reflect a broader trend in fungal evolution.

While the benefit of a more limited carbon metabolism is not immediately clear, it may also

reflect the narrow niche occupied by P. destructans. Unlike more versatile fungal

pathogens such as A. fumigatus, C. albicans and C. neoformans which can colonize

multiple niches within the host, P. destructans is restricted to dermal tissue. Interestingly,

Malassezia10 species and dermatophytes11, fungal pathogens that are similarly restricted

to infection of dermal tissue, also exhibit a limited carbon metabolism with large reductions

in genes related to carbohydrate utilization. In Malassezia and dermatophytes a loss of

carbohydrate genes is associated with a metabolic specialization for lipids10 and

protein42,43 respectively. Interestingly, I now show that P. destructans was able to utilize

several amino acids and lipid-like molecules as sole-carbon sources raising the possibility

that it has developed a similar metabolic specialization during its evolutionary transition to

an animal pathogen.

Carbon metabolism and cell wall composition in P. destructans

A relatively limited carbohydrate metabolism in P. destructans could also have a

significant impact on fungal cell wall composition. Polysaccharides including chitin, β- 127

glucans and mannans make up a large proportion of the fungal cell wall.44 The synthesis

of these molecules requires various monomeric sugars and is therefore dependent on

available carbon sources. As the fungal cell wall represents the primary barrier and contact

point for the host immune system45, differences in its composition could have important

implications for pathogenicity. In C. albicans, growth on different carbon sources results in dramatic changes to cell wall composition that impact cell wall integrity, and susceptibility to stress.46 Likewise, galactose metabolism is crucial for cell wall structure

and capsule formation, a key virulence factor in C. neoformans enabling immune evasion

and resistance to phagocytic killing.47 In A. fumigatus, galactose-containing cell wall

polymers also mediate immune evasion by suppressing neutrophil recruitment and

eliciting other immunosuppressive effects48 and their disruption leads to attenuated

virulence49. My results suggest that P. destructans may be lacking the fungal-specific

galactose catabolic pathway. However, P. destructans was capable of slow growth on

medium containing galactose as the sole carbon source. If P. destructans has, in fact, lost

the fungal-specific galactose pathway, it is therefore likely it has retained function of the

conserved Leloir pathway. Interestingly, the Leloir galactose pathway provides UDP-

galactose, the monomer used in synthesis of galactose-containing cell wall polymers.

Could the putative loss of the alternative galactose pathway in P. destructans therefore

lead to increased flux through the Leloir pathway and a subsequent increase in galactose-

containing cell wall components? It is an intriguing question given the immunosuppressive

properties of galactose polymers in other fungi as well as the observed lack of neutrophil

recruitment in bats infected with P. destructans.50-52

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Mitochondrial function in fungi and implications for pathogenicity

Based on experiments thus far, the mechanism and biological implications of higher tetrazolium dye reduction by P. destructans remain unclear. However, this phenotypic difference suggests interesting, potentially virulence-relevant, differences in metabolism. The dependence of tetrazolium reduction on mitochondrial ETC function in other species and the elevated sensitivity of P. destructans to ETC inhibitors suggests that there may be key differences in mitochondrial activity between this fungal pathogen and its non-pathogenic relatives. Mitochondrial function is crucial for virulence in several mammalian fungal pathogens53 through its close involvement with key processes including

morphological transition54, cell wall synthesis55 and stress tolerance56. Interestingly,

hypervirulent strains of C. gattii involved in the Vancouver Island outbreak show upregulation of various mitochondrial ETC genes and distinct mitochondrial morphology compared to less pathogenic strains. This elevated mitochondrial activity was associated with an increased ability to proliferate within macrophages and may be the key driver of the hypervirulence exhibited by these strains.57 These studies provide further motivation

to investigate mitochondrial function in P. destructans and non-pathogenic relatives and

future experiments will include microscopy to assess morphology, localization and activity

of mitochondria as well as cellular respiration assays to compare mitochondrial function.

Conclusions

Together, the results outlined in this chapter indicate the development of pathogenicity in P. destructans is associated with fundamental changes to primary carbon metabolism. Like Mallasezzia sp. and Dermatophyte fungi, P. destructans may have

129

evolved a primary carbon metabolism specialized for growth on host skin, having lost the

ability to utilize a wide array of carbon sources. Evidence presented here also suggests

that the metabolism of P. destructans may be more dependent on mitochondrial function.

While similar findings have been obtained in other pathogenic fungi, the relationship

between mitochondria and virulence within the Pseudogymnoascus genus remains

unclear. However, the observations described in this chapter suggest a strong connection

exists between metabolism and virulence in P. destructans. Clarifying this connection will

motivate future studies and may help reveal how such a deadly pathogen arose from a

clade of harmless saprophytes.

Methods

Strains and culture conditions

Pseudogymnoascus strains Pseudogymnoascus destructans 20631-21 (ATCC stock:

MYA-4855), P. verrucosus, P. sp. 24MN13, 05NY8, 03VT05, 23342-1 and WSF3629

(CFMR culture collection14) as well as P. sp. FL204, FL590, BL308, BL549, and BL578 12

were maintained on Yeast Extract Peptone Dextrose (YPD) agar. ATMT-transformed P.

destructans strains (24MN13-LxrA construct 1, 3VT5-LxrA construct 2 and 3VT5-LxrA

construct 3) were maintained on YPD + hygromycin. P. destructans cultures were

maintained at 13 °C while all other Pseudogymnoascus strains were maintained at room

temperature. Spores were harvested from solid medium by washing culture plates with

0.05% Tween 20 solution and filtration with MiraclothTM (Millipore-Sigma) to remove larger hyphae and mycelium.

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Biolog Phenotype Microarray for carbon source utilization

P. destructans spores were harvested, washed in PBS, counted, and adjusted to 8x105

cells/mL in PM assay medium (IFY-0 + 5mM glutamic acid, 5mM potassium phosphate,

2mM sodium sulfate and trace amounts (10-50 µM) of adenine, uracil and amino acids)

with or without Biolog Dye mix “D”. 100 µL of the spore solution in PM assay medium

was used to inoculate each well of PM plates 1 and 2A (Biolog, Hayward, CA)30. Each well contains one of 190 different carbon-containing compounds with 1 well in each plate serving as a no carbon source control (See Supplementary Tables 1-3 in Appendix D).

Plates were incubated at 13°C or 18°C and growth in each well was measured daily by optical density (OD560 nm) in a Synergy HT plate reader (Biotek, Winooski, VT) for 15 days. OD readings were compiled to construct growth curves which were analyzed using DuctApeTM software58 to convert each growth curve into an “activity index” (0-9)

representing relative ability to utilize each carbon source present in the PM panel.

Biolog Dye assays, ETC inhibitors and SOD experiments

Experiments examining conditions for Biolog Dye reduction and the impact of ETC

inhibitors / SOD on growth / dye reduction were conducted in PM assay medium

supplemented with glucose (1.8% mass/volume). Spores collected from each strain

were adjusted to 8 x 105/mL in PM medium for all experiments. Biolog Dye mix “D” at a

1: 75 dilution was used in all experiments examining dye reduction and was excluded

from assays measuring growth only. All assays were conducted in clear 96-well assay

plates and absorbance (OD560) was measured using a Synergy HT plate reader after

the indicated incubation time (Biotek). Concentrations of inhibitors / SOD used in each

experiment are provided in figure legends.

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NAD+/H assay

The indicated Pseudogymnoascus strains were cultured in 5mL YPD liquid at 13°C for

72 hours. mycelium was harvested from each culture and grinded with a plastic syringe plunger against a cell strainer (70 µm, Fisher) in a 6-well tissue culture plate. Ground mycelium was then transferred to a 1.5 mL screw-top tube with 500 µL 0.1M NaOH on ice and sonicated 3 times (10 sec @ 20% amplitude) to break up larger clumps. The sonicated samples were then lysed by adding an equal volume of glass beads to each tube and shaking 6 times for 30 sec each in a Mini-Beadbeater (Biospec). 100 µL of each lysate was transferred to two separate 1.5 tubes for separate detection of NAD+ and NADH. 50 µL of 0.4 M HCl was added to NAD+ samples and both sets of samples were incubated at 60°C for 15 minutes and allowed to cool to room temperature. 50 µL

0.5 M tris base solution was then added to the NAD+ samples and 100 µL of Tris-HCl solution (equal volumes 0.5M Tris base and 0.4M HCl) was added to NADH samples.

NADH and NAD+ concentrations were then measured in the corresponding samples using the NAD+/H glo assay kit (Promega) according to the product manual. Briefly, 25

µL of each sample/standard was mixed with an equal volume of the prepared detection reagent in a black 96-well assay plate (4 wells/sample) and resulting luminescence was measured after 30 min in a plate reader. Standards (0, 50, 100, 200, 300 and 400 nM) were prepared from freshly made suspensions of NAD+ or NADH in dilution buffer (2:1,

0.1M NaOH and Tris-HCl).

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Figures and Tables

Figure 1. Carbon source utilization in P. destructans and non-pathogenic relatives (A, B) Heatmaps of Activity Index (AI) values generated by Pseudogymnoascus species (columns) across each tested carbon source (rows) at 13°C (A) and 18°C (B), constructed using Morpheus webtool: (https://software.broadinstitute.org/morpheus). (C) Mean AI

133 values for each species across each class of carbon sources present in PM panel. Error bars represent SD.

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Figure 2. Genomic alignment of putative LxrA ortholog in Pseudogymnoascus species

Figure 3. Fungal arabinose and alternative galactose catabolic pathways.

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Fungal arabinose (A) and galactose (B) pathways based on studies in Aspergillus sp. And T. reesei. Arrows indicate enzymatic steps, with responsible enzymes labeled adjacent in blue (Aspergillus) and purple (T. reesei). Differentially-colored bars underlying pathway substrates represent AI values from PM carbon source analysis in each Pseudogymnoascus species with P. destructans represented by the right-most, enlarged bar and non-pathogenic species represented by smaller bars to the left.

Figure 4. Carbon source utilization in presence of a metabolic indicator.

(A) Heatmap displaying Activity Index (AI) of Pseudogymnoascus species (columns) across each carbon source in PM panel (rows) at 13°C in the presence of the Biolog redox dye. (B) Biolog dye reduction across various Pseudogymnoascus species cultured in PM medium + glucose. (NT= not tested)

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Figure 5. Metabolic features relevant to Biolog dye reduction

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(A) NAD+/H levels across Pseudogymnoascus species cultured in rich glucose medium. (B) Sensitivity of Pseudogymnoascus to 3 different mitochondrial ETC inhibitors. (C) Sensitivity of Pseudogymnoascus species to several concentrations of antimycin. For B and C % growth is relative to growth in same medium lacking inhibitor. (D) Biolog dye reduction by P. destructans under several concentrations of antimycin +/- SOD. References:

1 Amich, J. & Krappmann, S. Deciphering metabolic traits of the fungal pathogen Aspergillus fumigatus: redundancy vs. essentiality. Front Microbiol 3, 414, doi:10.3389/fmicb.2012.00414 (2012).

2 Brock, M. Fungal metabolism in host niches. Curr Opin Microbiol 12, 371-376, doi:10.1016/j.mib.2009.05.004 (2009).

3 Ene, I. V., Brunke, S., Brown, A. J. & Hube, B. Metabolism in fungal pathogenesis. Cold Spring Harb Perspect Med 4, a019695, doi:10.1101/cshperspect.a019695 (2014).

4 Krappmann, S. How to invade a susceptible host: cellular aspects of aspergillosis. Curr Opin Microbiol 34, 136-146, doi:10.1016/j.mib.2016.10.002 (2016).

5 Kronstad, J. et al. Adaptation of Cryptococcus neoformans to mammalian hosts: integrated regulation of metabolism and virulence. Eukaryot Cell 11, 109-118, doi:10.1128/EC.05273-11 (2012).

6 Sugui, J. A. et al. Genes differentially expressed in conidia and hyphae of Aspergillus fumigatus upon exposure to human neutrophils. PLoS One 3, e2655, doi:10.1371/journal.pone.0002655 (2008).

7 Chen, Y. et al. The Cryptococcus neoformans transcriptome at the site of human meningitis. MBio 5, e01087-01013, doi:10.1128/mBio.01087-13 (2014).

8 Lorenz, M. C., Bender, J. A. & Fink, G. R. Transcriptional response of Candida albicans upon internalization by macrophages. Eukaryot Cell 3, 1076-1087, doi:10.1128/EC.3.5.1076-1087.2004 (2004).

9 Saunders, C. W., Scheynius, A. & Heitman, J. Malassezia fungi are specialized to live on skin and associated with dandruff, eczema, and other skin diseases. PLoS Pathog 8, e1002701, doi:10.1371/journal.ppat.1002701 (2012).

10 Wu, G. et al. Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny, Physiology, and Niche Adaptation on Human Skin. PLoS Genet 11, e1005614, doi:10.1371/journal.pgen.1005614 (2015).

11 White, T. C. et al. Fungi on the skin: dermatophytes and Malassezia. Cold Spring Harb Perspect Med 4, doi:10.1101/cshperspect.a019802 (2014).

138

12 Reynolds, H. T. & Barton, H. A. Comparison of the white-nose syndrome agent Pseudogymnoascus destructans to cave-dwelling relatives suggests reduced saprotrophic enzyme activity. PLoS One 9, e86437, doi:10.1371/journal.pone.0086437 (2014).

13 Chaturvedi, V. et al. Morphological and molecular characterizations of psychrophilic fungus Geomyces destructans from New York bats with White Nose Syndrome (WNS). PLoS One 5, e10783, doi:10.1371/journal.pone.0010783 (2010). 14 Palmer, J. M., Drees, K. P., Foster, J. T. & Lindner, D. L. Extreme sensitivity to ultraviolet light in the fungal pathogen causing white-nose syndrome of bats. Nat Commun 9, 35, doi:10.1038/s41467-017-02441-z (2018).

15 Wilson, M. B., Held, B. W., Freiborg, A. H., Blanchette, R. A. & Salomon, C. E. Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans, the cause of white-nose syndrome in bats. PLoS One 12, e0178968, doi:10.1371/journal.pone.0178968 (2017).

16 Chaturvedi, V., DeFiglio, H. & Chaturvedi, S. Phenotype profiling of white-nose syndrome pathogen Pseudogymnoascus destructans and closely-related Pseudogymnoascus pannorum reveals metabolic differences underlying fungal lifestyles. F1000Res 7, 665, doi:10.12688/f1000research.15067.2 (2018).

17 Khosravi, C., Benocci, T., Battaglia, E., Benoit, I. & de Vries, R. P. Sugar catabolism in Aspergillus and other fungi related to the utilization of plant biomass. Adv Appl Microbiol 90, 1-28, doi:10.1016/bs.aambs.2014.09.005 (2015).

18 van den Brink, J. & de Vries, R. P. Fungal enzyme sets for plant polysaccharide degradation. Appl Microbiol Biotechnol 91, 1477-1492, doi:10.1007/s00253-011- 3473-2 (2011).

19 de Vries, R. P. & Visser, J. Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microbiol Mol Biol Rev 65, 497-522, table of contents, doi:10.1128/MMBR.65.4.497-522.2001 (2001).

20 Richard, P., Putkonen, M., Väänänen, R., Londesborough, J. & Penttilä, M. The Missing Link in the Fungall-Arabinose Catabolic Pathway, Identification of thel- Xylulose Reductase Gene†,‡. Biochemistry 41, 6432-6437, doi:10.1021/bi025529i (2002).

21 Metz, B. et al. A novel L-xylulose reductase essential for L-arabinose catabolism in Trichoderma reesei. Biochemistry 52, 2453-2460, doi:10.1021/bi301583u (2013).

22 Seiboth, B. & Metz, B. Fungal arabinan and L-arabinose metabolism. Appl Microbiol Biotechnol 89, 1665-1673, doi:10.1007/s00253-010-3071-8 (2011).

23 de Groot, M. J., Prathumpai, W., Visser, J. & Ruijter, G. J. Metabolic control analysis of Aspergillus niger L-arabinose catabolism. Biotechnol Prog 21, 1610- 1616, doi:10.1021/bp050189o (2005).

139

24 Mojzita, D., Vuoristo, K., Koivistoinen, O. M., Penttila, M. & Richard, P. The 'true' L-xylulose reductase of filamentous fungi identified in Aspergillus niger. FEBS Lett 584, 3540-3544, doi:10.1016/j.febslet.2010.06.037 (2010).

25 Kowalczyk, J. E. et al. Genetic Interaction of Aspergillus nidulans galR, xlnR and araR in Regulating D-Galactose and L-Arabinose Release and Catabolism Gene Expression. PLoS One 10, e0143200, doi:10.1371/journal.pone.0143200 (2015).

26 Filling, C. et al. Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem 277, 25677-25684, doi:10.1074/jbc.M202160200 (2002). 27 Holden, H. M., Rayment, I. & Thoden, J. B. Structure and function of enzymes of the Leloir pathway for galactose metabolism. J Biol Chem 278, 43885-43888, doi:10.1074/jbc.R300025200 (2003).

28 Seiboth, B., Pakdaman, B. S., Hartl, L. & Kubicek, C. P. Lactose metabolism in filamentous fungi: how to deal with an unknown substrate. Fungal Biology Reviews 21, 42-48, doi:10.1016/j.fbr.2007.02.006 (2007).

29 Berridge, M. V., Herst, P. M. & Tan, A. S. Biotechnology Annual Review 127- 152 (2005).

30 Bochner, B. R. et al. Assay of the multiple energy-producing pathways of mammalian cells. PLoS One 6, e18147, doi:10.1371/journal.pone.0018147 (2011).

31 Tachon, S. et al. Experimental conditions affect the site of tetrazolium violet reduction in the electron transport chain of Lactococcus lactis. Microbiology 155, 2941-2948, doi:10.1099/mic.0.029678-0 (2009).

32 Herst, P. M., Perrone, G. G., Dawes, I. W., Bircham, P. W. & Berridge, M. V. Plasma membrane electron transport in Saccharomyces cerevisiae depends on the presence of mitochondrial respiratory subunits. FEMS Yeast Res 8, 897-905, doi:10.1111/j.1567-1364.2008.00418.x (2008).

33 Tan, A. S. & Berridge, M. V. Evidence for NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated quinone-dependent redox cycling via plasma membrane electron transport: A sensitive cellular assay for NQO1. Free Radic Biol Med 48, 421-429, doi:10.1016/j.freeradbiomed.2009.11.016 (2010).

34 Knight, S. A. & Dancis, A. Reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)- 2H-tetrazolium-5-carboxanilide inner salt (XTT) is dependent on CaFRE10 ferric reductase for Candida albicans grown in unbuffered media. Microbiology 152, 2301-2308, doi:10.1099/mic.0.28843-0 (2006).

35 Berridge, M. V. & Tan, A. S. Trans-plasma membrane electron transport: A cellular assay for NADH- and NADPH-oxidase based on extracellular, superoxide-mediated reduction of the sulfonated tetrazolium salt WST-1. Protoplasma 205, 74-82, doi:10.1007/bf01279296 (1998).

140

36 Tan, A. S. & Berridge, M. V. Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1 to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents. J Immunol Methods 238, 59-68 (2000).

37 Hayashi, S. et al. Control of reactive oxygen species (ROS) production through histidine kinases in Aspergillus nidulans under different growth conditions. FEBS Open Bio 4, 90-95, doi:10.1016/j.fob.2014.01.003 (2014).

38 Mittal, M., Siddiqui, M. R., Tran, K., Reddy, S. P. & Malik, A. B. Reactive oxygen species in inflammation and tissue injury. Antioxid Redox Signal 20, 1126-1167, doi:10.1089/ars.2012.5149 (2014). 39 Butler, J., Koppenol, W. H. & Margoliash, E. Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion. J Biol Chem 257, 10747- 10750 (1982).

40 Lavigne, L. M. et al. Integrin Engagement Mediates the Human Polymorphonuclear Leukocyte Response to a Fungal Pathogen-Associated Molecular Pattern. The Journal of Immunology 178, 7276-7282, doi:10.4049/jimmunol.178.11.7276 (2007).

41 Shen, X. X. et al. Tempo and Mode of Genome Evolution in the Budding Yeast Subphylum. Cell 175, 1533-1545 e1520, doi:10.1016/j.cell.2018.10.023 (2018).

42 Monod, M. Secreted proteases from dermatophytes. Mycopathologia 166, 285- 294, doi:10.1007/s11046-008-9105-4 (2008).

43 Martinez-Rossi, N. M., Peres, N. T. & Rossi, A. Pathogenesis of Dermatophytosis: Sensing the Host Tissue. Mycopathologia 182, 215-227, doi:10.1007/s11046-016-0057-9 (2017).

44 Bowman, S. M. & Free, S. J. The structure and synthesis of the fungal cell wall. Bioessays 28, 799-808, doi:10.1002/bies.20441 (2006).

45 Hopke, A., Brown, A. J. P., Hall, R. A. & Wheeler, R. T. Dynamic Fungal Cell Wall Architecture in Stress Adaptation and Immune Evasion. Trends Microbiol 26, 284-295, doi:10.1016/j.tim.2018.01.007 (2018).

46 Ene, I. V. et al. Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen. Cell Microbiol 14, 1319-1335, doi:10.1111/j.1462-5822.2012.01813.x (2012).

47 Moyrand, F., Fontaine, T. & Janbon, G. Systematic capsule gene disruption reveals the central role of galactose metabolism on Cryptococcus neoformans virulence. Mol Microbiol 64, 771-781, doi:10.1111/j.1365-2958.2007.05695.x (2007).

48 Fontaine, T. et al. Galactosaminogalactan, a new immunosuppressive polysaccharide of Aspergillus fumigatus. PLoS Pathog 7, e1002372, doi:10.1371/journal.ppat.1002372 (2011).

141

49 Schmalhorst, P. S. et al. Contribution of galactofuranose to the virulence of the opportunistic pathogen Aspergillus fumigatus. Eukaryot Cell 7, 1268-1277, doi:10.1128/EC.00109-08 (2008).

50 Bandouchova, H. et al. Pseudogymnoascus destructans: evidence of virulent skin invasion for bats under natural conditions, Europe. Transbound Emerg Dis 62, 1-5, doi:10.1111/tbed.12282 (2015).

51 Field, K. A. et al. The White-Nose Syndrome Transcriptome: Activation of Anti- fungal Host Responses in Wing Tissue of Hibernating Little Brown Myotis. PLoS Pathog 11, e1005168, doi:10.1371/journal.ppat.1005168 (2015).

52 Moore, M. S. et al. Energy conserving thermoregulatory patterns and lower disease severity in a bat resistant to the impacts of white-nose syndrome. J Comp Physiol B 188, 163-176, doi:10.1007/s00360-017-1109-2 (2018). 53 Shingu-Vazquez, M. & Traven, A. Mitochondria and fungal pathogenesis: drug tolerance, virulence, and potential for antifungal therapy. Eukaryot Cell 10, 1376- 1383, doi:10.1128/EC.05184-11 (2011).

54 Martins, V. P. et al. Involvement of an alternative oxidase in oxidative stress and mycelium-to-yeast differentiation in Paracoccidioides brasiliensis. Eukaryot Cell 10, 237-248, doi:10.1128/EC.00194-10 (2011).

55 Dagley, M. J. et al. Cell wall integrity is linked to mitochondria and phospholipid homeostasis in Candida albicans through the activity of the post-transcriptional regulator Ccr4-Pop2. Mol Microbiol 79, 968-989, doi:10.1111/j.1365- 2958.2010.07503.x (2011).

56 Bambach, A. et al. Goa1p of Candida albicans localizes to the mitochondria during stress and is required for mitochondrial function and virulence. Eukaryot Cell 8, 1706-1720, doi:10.1128/EC.00066-09 (2009).

57 Ma, H. et al. The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation. Proc Natl Acad Sci U S A 106, 12980-12985, doi:10.1073/pnas.0902963106 (2009).

58 Galardini, M. et al. DuctApe: a suite for the analysis and correlation of genomic and OmniLog Phenotype Microarray data. Genomics 103, 1-10, doi:10.1016/j.ygeno.2013.11.005 (2014).

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CHAPTER 5: Discussion

143

Overview

Given the marked virulence of P. destructans, with lethality in some hosts such as

M. lucifugus surpassing 90%, investigations of fungal virulence traits represent an

important avenue of research. The identification of individual virulence factors may

suggest effective strategies of intervention against P. destructans. Additionally, a better

understanding of virulence in P. destructans may provide insight into the pathogenicity of

invasive fungal pathogens as a whole, which represent a growing global threat to

biodiversity.1 The status of P. destructans, a unique animal pathogen within a genus of saprophytic fungi, also raises an important and interesting question: what separates P. destructans from its non-pathogenic relatives? The answer is likely complex and multifactorial, however direct cross-species comparisons of fungal proteins and phenotypic traits may enable a clearer understanding. In the work presented here we have made significant strides to advance our understanding of virulence in P. destructans. In

Chapter 2 we conducted a comparative analysis of fungal secretomes revealing notable differences between P. destructans and its non-pathogenic relatives, including the unique presence of secreted peptidases. One of these, PdCP1, was isolated and characterized in detail, revealing its biochemical properties, effective inhibitors and by proxy, its potential to contribute to host colonization. In Chapter 3, we established an invertebrate infection model for P. destructans which will enable testing of putative virulence factors in a high- throughput manner. In Chapter 4 we conducted further comparative analyses of P. destructans and its non-pathogenic relatives, including evaluating differences in primary fungal metabolism. These studies may improve our understanding of the transition of P. destructans from saprophyte to deadly mammalian pathogen and reveal important traits contributing to its virulence.

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Similarities in secretome composition

Our comparative analysis of secreted proteins from P. destructans and non-

pathogenic relatives in Chapter 2 revealed a common abundance of peptidases,

glycosidases and redox enzymes. The presence of these three classes of enzymes is not

surprising as they make up a core set of secretome components in other saprophytic and

animal-pathogenic fungi. Glycosidases, peptidases and other hydrolytic enzymes are

abundant in secretomes from species including Cryptococcus neoformans/gattii2,

Aspergillus fumigatus3 and Histoplasma capsulatum4. Like P. destructans, each of these species grow as saprophytes but also act as mammalian pathogens. Glycosidases in these fungi likely support saprophytic growth, enabling the break-down of plant cell wall components, a major source of environmental carbon/nitrogen. In A. fumigatus, a species particularly well adapted for saprophytic growth5,6, over half of the secretome is made up

of proteins annotated with functions in carbohydrate hydrolysis/metabolism.3

Glycosidases ranged from 20-30% of each secretome analyzed here, demonstrating that

Pseudogymnoascus species are also equipped for saprophytic growth. Redox enzymes,

also prevalent in the Pseudogymnoascus secretomes, could further support saprophytic

growth and polysaccharide degradation. Lytic polysaccharide monooxygenases (LPMOs),

identified in each of the secretomes here have recently been recognized for their key role

in the degradation of cellulose and other plant polysaccharides by saprophytic fungi.

LPMOs accept electrons from a variety of other redox enzymes or oxygen radicals and

utilize them in oxidative cleavage of glycosidic bonds7-9. The presence of LPMOs in each

of the Pseudogymnoascus secretomes likely adds to the capability of these fungi to grow

as saprophytes. Additional redox enzymes present in the Pseudogymnoascus secretomes may function as electron donors for LPMOs. However, many redox enzymes detected here including catalases, peroxidases, superoxide dismutases (SODs) and thioredoxins

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likely serve general functions to protect against reactive oxygen species (ROS).

Catalases, SODs and thioredoxins serve as virulence factors in some fungal species by

providing protection from host immune-generated ROS10-13. These ROS-scavenging enzymes are also common to secretomes of saprophytic and pathogenic fungal species with some such as thioredoxin being largely conserved across all fungal lineages suggesting they are also generally important to fungal biology/metabolism14.

The P. destructans secretome and pathogenicity: is less more?

Given the substantial contributions of peptidases, glycosidases and redox

enzymes to each of the Pseudogymnoascus secretomes, the overall profiles of secreted enzymes are largely similar. This is not entirely surprising as secretome composition across the fungal kingdom correlates strongly with phylogeny 14. The broad similarities in

secretome composition observed here therefore likely reflect the close phylogenic

relationship of these species. However, within fungal taxa, differences in secretome

composition have been linked to lifestyle 2,14,15. Accordingly, we did observe features of the P. destructans secretome that distinguish it from those of its non-pathogenic relatives.

First, the secretome of P. destructans contained a smaller number of proteins than each of its non-pathogenic relatives. This observation is consistent with results from other fungal mammalian pathogens; smaller secretomes are associated with virulence as animal pathogens generally have smaller, less diverse secretomes compared to strictly saprophytic fungi 14,15. While the selective pressures driving secretome reduction are not fully understood, it may reflect an evolutionary transition towards a more specialized pathogenic lifestyle where proteins serving roles in saprophytic growth are no longer required. Interestingly, microsporidia, perhaps the most specialized fungal animal-

pathogens and rare examples of fungal obligate parasites, have the smallest number of

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secreted proteins and smallest genomes of all fungi, supporting this idea 16-18. Reduced secretomes in pathogenic fungi may also be an adaptation to avoid immune-recognition by reducing the number of potentially antigenic proteins exposed to the host 14. A recent

genomic analysis in Aspergillus species revealed that secreted proteins are enriched for

predicted antigenic regions lending further support to this hypothesis 3.

Secreted peptidases: conservation of important virulence factors?

Despite this reduction in size, the P. destructans secretome contained a higher proportion of secreted enzymes including the highest proportion of peptidases. The retainment of these enzymes suggests they may serve important functions in this fungal pathogen. A higher proportion of secreted enzymes, including peptidases, is also a feature present of secretomes from other mammalian pathogens. In a comparative analysis of C. neoformans strains, enzymes (including peptidases) made up over 50% of hypervirulent secretomes and less than 20% of hypovirulent secretomes.2 In the dermatophyte T. rubrum, a specialized pathogen of mammalian skin, hydrolytic enzymes make up over two-thirds of the secretome, most of which are peptidases.19 Fungal peptidases contribute

to virulence in fungi by promoting adhesion and invasion of host tissues 20-22, degrading

immune proteins23-25 and releasing amino acid carbon/nitrogen sources to support growth

within the host26,27. Here, we have identified and characterized a secreted carboxypeptidase, PdCP1 (Chapter 2) that was unique to the P. destructans secretome as well as an abundant endopeptidase, Destructin-1 (Appendix A), capable of degrading collagen and elastin. The potential for these enzymes to contribute to virulence will be a focus of future investigations within our lab. The establishment of an invertebrate infection model outlined in Chapter 3 of this thesis in conjunction with current work in our lab to

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develop effective gene-targeting methods in P. destructans will greatly facilitate these efforts.

Galleria mellonella: A new model system to evaluate virulence in P. destructans

Our development of G. mellonella as an infection model for P. destructans

(Chapter 3) may enable future assessment of putative virulence factors including

peptidases and other secreted proteins identified in Chapter 2. G. mellonella has been

utilized to study virulence across a wide range of mammalian fungal pathogens28 including

C. neoformans29-31, A. fumigatus32-35 and C. albicans36,37. Importantly, many virulence

factors used by these fungi during infection of G. mellonella are also important for infection

of a mammalian host. A study of 6 A. fumigatus mutants lacking individual genes required

for full virulence in mice, found that the virulence of each of these strains towards G.

mellonella was also attenuated 35. Similarly, a study of 10 C. albicans mutants, with varied virulence in mice showed matching patterns of virulence in G. mellonella 37. A larger

analysis in C. neoformans involving 66 mutant strains with attenuated virulence in mice

found that ~70% of these also showed reduced virulence in G. mellonella 29.

Given these findings, G. mellonella is likely to serve as a valid model for initial

evaluation of putative virulence factors in P. destructans, including PdCP1 (Chapter 2),

Destructin-1 (Appendix A) and other secreted peptidases. However, my initial

experiments indicate that administration of individual peptidase inhibitors to P.

destructans-infected G. mellonella does not significantly reduce larval mortality (Figure

1A). This result could be partially explained by degradation or larval metabolism of inhibitors over the course of the experiment (up to 2 months). In this case, deletion of peptidase genes may be a better approach once an effective gene-targeting method is established in P. destructans. However, the multiplicity of secreted peptidases within the

148

P. destructans secretome may also complicate this strategy. In other fungal pathogens

functional/genetic redundancy of secreted peptidases has been circumvented by deletion

of multiple genes in a single strain38,39, or by disruption of transcriptional activators of

peptidase expression 40,41. Such approaches to evaluate the importance of peptidases during infection by P. destructans may also be possible in the future. However, other proteins identified within the P. destructans secretome in Chapter 2 also represent

putative virulence factors. Lipases, redox enzymes and phosphatases, present in the P.

destructans secretome, are established virulence factors in a number of other fungi 10,11,42-

45. G. mellonella has been used to evaluate secreted virulence factors in diverse pathogens 33,46-48, suggesting that this infection model may enable similar investigation of proteins secreted by P. destructans.

The G. mellonella model may also be limited in its ability to evaluate virulence across Pseudogymnoascus species as at least one non-pathogenic species (24MN13)

was able to kill larvae more rapidly than P. destructans (Figure 1B). This result is likely

due to differences in the host environment and/or immune response as mammalian

infection presents additional barriers, including an adaptive immune system, and indicates

limitations of the model. Similar findings have been obtained in other fungal genera such

as Aspergillus, where virulence across individual species towards G. mellonella does not

correlate with virulence towards mammalian hosts.28 However, this model may still provide

a means to evaluate the role of fungal metabolism and virulence in P. destructans.

Regulation of metabolism and the ability to effectively utilize available nutrients is closely

linked to pathogenesis across virtually all mammalian fungal pathogens 49-52. Interestingly,

disruption of genes regulating amino acid metabolism, gluconeogenesis and iron

acquisition in A. fumigatus also reduces larval killing during infection of G. mellonella,32,53

suggesting that these metabolic processes are important for colonization in this model.

149

The PM analysis described in Chapter 4 revealed that P. destructans possesses a much

more limited carbon metabolism than non-pathogenic Pseudogymnoascus species.

However, P. destructans was able to utilize multiple lipid and peptide carbon sources that

are abundant in host tissue. In future experiments it may be possible to disrupt genes

involved in lipid/peptide catabolism in P. destructans in order determine if the utilization of

these carbon sources is required for host colonization and larval killing in the G. mellonella

infection model developed here.

Conclusion

With millions of bats already lost to WNS, the impacts of this devastating disease

are likely to be severe and far-reaching. The losses suffered by these important species

will not only damage native ecosystems but also threaten food security and our

economy.54,55 Despite the damage already inflicted, WNS remains an ongoing threat with few signs of slowing its rapid spread across N. America. With each hibernation season since its discovery in 2006 the geographic range of WNS has broadened, recently detected as far west as Washington state. Yet its full destructive potential has not been realized as many locations populated by susceptible species such as M. lucifugus remain

WNS-free. Some hope lies in recent studies demonstrating evidence of resistance to WNS developing in remnant M. lucifugus populations.56,57 However, given the massive declines

of this species, it remains to be seen if the remaining populations can support a true

recovery.

The work outlined above represents some of the first investigations of potential

virulence factors in P. destructans. In Chapter 3 we also developed an invertebrate

infection model that will enable the impact of putative virulence factors to be assessed in

future experiments. Additionally, our lab is currently focused on developing genetic

150 methods in P. destructans that will enable efficient gene targeting in this fungus. My initial experiments identified conditions promoting successful formation and regeneration of protoplasts in P. destructans, a process that is widely used for genetic transformation of filamentous fungi. Current work by other lab members has demonstrated preliminary success, accomplishing efficient gene deletion in P. destructans protoplasts using a

CRISPR-Cas9 based system. An efficient method of gene disruption in conjunction with the newly established G. mellonella infection model would enable rapid evaluation of putative virulence genes. This will allow us to directly assess the contribution of secreted peptidases, other secreted enzymes and metabolic processes to virulence in P. destructans. Identification of virulence factors may reveal novel avenues of intervention against this deadly fungus and will inform future efforts to control the rising threat of emerging fungal pathogens.

151

Figures

Figure 1. Evaluation of peptidase inhibitor treatments and virulence of multiple Pseudogymnoascus species using Galleria mellonella larvae. (A) Survival curves from Galleria larvae injected with P. destructans spores (106/larva) resuspended in PBS alone or spores resuspended in PBS containing Chymostatin or Antipain. (B) Survival curves from Galleria larvae injected with live or heat-killed (HK) spores (106/larva) from P. destructans, 24MN13, or P. verrucosus.

152

References

1 Fisher, M. C. et al. Emerging fungal threats to animal, plant and ecosystem health. Nature 484, 186-194, doi:10.1038/nature10947 (2012).

2 Campbell, L. T. et al. Cryptococcus strains with different pathogenic potentials have diverse protein secretomes. Eukaryot Cell 14, 554-563, doi:10.1128/EC.00052-15 (2015).

3 Vivek-Ananth, R. P. et al. Comparative systems analysis of the secretome of the opportunistic pathogen Aspergillus fumigatus and other Aspergillus species. Sci Rep 8, 6617, doi:10.1038/s41598-018-25016-4 (2018).

4 Holbrook, E. D., Edwards, J. A., Youseff, B. H. & Rappleye, C. A. Definition of the extracellular proteome of pathogenic-phase Histoplasma capsulatum. J Proteome Res 10, 1929-1943, doi:10.1021/pr1011697 (2011).

5 Askew, D. S. Aspergillus fumigatus: virulence genes in a street-smart mold. Curr Opin Microbiol 11, 331-337, doi:10.1016/j.mib.2008.05.009 (2008).

6 Tekaia, F. & Latge, J. P. Aspergillus fumigatus: saprophyte or pathogen? Curr Opin Microbiol 8, 385-392, doi:10.1016/j.mib.2005.06.017 (2005).

7 Kracher, D. et al. Extracellular electron transfer systems fuel cellulose oxidative degradation. Science 352, 1098-1101, doi:10.1126/science.aaf3165 (2016).

8 Agger, J. W. et al. Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation. Proc Natl Acad Sci U S A 111, 6287-6292, doi:10.1073/pnas.1323629111 (2014).

9 Johansen, K. S. Lytic Polysaccharide Monooxygenases: The Microbial Power Tool for Lignocellulose Degradation. Trends Plant Sci 21, 926-936, doi:10.1016/j.tplants.2016.07.012 (2016).

10 Cox, G. M. et al. Superoxide Dismutase Influences the Virulence of Cryptococcus neoformans by Affecting Growth within Macrophages. Infection and Immunity 71, 173-180, doi:10.1128/iai.71.1.173-180.2003 (2003).

11 Frohner, I. E., Bourgeois, C., Yatsyk, K., Majer, O. & Kuchler, K. Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance. Mol Microbiol 71, 240- 252, doi:10.1111/j.1365-2958.2008.06528.x (2009).

12 Holbrook, E. D., Smolnycki, K. A., Youseff, B. H. & Rappleye, C. A. Redundant catalases detoxify phagocyte reactive oxygen and facilitate Histoplasma capsulatum pathogenesis. Infect Immun 81, 2334-2346, doi:10.1128/IAI.00173- 13 (2013).

153

13 da Silva Dantas, A. et al. Thioredoxin regulates multiple hydrogen peroxide- induced signaling pathways in Candida albicans. Mol Cell Biol 30, 4550-4563, doi:10.1128/MCB.00313-10 (2010).

14 Krijger, J. J., Thon, M. R., Deising, H. B. & Wirsel, S. G. Compositions of fungal secretomes indicate a greater impact of phylogenetic history than lifestyle adaptation. BMC Genomics 15, 722, doi:10.1186/1471-2164-15-722 (2014).

15 Lowe, R. G. & Howlett, B. J. Indifferent, affectionate, or deceitful: lifestyles and secretomes of fungi. PLoS Pathog 8, e1002515, doi:10.1371/journal.ppat.1002515 (2012).

16 Katinka, M. D. et al. Genome sequence and gene compaction of the eukaryote parasite Encephalitozoon cuniculi. Nature 414, 450-453, doi:10.1038/35106579 (2001).

17 Corradi, N., Pombert, J. F., Farinelli, L., Didier, E. S. & Keeling, P. J. The complete sequence of the smallest known nuclear genome from the microsporidian Encephalitozoon intestinalis. Nat Commun 1, 77, doi:10.1038/ncomms1082 (2010).

18 Pombert, J. F., Haag, K. L., Beidas, S., Ebert, D. & Keeling, P. J. The Ordospora colligata genome: Evolution of extreme reduction in microsporidia and host-to- parasite horizontal gene transfer. MBio 6, doi:10.1128/mBio.02400-14 (2015).

19 Giddey, K. et al. Comprehensive analysis of proteins secreted by Trichophyton rubrum and Trichophyton violaceum under in vitro conditions. J Proteome Res 6, 3081-3092, doi:10.1021/pr070153m (2007).

20 Naglik, J. R., Challacombe, S. J. & Hube, B. Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis. Microbiology and Molecular Biology Reviews 67, 400-428, doi:10.1128/mmbr.67.3.400-428.2003 (2003).

21 Ollert, M. W. et al. Mechanisms of adherence of Candida albicans to cultured human epidermal keratinocytes. Infect Immun 61, 4560-4568 (1993).

22 Baldo, A. et al. Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes. J Med Microbiol 57, 1152-1156, doi:10.1099/jmm.0.47827-0 (2008).

23 Thekkiniath, J. C., Zabet-Moghaddam, M., San Francisco, S. K. & San Francisco, M. J. A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis is induced by thyroid hormone and degrades antimicrobial peptides. Fungal Biol 117, 451-461, doi:10.1016/j.funbio.2013.05.002 (2013).

24 Behnsen, J. et al. Secreted Aspergillus fumigatus protease Alp1 degrades human complement proteins C3, C4, and C5. Infect Immun 78, 3585-3594, doi:10.1128/IAI.01353-09 (2010).

25 Kaminishi, H. et al. Degradation of humoral host defense by Candida albicans proteinase. Infect Immun 63, 984-988, doi:0019-9567 (1995).

154

26 Monod, M. Secreted proteases from dermatophytes. Mycopathologia 166, 285- 294, doi:10.1007/s11046-008-9105-4 (2008).

27 Martinez-Rossi, N. M., Peres, N. T. & Rossi, A. Pathogenesis of Dermatophytosis: Sensing the Host Tissue. Mycopathologia 182, 215-227, doi:10.1007/s11046-016-0057-9 (2017).

28 Binder, U., Maurer, E. & Lass-Florl, C. Galleria mellonella: An invertebrate model to study pathogenicity in correctly defined fungal species. Fungal Biol 120, 288- 295, doi:10.1016/j.funbio.2015.06.002 (2016).

29 Desalermos, A. et al. A multi-host approach for the systematic analysis of virulence factors in Cryptococcus neoformans. J Infect Dis 211, 298-305, doi:10.1093/infdis/jiu441 (2015).

30 Firacative, C., Duan, S. & Meyer, W. Galleria mellonella model identifies highly virulent strains among all major molecular types of Cryptococcus gattii. PLoS One 9, e105076, doi:10.1371/journal.pone.0105076 (2014).

31 Mylonakis, E. et al. Galleria mellonella as a model system to study Cryptococcus neoformans pathogenesis. Infect Immun 73, 3842-3850, doi:10.1128/IAI.73.7.3842-3850.2005 (2005).

32 Beckmann, N. et al. Characterization of the Link between Ornithine, Arginine, Polyamine and Siderophore Metabolism in Aspergillus fumigatus. PLoS One 8, e67426, doi:10.1371/journal.pone.0067426 (2013).

33 Reeves, E. P. et al. A nonribosomal peptide synthetase (Pes1) confers protection against oxidative stress in Aspergillus fumigatus. FEBS J 273, 3038-3053, doi:10.1111/j.1742-4658.2006.05315.x (2006).

34 Renwick, J., Daly, P., Reeves, E. P. & Kavanagh, K. Susceptibility of larvae of Galleria mellonella to infection by Aspergillus fumigatus is dependent upon stage of conidial germination. Mycopathologia 161, 377-384, doi:10.1007/s11046-006- 0021-1 (2006).

35 Slater, J. L., Gregson, L., Denning, D. W. & Warn, P. A. Pathogenicity of Aspergillus fumigatus mutants assessed in Galleria mellonella matches that in mice. Med Mycol 49 Suppl 1, S107-113, doi:10.3109/13693786.2010.523852 (2011).

36 Cowen, L. E. et al. Harnessing Hsp90 function as a powerful, broadly effective therapeutic strategy for fungal infectious disease. Proc Natl Acad Sci U S A 106, 2818-2823, doi:10.1073/pnas.0813394106 (2009).

37 Brennan, M., Thomas, D. Y., Whiteway, M. & Kavanagh, K. Correlation between virulence ofCandida albicansmutants in mice andGalleria mellonellalarvae. FEMS Immunology & Medical Microbiology 34, 153-157, doi:10.1111/j.1574- 695X.2002.tb00617.x (2002).

155

38 Sanglard, D., Hube, B., Monod, M., Odds, F. C. & Gow, N. A. A triple deletion of the secreted aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes attenuated virulence. Infect Immun 65, 3539-3546 (1997).

39 Schaller, M. et al. Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis. Mol Microbiol 34, 169-180 (1999).

40 Shemesh, E. et al. Phenotypic and Proteomic Analysis of the Aspergillus fumigatus DeltaPrtT, DeltaXprG and DeltaXprG/DeltaPrtT Protease-Deficient Mutants. Front Microbiol 8, 2490, doi:10.3389/fmicb.2017.02490 (2017).

41 Bertuzzi, M. et al. The pH-responsive PacC transcription factor of Aspergillus fumigatus governs epithelial entry and tissue invasion during pulmonary aspergillosis. PLoS Pathog 10, e1004413, doi:10.1371/journal.ppat.1004413 (2014).

42 Collopy-Junior, I. et al. An ectophosphatase activity in Cryptococcus neoformans. FEMS Yeast Res 6, 1010-1017, doi:10.1111/j.1567-1364.2006.00105.x (2006).

43 Kiffer-Moreira, T. et al. An ectophosphatase activity in Candida parapsilosis influences the interaction of fungi with epithelial cells. FEMS Yeast Res 7, 621- 628, doi:10.1111/j.1567-1364.2007.00223.x (2007).

44 Garfoot, A. L. & Rappleye, C. A. Histoplasma capsulatum surmounts obstacles to intracellular pathogenesis. FEBS J 283, 619-633, doi:10.1111/febs.13389 (2016).

45 Gacser, A. et al. Lipase 8 affects the pathogenesis of Candida albicans. Infect Immun 75, 4710-4718, doi:10.1128/IAI.00372-07 (2007).

46 Lev, S. et al. Identification of Aph1, a phosphate-regulated, secreted, and vacuolar acid phosphatase in Cryptococcus neoformans. MBio 5, e01649-01614, doi:10.1128/mBio.01649-14 (2014).

47 Andrejko, M. & Mizerska-Dudka, M. Effect of Pseudomonas aeruginosa elastase B on level and activity of immune proteins/peptides of Galleria mellonella hemolymph. J Insect Sci 12, 88, doi:10.1673/031.012.8801 (2012).

48 Fedhila, S., Nel, P. & Lereclus, D. The InhA2 Metalloprotease of Bacillus thuringiensis Strain 407 Is Required for Pathogenicity in Insects Infected via the Oral Route. Journal of Bacteriology 184, 3296-3304, doi:10.1128/jb.184.12.3296- 3304.2002 (2002).

49 Beattie, S. R. et al. Filamentous fungal carbon catabolite repression supports metabolic plasticity and stress responses essential for disease progression. PLoS Pathog 13, e1006340, doi:10.1371/journal.ppat.1006340 (2017).

50 Brock, M. Fungal metabolism in host niches. Curr Opin Microbiol 12, 371-376, doi:10.1016/j.mib.2009.05.004 (2009).

156

51 Ene, I. V. et al. Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen. Cell Microbiol 14, 1319-1335, doi:10.1111/j.1462-5822.2012.01813.x (2012).

52 Ene, I. V., Brunke, S., Brown, A. J. & Hube, B. Metabolism in fungal pathogenesis. Cold Spring Harb Perspect Med 4, a019695, doi:10.1101/cshperspect.a019695 (2014).

53 Liu, H. et al. Aspergillus fumigatus AcuM regulates both iron acquisition and gluconeogenesis. Mol Microbiol 78, 1038-1054, doi:10.1111/j.1365- 2958.2010.07389.x (2010).

54 Maine, J. J. & Boyles, J. G. Bats initiate vital agroecological interactions in corn. Proc Natl Acad Sci U S A 112, 12438-12443, doi:10.1073/pnas.1505413112 (2015).

55 Boyles, J. G., Cryan, P. M., McCracken, G. F. & Kunz, T. H. Conservation. Economic importance of bats in agriculture. Science 332, 41-42, doi:10.1126/science.1201366 (2011).

56 Lilley, T. M. et al. White-nose syndrome survivors do not exhibit frequent arousals associated with Pseudogymnoascus destructans infection. Front Zool 13, 12, doi:10.1186/s12983-016-0143-3 (2016).

57 Reichard, J. D. et al. Interannual Survival of Myotis lucifugus (Chiroptera: Vespertilionidae) near the Epicenter of White-Nose Syndrome. Northeast Nat (Steuben) 21, N56-N59, doi:10.1656/045.021.0410 (2014).

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APPENDIX A: Destructin-1 is a Collagen-Degrading Endopeptidase Secreted by P. destructans, the Causative Agent of White-Nose Syndrome

This appendix was published in PNAS (2015 Jun 16; 112(24): 7478–7483). I performed the following experiments in contribution to this publication: recombinant expression and purification of Destructin-1 from P. pastoris, collagenase activity assays in figures 4A and 5B, and experiments represented in supplementary figure S3.

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Destructin-1 is a Collagen-Degrading Endopeptidase Secreted by P. destructans, the Causative Agent of White-Nose Syndrome

Running Title: Collagen-Degrading Peptidase Destructin-1

Anthony J. O’Donoghue1#, Giselle M. Knudsen1#, Chapman Beekman2, Jenna Perry2,

Alexander D. Johnson3, Joseph L. DeRisi3, Charles S. Craik1, and Richard J. Bennett2*

1Department of Pharmaceutical Chemistry, University of California San Francisco (UCSF),

San Francisco, California, United States of America.

3Department of Biochemistry and Biophysics, University of California San Francisco

(UCSF),

San Francisco, California, United States of America, and the Howard Hughes Medical

Institute, San Francisco, CA.

2Brown University, 171 Meeting St, Providence, RI 02912.

#These authors contributed equally to the work

*To whom correspondence should be addressed. E-mail: [email protected]

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Abstract

P. destructans is the causative agent of White-Nose Syndrome (WNS), a

devastating disease that has caused the deaths of millions of bats in North America. This

psychrophilic fungus targets hibernating bats, resulting in their premature arousal from

stupor with catastrophic consequences. Despite the impact of WNS, little is known about

the fungus or how it mediates infection of the mammalian host. P. destructans is not

amenable to genetic manipulation, and therefore understanding the proteins involved in

infection requires alternative approaches. Here, we identify a set of proteolytic enzymes

that are a part of a broad arsenal of hydrolytic enzymes secreted by P. destructans.

Collagen, the major structural protein in mammals, was degraded by secreted peptidases from this fungus, and we therefore used a novel and unbiased substrate profiling technique to define active peptidases in the P. destructans secretome. These experiments revealed that endopeptidases are the major proteolytic activities secreted by

P. destructans. A serine endopeptidase, hereby-named Destructin-1, was subsequently identified, and a recombinant form overexpressed and purified. Biochemical analysis of

Destructin-1 showed that it mediated collagen degradation, and a potent inhibitor of peptidase activity was identified. Treatment of P. destructans conditioned media with this antagonist blocked collagen degradation and facilitated the detection of additional secreted proteolytic activities, including aminopeptidases and carboxypeptidases. These results provide the first molecular insights into the secretome of P. destructans, and identify serine endopeptidase(s) that have the clear potential to facilitate tissue invasion and pathogenesis in the mammalian host.

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Significance Statement

This work is the first to identify molecular factors produced by the fungus P. destructans, the causative agent of White-Nose Syndrome in bats. Our study reveals the repertoire of redox enzymes and hydrolytic enzymes secreted by P. destructans. We establish that a secreted serine peptidase, Destructin-1, is a major component of the P. destructans secretome. This peptidase was purified and shown to degrade collagen, the major structural protein in mammalian connective tissue. Furthermore, chemical inhibition of Destructin-1 blocked collagen degradation in conditioned media from P. destructans.

We therefore propose that serine endopeptidase(s) aid in invasive growth and tissue destruction by the fungus, and represent potential targets for therapeutic intervention in

WNS.

161

Introduction

White-Nose Syndrome (WNS) has caused the deaths of more than 6 million bats

in North America since its discovery in a New York cave in 2006 (1, 2). It has spread to

22 US states and 5 Canadian provinces, with nearly 100% mortality observed in some

locations (3). This represents one of the most precipitous declines in North American

wildlife seen in the past century (1). If current trends continue, 25 species of hibernating

bats in the US will be threatened, with some previously common species becoming extinct

(4). In addition to the devastating impact on bat populations, the disease is an economical

threat to the North American agricultural industry, where the loss of bats could cost the

industry more than 3 billion dollars a year (5).

The causative agent of WNS is the fungus Pseudogymnoascus destructans

(formerly Geomyces destructans) (6), which grows as a white layer on the muzzle, wings

and ears of bats (7). P. destructans is a psychrophilic fungus that belongs to the family

Pseudeurotiaceae, and appears to be an invasive species with no close relatives in the

hibernacula of North America (6). P. destructans targets hibernating bats whose immune

function is reduced and whose body temperatures are lowered. The fungus grows

optimally at these lower temperatures, with maximal growth between 12°C and 16°C (8).

The injuries associated with fungal infections result in increased arousal in hibernating

bats and the premature use of fat storage, with the outcome that bats are emaciated and

die before the end of hibernation. Infection involves deep penetration of the subcutaneous

tissue by fungal hyphae, causing ulcerative necrosis and tissue destruction (7, 9-11). P.

destructans typically forms more superficial infections in European bat populations, with

no evidence for associated mortality (9, 12), although a recent study also found evidence

of invasive WNS lesions in European bats (13). Current models suggest that P.

162

destructans is an invasive species that originated in Europe, where native bat species may

be more resistant to the most debilitating forms of the disease (9).

There is currently little information as to the mechanism by which P. destructans

causes tissue invasion or infection in bats. To begin to address the properties of P.

destructans associated with WNS, we focused on secreted enzymes produced by this

fungus. Many fungal pathogens secrete a number of important enzymes that promote

pathogenesis, of which proteolytic activities have been the most intensively studied (14,

15). Peptidases play diverse roles in fungal disease as illustrated by the SAP family of

aspartyl peptidases produced by pathogenic Candida species. In Candida albicans, the

most common human fungal pathogen, these enzymes are implicated in multiple

processes including adhesion to epithelial cells, degradation of host proteins, survival and

escape from immune cells, and invasion of mucosal tissues (16). Aspartyl and serine

peptidases are also associated with dermatophytes that infect the stratum corneum, nails,

and hair of animals. Here, they are implicated in promoting adherence to host cells and

keratin degradation during tissue invasion (17, 18). Both Candida species and

dermatophytes display expanded protein families of peptidases, supporting the contention

that these factors are key virulence factors (15, 18). Given their central role in

pathogenesis, there is also now considerable interest in identifying inhibitors of fungal

peptidases as potential therapeutic drugs (19). Other virulence factors secreted by

mammalian fungal pathogens include lipolytic enzymes (lipases and phospholipases) that

can further mediate the destruction of epithelial tissues (20).

In this work, we analyzed the secretome of P. destructans and found that most proteins are predicted to have hydrolytic activity, including a number of peptidases, lipases and glycosidases, or are redox enzymes such as catalase peroxidase. Secreted peptidases included those with the ability to degrade collagen, the major component of

163 mammalian connective tissue. To address global proteolytic activity, an unbiased substrate profiling assay was performed, and revealed that endopeptidases are the major proteolytic activities secreted by P. destructans. Using conventional chromatography and an internally quenched fluorescence reporter substrate, the major endopeptidase activity was isolated and shown to be associated with a serine endopeptidase, hereby named

Destructin-1. Recombinant Destructin-1 was overexpressed and purified, and shown to actively degrade collagen. Significantly, Destructin-1 activity was potently blocked by the serine peptidase inhibitor chymostatin, and treatment of conditioned media with this inhibitor blocked collagen degradation. Destructin-1 therefore represents a novel virulence factor for P. destructans, with the ability to promote tissue damage and invasion in the mammalian host.

Results

Hydrolytic enzymes are the major proteins secreted by P. destructans

In order to identify proteins secreted by P. destructans, fungal cells were grown in

RPMI medium at 13°C for 7 days. Proteins from the conditioned medium were analyzed by peptide sequencing using liquid chromatography-tandem mass spectrometry (LC-

MS/MS), and targets searched against the P. destructans genome. In total, 44 proteins were identified in the secretome, of which 33 were found in at least 2 of 3 independent experiments (Tables S1-S3). Many of these proteins were predicted to have enzymatic activity based on sequence analysis and were broadly grouped as hydrolytic enzymes, glycosyl transferases, or redox enzymes. The hydrolytic enzymes included 13 glycosidases, 6 peptidases, 2 lipases and 1 amidase (Fig. 1A). The diversity of hydrolytic enzymes present is consistent with previous reports of multiple hydrolytic activities in P. destructans cultures, although the proteins responsible for these activities were not

164

determined (21, 22). Many of these enzymes are likely to play a role in supporting saprophytic growth, but fungal peptidases can also function in supporting host-pathogen

interactions (14, 15).

The P. destructans secretome included three serine endopeptidases, two serine carboxypeptidases, and an aspartyl endopeptidase (Fig. 1B). The aspartyl endopeptidase shared 21% to 26% sequence identity with the C. albicans Sap protein family (23). The

two carboxypeptidases were GMDG_06096, which is closely related to carboxypeptidase

Y from Saccharomyces cerevisiae (56% sequence identity), and GMDG_05452, which is

similar to carboxypeptidase II from Aspergillus niger (58% sequence identity). The three serine endopeptidases exhibited similarity to cuticle-degrading enzymes secreted by entomopathogenic fungi (24). These included GMDG_06417 and GMDG_08491, which share 90% amino acid identity and are hereby named Destructin-1 and Destructin-2,

respectively. A third serine peptidase, GMDG_04447, showed 56% identity to Destructin-

1 and was named Destructin-3 (Fig. S1).

Collagen and synthetic peptides are degraded by secreted peptidases

One of the primary sites of infection by P. destructans is the membranous skin of bats’ wings, where it causes extensive invasion and tissue damage (25). To test whether peptidases in the secretome could contribute to wing damage and tissue invasion, conditioned media was incubated with azo dye-impregnated collagen. We observed a

time-dependent release of dye over a 54 hour time course (Fig. 2A). This finding led us

to perform a comprehensive analysis of the proteolytic activity secreted from P.

destructans with the goal of identifying and characterizing peptidase(s) responsible for

collagen degradation. We used a global and unbiased substrate profiling assay to uncover

165

the secreted proteolytic signature of this fungus. This assay consists of a mixture of 124

physiochemically diverse peptides that are each 14-residues in length. Cleavage at any

one of the 1612 peptide bonds within these peptides can be readily detected by LC-

MS/MS sequencing (Fig. 2B) (26). Co-incubation with the P. destructans secretome

resulted in 137 cleavage sites detected after 1-hour incubation and 308 cleavage events

after 20 hours incubation. The complexity of these hydrolytic events is illustrated in three

example peptides where multiple cleavage sites were often detected within each peptide

(Fig. 2C). Using iceLogo software (27), a substrate signature was generated

corresponding to the global specificity of the peptidases in the media. These peptidases

exhibited a preference for hydrophobic residues at P4, Ile and norleucine at P2, Gln, Phe

and Trp at P1, and Ile at P2ʹ (Fig. 2D). In addition, the detected peptidases showed a low

tolerance for Glu in almost all positions and Val, Pro and Gly at P1. Time-dependent trimming of amino acids from the termini of these peptides was not evident, indicating that exopeptidase activity was rare and that the major activity was due to one or more endopeptidases.

Endopeptidase activity from P. destructans can be monitored with fluorescent substrates

A diverse set of 15 internally quenched (IQ) fluorescent peptides (Table S5) was screened to identify substrates that could be used to monitor endopeptidase activity in P. destructans conditioned media. Two of the 15 peptides were efficiently cleaved (Fig. 3A) and the sites of cleavage determined by MALDI-TOF mass spectrometry (Fig. S2). These substrates consisted of tQAS↓SRS (IQ8) and PKRLSAL↓L (IQ12), where t represents tert butyl glycine and ↓ the position of cleavage. Analysis of these cleavage sites revealed the presence of a hydrophobic residue at P4 and Ala at P2 in both substrates, consistent with

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the global iceLogo substrate signature (Fig. 2D). However, these initial experiments did

not determine whether the endopeptidase activity is derived from one or multiple enzymes.

Purification and identification of endopeptidases from P. destructans

To isolate the peptidase(s) responsible for cleavage of IQ8 and IQ12 peptides, conditioned P. destructans medium was applied to a DEAE sepharose column and eluted

fractions assayed for proteolytic activity (Fig. 3B). Fractions with activity were pooled,

applied to a Phenyl sepharose column, and eluted fractions assayed again using IQ8 and

IQ12 (Fig. 3C). Proteolytic activity on these substrates was found to co-purify, and active fractions pooled and subjected to gel filtration chromatography. Activity from the gel filtration column identified a peptidase with a molecular weight of ~25 kDa (Fig. 3D).

Analysis of protein from the active fractions showed two major bands on a silver-stained

SDS-PAGE gel (Fig. 3D, inset). These bands were excised and analyzed by LC-MS/MS, and the upper band shown to represent Destructin-1 (GMDG_06417). The lower, minor band was GMDG_08104, a highly abundant protein in the secretome that contains a WSC domain. A number of unique peptides support the specific identification of Destructin-1

(Fig. S1 and Table S4); however, due to the high sequence conservation with Destructin-

2 it is not possible to exclude its presence at lower abundance. Indeed, analysis of individual protein bands excised after SDS-PAGE analysis of the Phenyl sepharose eluate

showed the presence of Destructin-2-specific peptides (Table S4).

These results suggest that Destructin-1 encodes the major proteolytic activity

responsible for cleavage of both IQ8 and IQ12 substrates. This enzyme shares 50-52%

amino acid identity with secreted cuticle-degrading peptidases from nematode-trapping fungi such as Dactylella varietas and Arthrobotrys conoides (DvS8 and AcAC1, Fig. S1)

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(28, 29). In addition, Destructin-1 shares 46% identity with EaS8 (Fig. S1), a broad-

spectrum endopeptidase from Engyodontium album that is stable in SDS, urea, chelating

agents and sulfhydryl reagents, and is commercially marketed as “Proteinase K”. These

enzymes utilize a catalytic triad of aspartic acid, histidine, and serine residues (30), which

are conserved in Destructin-1 at positions 160, 192, and 345, respectively (Fig. S1).

Destructin-1, -2, and -3 contain an N-terminal signal sequence and a pro-domain

that are predicted to be removed during secretion and catalytic maturation, respectively.

Analysis of the N-terminus of Destructin-1 using SignalP 4.0 (31) identified a signal peptide

(residues 2-20), that was highly conserved with Destructin-2 and Destructin-3 (Fig. S1).

Protein alignment with other fungal enzymes predicted auto-catalytic processing of the

Destructin-1 pro-domain occurs after Asn119 to yield a mature peptidase of 27.7 kDa, which correlates with its elution size from gel filtration (Fig. 3D). Peptide sequencing showed coverage exclusively within the mature peptidase domain (highlighted in Fig. S1) and the absence of tryptic peptides corresponding to the pro-domain (Ala21-Asn119). This establishes that the protein species detected here is the activated form.

Expression and characterization of recombinant Destructin-1

To further characterize the activity of Destructin-1, a recombinant form of the pro- enzyme was expressed with a C-terminal hexahistidine tag and purified from Pichia pastoris (Fig. S3A). The resulting major band on a SDS-PAGE gel was excised and analyzed by MS sequencing and Edman degradation. These results established the identity of recombinant Destructin-1 and confirmed that the pro-enzyme is auto-processed

between Asn119 and Ala120 (Fig. S1). The recombinant Destructin-1 hydrolyzed IQ8 and

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IQ12 substrates with optimal activity between pH 9 and 10, and no activity was evident below pH 4.2 (Fig. S3B).

Degradation of collagen by Destructin-1

Destructin-1 was assayed with azo dye-impregnated collagen for 72 hours and shown to release dye in a time-dependent manner (Fig. 4A). The recombinant enzyme was also incubated with soluble rat-tail collagen and the hydrolytic products assessed by

SDS-PAGE and coomassie staining. As shown in Fig. 4B, collagen consists of several major protein bands; the lower molecular weight α-bands at ~120 kDa consist only of triple helical protein while the higher molecular weight β-bands contain additional non-helical regions. Destructin-1 rapidly degraded the β-bands but did not cleave the alpha bands, even after extended incubation. These experiments reveal that Destructin-1 readily degrades the non-helical regions of collagen that function in the cross-linking of the helical components.

Rational design of optimal fluorescent substrates for Destructin-1

The substrate specificity of recombinant Destructin-1 was further investigated using an expanded MSP-MS assay containing 228 tetradecapeptides. Using 10 nM of enzyme, 197 peptide bonds were cleaved within 5 minutes, with a preference for Phe, Gln and Tyr at P1. Hydrophobic residues were preferred at P4 and P2, with positively charged or bulky residues at P3. On the prime side of the scissile bond Lys and Thr were preferred at P1ʹ and Ile, Trp and Tyr at P2ʹ (Fig. 4C).

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The MSP-MS assay was validated as a tool for defining the substrate specificity of

recombinant PdSP1 by direct comparison with specificity data generated using a

positional scanning synthetic combinatorial library (PS-SCL). The PS-SCL assay has

been used to profile the P1 to P4 substrate specificity of more than 90 endopeptidases,

most of which are serine and cysteine peptidases (32). This assay consists of 80 sub-

libraries each containing 8,000 unique tetrapeptides linked to a fluorogenic 7-amino-4-

carbamoylmethylcoumarin group on the C-terminus. This assay cannot be used to

characterize complex protease mixtures such as conditioned media due to an inability to

detect aminopeptidase and carboxypeptidases activity and a requirement for >5 µg of

each peptidase. As was observed in the MSP-MS assay, PdSP1 preferentially cleaved

substrates containing hydrophobic residues at P4, positively charged residues at P3, small

or flexible residues at P2, and large, bulky residues at P1 (Fig. 4E). Both assays showed

a strong positive correlation of 0.86, 0.93, 0.54 and 0.73 (Pearson chi-squared test) at

positions P4, P3, P3 and P1, respectively (Table S6). This substrate signature represents

the most detailed specificity profile of a peptidase from a fungal species to date.

Based on the substrate specificity data, we predicted that IQ8 and IQ12 were

suboptimal substrates for Destructin-1. We have previously synthesized improved

substrates for peptidases based on the auto-activation site of the enzyme (33) or on the

optimal sequences found in the substrate specificity profile (34). An IQ substrate was

therefore synthesized corresponding to the P4 to P4ʹ residues at the pro-Destructin-1 auto-

activation site (VQAN-SLET) with flanking methylcoumarin and dinitrophenol groups (IQ-

Pro). An additional IQ substrate was synthesized corresponding to the preferred residues

in the P4 to P4ʹ positions from the MSP-MS assay (IQ-Opt). IQ-Opt was the most

efficiently cleaved substrate with a kcat/Km of 14.3 x 106 M-1 s-1, which is a 10-fold improvement over IQ8 and 6-fold more efficient than IQ-Pro (Fig. 4D). Both IQ-Pro and

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IQ-Opt could be accommodated into a homology model for the destructin-1 structure (Fig.

4F and Fig. S4). In the homology model, P3’ and P4’ positions of the peptide do not significantly interact with the enzyme, but there are deep hydrophobic S1 and S2 pockets on the enzyme that could bind to F,Y,Q and n,I,V, respectively, consistent with the substrate recognition motif shown in Fig. 4C. These data highlight the use of specificity profiling to develop optimized peptide substrates that can serve as highly sensitive biochemical probes, even when compared to natural peptide substrates.

Contribution of Destructin-1 to global proteolytic activity in the P. destructans secretome

In order to determine the contribution of Destructin-1 and related serine peptidases to global proteolytic activity, we tested known protease inhibitors for inhibition of

Destructin-1 activity. Using the IQ8 substrate, we found that the serine inhibitors PMSF, antipain, and chymostatin were antagonists of Destructin-1 activity with IC50 values of

46.1 µM, 85 nM, and 7.5 nM, respectively (Fig. 5A). Addition of the potent agonist chymostatin to P. destructans conditioned media resulted in a 77% reduction in collagen degradation at 54 hours (Fig. 5B). This confirms that Destructin-1, together with its close homologs, is the dominant collagen-degrading activity secreted by P. destructans.

The contribution of the chymostatin-sensitive serine endopeptidases to the global secreted proteolytic activity of P. destructans was evaluated using the MSP-MS assay.

Conditioned media was treated with either DMSO or chymostatin and incubated with the peptide library. The appearance of cleavage products was assessed after 15 minutes and

1, 4 and 20 hours. Media that was treated with chymostatin resulted in a loss of 74% or more of the cleavage sites that were detected in the DMSO control (Fig. 5C-D). This

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indicated that Destructin-1 and its homologs are the source of most of the peptidase

activity secreted from P. destructans. Interestingly many of the cleavage sites that were

resistant to chymostatin were located at the amino and carboxyl terminus. In fact,

treatment with the inhibitor resulted in the appearance of additional cleavage sites at each

termini (Fig. 5E). These sites were not detected in the control assay because the 14-mer

substrates were rapidly degraded into short oligopeptides by the serine endopeptidases.

The enzymes responsible for generation of cleavage sites at the termini are likely to be

the exopeptidases detected in the proteomic study (Fig. 1). Together, this data indicates

that chymostatin-resistant aminopeptidases and carboxypeptidases are present in the

conditioned media, and are revealed upon inhibition of the dominant serine

endopeptidases.

Discussion

White-Nose Syndrome is a devastating disease that has targeted bat populations

in North America over the last decade. The disease is caused by P. destructans, a fungus

that infects hibernating bats and causes extensive tissue damage, particularly to the fragile

membranous wings (1). Connective tissue, vascular structures, and muscle fibers are degraded during infection, suggesting that hydrolytic enzymes are used by the invading pathogen (25). Secreted hydrolytic activities have been described by monitoring growth of P. destructans on a wide range of in vitro substrates (21, 22), but the fungal proteins responsible for these activities have not been elucidated.

In this work, we analyzed the secretome of P. destructans, and identified a number

of prevalent hydrolytic and redox enzymes. The array of secreted proteins shows

172 similarities to those described in other fungal species, including the human pathogens C. albicans and A. fumigatus (35, 36). These fungi produce multiple hydrolytic enzymes that target host cells, including peptidases that function in tissue degradation, nutrient acquisition and host invasion (37). P. destructans secretes two serine carboxypeptidases

(S10 family), an aspartyl peptidase (A1 family) and three serine endopeptidases (S8 family). Our functional studies determined that one or more of these peptidases degrades collagen, the major structural protein in mammalian tissue (38). Therefore, we surmised that uncovering the peptidase(s) responsible for degradation of this protein would be a valuable step towards understanding bat tissue invasion by P. destructans.

A global and unbiased substrate profiling technology (26) was used to determine that endopeptidase-type activities dominate the P. destructans secretome. Using a set of fluorescent reporter substrates, a serine endopeptidase, Destructin-1, was identified as the principal proteolytic activity present in P. destructans cultures. A recombinant form of the enzyme was purified and shown to be capable of degrading collagen. In contrast, no cleavage was observed by Destructin-1 on keratin and only very weak activity on elastin

(data not shown). Collagen consists of a core triple helix structure linked together by non- helical cross-links to form a collagen fiber (38). Collagenases such as those produced by

Clostridium species readily degrade the helical regions of collagen (39). In contrast, however, Destructin-1 specifically cleaved the non-helical cross-links between alpha 1 and

2 proteins. This disrupts the integrity of collagen and may allow the fungus to penetrate further into the host tissue, possibly in combination with other peptidase activities.

An in-depth study of recombinant Destructin-1 activity was performed using an expanded MSP-MS assay containing 228 tetradecapeptides and a fluorescent library of

160,000 tetrapeptides. Destructin-1 was shown to readily cleave on the C-terminal side of Gln, Tyr and Phe residues, particularly when hydrophobic residues were present at the

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P4 position and Nle, Ile or Val were present at the P2 position. This study represents the

most detailed substrate specificity profile performed on a fungal peptidase to date, and

allowed us to design a synthetic peptide that was a more efficient substrate than one

corresponding to the pro-Destructin-1 auto-activation site.

The recombinant enzyme was potently inhibited by the serine peptidase antagonist chymostatin, with an IC50 of 7.5 nM. Treatment of P. destructans conditioned media with chymostatin established that Destructin-1 and its close homologs were responsible for collagen degradation; inhibition of these endopeptidases resulted in a loss of 85% of the peptide cleavage sites in the MSP-MS assay compared to a vehicle-treated control.

Interestingly, because inhibitor treatment prevented the breakdown of many substrates in the MSP-MS assay, proteolytic activities derived from other peptidases could now be detected. Analysis of the proteolytic activities uncovered by chymostatin treatment revealed that aminopeptidases and carboxypeptidases were present in the media. The potential synergy between endopeptidases and exopeptidases is intriguing, as Destructin-

1 may cleave intact proteins in the bat tissue, resulting in the appearance of neo-termini that are then substrates for trimming by exopeptidases.

The closest homologs of Destructin-1 are cuticle-degrading subtilisin peptidases found in nematophagous fungi such as A. conoides and D. varietas. Nematophagous fungi use a variety of methods to capture and kill nematodes, which are subsequently digested by the fungi (24). The subtilisin-type peptidases promote penetration and digestion of nematode cuticles, and are key enzymes in nematophagous species for killing of their prey (24, 29, 40-42). Interestingly, a subtilisin-like serine peptidase was also recently identified in Batrachochytrium dendrobatidis, a chytrid fungus responsible for a global decline in amphibian species. This peptidase was shown to cleave anti-microbial peptides produced by frog skin, and is thus implicated in fungal survival and pathogenesis

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(43). Furthermore, the kexin gene in C. albicans encodes a subtilisin-type protease that

is necessary for virulence due to its role in processing of proproteins (44). This suggests

that the family of subtilisin-type peptidases can play diverse roles as fungal virulence

factors.

In summary, this work details the composition of the P. destructans secretome and identifies the serine peptidase Destructin-1 as the major extracellular, collagen-degrading endopeptidase. Future studies will further address the potential role of Destructin-1 and its homologs as novel virulence factors, and will determine the role of other secreted proteins in promoting infection of epithelial tissues. It is expected that a combination of hydrolytic activities are used by P. destructans to invade and destroy bat tissues. As such, limiting these hydrolytic activities is predicted to be a successful approach for the prevention or treatment of WNS in bats.

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Materials and Methods

Proteomic Analysis of the P. destructans secretome

P. destructans was grown in multiple 5 ml RPMI cultures in a roller drum at 13°C

for 7 days. Cultures were centrifuged and the supernatant filtered (0.45 µM). The

supernatant was concentrated and buffer exchanged into PBS using Amicon centrifuge filters. Protein identification in protease-enriched samples from P. destructans secretions was performed using peptide sequencing by mass spectrometry. For analysis of the secretions, protein samples were processed by trypsin digestion following a previously published method (1). Peptides were extracted and desalted using C18 zip-tips, then lyophilized and stored at -80 °C for later analysis.

Peptide sequencing was performed using either an LTQ-Orbitrap XL (Thermo) or an LTQ-Orbitrap Velos mass spectrometer, equipped with 10,000 psi system nanoACUITY (Waters) ultra-performance liquid chromatography (UPLC) instruments for reversed phase chromatography with a C18 column (BEH130, 1.7 µm bead size, 100 µm x 100 mm). The LC was operated at 600 nL/min flow rate, and peptides were separated using a linear gradient over 42 min from 2% to 30% acetonitrile in 0.1% formic acid, as previously reported (1).

For MS/MS analysis on the LTQ OrbitrapXL, survey scans were recorded over a

350-1500 m/z range, and peptide fragmentation was induced with collision-induced dissociation (CID) for MS/MS. CID fragmentation was performed on the six most intense precursor ions, with a minimum of 1,000 counts, using an isolation width of 2.0 amu, with

35% normalized collision energy. On the LTQ Orbitrap Velos, survey scans were recorded over a 350-1400 m/z range, and MS/MS was performed using higher-energy collisional dissociation (HCD) activation on the ten most intense precursor ions, with minimum signal of 2000 counts, using an isolation width of 2.5 amu, and 30% normalized collision energy 176

over a mass range of 350-1500 m/z. Internal recalibration to a polydimethylcyclosiloxane

(PCM) ion with m/z = 445.120025 was used for both MS and MS/MS scans on both

instruments (2).

Data were analyzed as previously reported using in-house PAVA software to

generate centroid peak lists and Protein Prospector software v. 5.10.15 for data searches

(3). For protein identification, searches were performed against the Geomyces destructans 20631-21 database from the Broad Institute of Harvard and MIT

(http://www.broadinstitute.org/, downloaded March 21, 2013) containing 9,153 entries.

This database was concatenated with a fully randomized set of 9,153 entries for estimation

of false discovery rate (Elias, 2007). Data were also searched against the SwissProt

database containing 449,080 entries (downloaded January 11, 2012) for identification of

common contaminant proteins. Peptide sequences were matched as tryptic peptides with

up to 2 missed cleavages, and carbamidomethylated cysteines as a fixed modification.

Variable modifications included: oxidation of methionine, N-terminal pyroglutamate from

glutamine, start methionine processing, and protein N-terminal acetylation. Mass accuracy tolerance was set to 20 ppm for parent and 0.6 Da fragment errors for CID data, or 20 ppm parent and 30 ppm fragment errors for HCD data.

For reporting of protein identifications from these database searches, score thresholds were selected that resulted in a protein false discovery rate of less than 1%.

The specific Protein Prospector parameters were: minimum protein score of 22, minimum peptide score of 15, and maximum expectation values of 0.02 for protein and 0.05 for peptide matches. Protein identifications are reported with a spectral count as an approximation of protein abundance, percent sequence coverage, and expectation values for the confidence of the peptide and protein identification (Table S1) (4). Protein identifications supported by single peptide identifications were manually verified.

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Supporting MS sequencing search results are provided in a peptide report for all P.

destructans protein identifications in the secretome (Supplemental Table 2) or in purified

proteolytically active fractions (Supplemental Table 3).

Gene Ontology Searches

Algorithms within the Blast2GO program (v. 2.7.2) were used to search for proteins

with shared sequence features to P. destructans secretome proteins (5). BLASTp searches of P. destructans secretome protein sequences were performed against the

SwissProt or NCBInr databases, using the NCBI server (July 1, 2014,

http://blast.ncbi.nlm.nih.gov/Blast.cgi) using default parameters: expectation value cutoff

of 1e6 and a maximum of 20 BLASTp hits reported. The top ranking BLASTp hit for each

P. destructans secretome protein from SwissProt or NCBInr is reported in Supplemental

Table 1, with associated expectation values and the number of BLAST hits identified.

Gene Ontology (GO) terms and Enzyme Code (EC) numbers associated with these

BLASTp hits were retrieved from the Blast2GO public database (b2g_Sept13).

Internally quenched peptide assays

Unless otherwise stated, all assays were performed at room temperature in Dulbecco’s-

Phosphate-Buffered Saline (D-PBS) either with or without 0.01% Tween-20. Assays were

performed in triplicate in round-bottom 96-well plates in a Biotek Synergy HT or H4 using

a λex 328nm and λem 393 nm. Initial velocities in relative fluorescent units per second were

calculated using Gen5 software (Biotek) or Excel. Protein from P. destructans conditioned

media was assayed with a set of internally quenched fluorescent substrates (40 µM each).

Each IQ substrate consisted of a 7 or 8-mer peptide flanked with 2,4-dinitrophenyl-L-lysine

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(AnaSpec) on the carboxyl terminus and either 7-Methoxycoumarin-4-acetic acid or 7- methoxycoumarin-4-yl-acetyl-L-lysine (AnaSpec) on the amino terminus. For assays on chromatography elutions, each fraction was diluted into assay buffer containing either IQ8 or IQ12. Inhibition studies were performed using 1 nM of recombinant enzyme, 12.5 µM of IQ8 (Km = 40.9 µM) and a 1 in 4 serial dilution of chymostatin (Sigma C7268), antipain

(Sigma A6191) or PMSF (Sigma P7626). Internally quenched substrates IQ-Pro and IQ-

Opt were synthesized by C S Bio (Menlo Park, CA). These substrates displayed poor solubility in assay buffer above 5 µM, therefore, kcat/km was calculated directly from the progress curves using 0.2 nM of Destructin-1 and 1 µM of each substrate. Progress curves were modeled using the first-order kinetics formula Y = exp(–t × kcat/KM × [E0]), where E0 is the total enzyme concentration, and fitted using the Marquardt method for nonlinear least-squares fitting to the enzyme kinetics model in GraphPad Prism v.5.

Catalytic efficiency was solved from the overall rate by estimating total enzyme concentration and is reported as kcat/KM.

Mass Spectrometry analysis of IQ digestion products

The cleavage sites of IQ8 and IQ12 were determined using matrix-assisted laser desorption ionization-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS). A

4700 mass analyzer (Applied Biosystems) was calibrated using a peptide mass calibration kit (Sigma) for a mass range of 500-4500 Da, and data were acquired in this same range using an average of 100 shots. Spectra were analyzed using Data Explorer software

(Applied Biosystems).

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Purification of peptidase activity from P. destructans

P. destructans was grown in RPMI medium for 7 days at 13°C and 200-300 ml of conditioned medium collected and filtered through a 0.45 µM filter. Medium was concentrated to ~1 ml using Amicon Ultra-15 centrifugal filters (10,000 NMWL, Millipore) and buffer exchanged into PBS. The sample was then diluted in 30 ml Buffer A (10 mM

Tris, pH 9.0, 10 mM NaCl) and applied to a 1 ml HiTrap Capto DEAE column. The column was washed with 10 ml Buffer A and activity eluted with a gradient of increasing concentrations of Buffer B (10 mM Tris, pH 9.0, 250 mM NaCl).

Fractions were assayed for proteolytic activity using IQ8 and IQ12 internally quenched peptide substrates, and active fractions combined (~2 ml total). The pooled activity was diluted with 10 ml Buffer C (10 mM Tris, pH 9.0, 1.5 M NH4SO4) and applied to a 1 ml Phenyl sepharose FF (low subl) column. The column was washed with 10 ml of

Buffer C and activity eluted with an increasing gradient of Buffer A. Activity was monitored using IQ8/IQ12 substrates and active fractions pooled. The sample was concentrated and buffer exchanged into PBS using an Amicon Ultra-0.5 ml centrifugal filter (10,000 NMWL,

Millipore). The sample was then applied to a Sephadex 75 gel filtration column and fractions assayed for activity. Activity assays and SDS-PAGE analysis indicated that the isolated peptidase fraction had poor stability at pH 7.4, most likely due to autodegradation.

To counter this, the pH of the isolated enzyme was adjusted to pH 4 where activity was minimal, but from which activity could be recovered upon raising the pH (data not shown).

Pooled fractions were therefore collected, pH adjusted to ~4.0 using 0.5 N HCl, and concentrated in an Amicon Ultra-0.5 ml filter. Samples were analyzed by SDS-PAGE and silver staining (Thermo Scientific Kit#24612).

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Cloning of Destructin-1 into P. pastoris expression system

Destructin-1 cDNA was generated by fusion of two synthetic fragments of the

Destructin-1 gene that were generated as GeneArt fragments (Life Technologies). The

gene was constructed with flanking sequences for ligase-independent cloning into the

pPINKalphaHC vector (Life Technologies) as well as a 6 x Histidine tag at the C terminus.

Flanking sequences were 5’- GAAGGGGTATCTCTCGAGAAAAGGCCT-3’ and 5’-

CCTGTATTTAAATGGCCGGCCGGTACCTTAATGATGATGATGATG – 3’. In addition,

the putative Destructin-1 signal sequence was removed from the gene sequence, as there

is an efficient signal sequence already present in the pPINKalphaHC vector. The two

synthetic fragments of Destructin-1 were cloned into pPINKalphaHC (linearized with StuI)

using a CPEC strategy (6). The resulting vector, pRB396, contains the Destructin-1 gene under the control of the P. pastoris AOX1 promoter.

Expression of recombinant Destructin-1 in Pichia pastoris

The expression vector pRB396 was transformed into P. pastoris PichiaPinkTM

Strain 2 using the PichiaPink Yeast Expression System from Life Technologies. A single

P. pastoris transformant was selected and patch-streaked onto adenine-deficient selective agar and incubated 24 hours at 30°C. The resulting colony was used to inoculate four separate 25 ml buffered glycerol-complex media (BMGY) cultures which were incubated at 29°C in a shaking incubator (~220 rpm). Once culture growth had reached an OD600 of 2-6 (~24 h), the 25 ml cultures were used to inoculate 4 x 1 L BMGY cultures which were incubated for a further 24 hours. Cultures were centrifuged and cells re-suspended in 400 ml buffered methanol-complex media (BMMY). Cultures were incubated for an additional 48 h at 29°C. 2 ml of 100% methanol was added to each 400 ml culture (0.5%)

181 after 24 hours to replace what had been consumed. Cells were removed by centrifugation and culture supernatants collected.

The conditioned supernatant (~4 L) was concentrated to 50 ml using a 400 ml stirred cell vacuum filtration unit (Amicon) and 10 kDa filter membrane (Millipore). The sample was diluted 1:10 in Binding buffer (50 mM sodium-phosphate buffer, pH 8.0, 300 mM NaCl, 5 mM Imidazole) and re-concentrated to a final volume of 50 ml. Concentrated supernatant was incubated with 1.5 ml His-Pur Ni-NTA resin (Thermo-Scientific) prewashed in Binding buffer for 16 hours at 4°C on a rotator. The slurry was applied to a

10 ml gravity-flow column (BioRad) and resin washed with 10 volumes of Binding buffer.

The Destructin-1 protein was eluted with 3 column volumes of Elution buffer (50 mM sodium-phosphate buffer, pH 8.0, 300 mM NaCl, 100 mM Imidazole). The Ni-NTA eluent was dialyzed into 250 ml of Buffer A and applied to a 1 ml DEAE-conjugated sepharose bead column (GE Healthcare) prewashed in Buffer A. The column was washed with 10 volumes of Buffer A and eluted with Buffer A containing increasing concentrations of NaCl.

Activity eluted in fractions containing 50 mM and 100 mM NaCl. Samples were then concentrated using Amicon Ultra 0.5 ml centrifugal filters (Millipore) and stored at -80°C in Buffer A supplemented with 10% glycerol. Edman sequencing (UC Davis Genome

Center) was performed using 2 µg of the recombinant enzyme following SDS-PAGE analysis and transfer to PVDF. Optimal activity was determined at room temperature using IQ12 in phosphate-citrate, Tris-HCl, and glycine buffers ranging from pH 4 to 10.5.

Multiplex Peptide Cleavage Assay

The MSP-MS assay was performed as previously described (1), with minor modifications. Briefly, secreted P. destructans protein was pre-incubated for 15 minutes

182 at room temperature with chymostatin or DMSO in D-PBS. Each reaction was split into 2 tubes containing an equimolar mixture of 64 peptides in D-PBS (124 total). The final assay consisted of 500 nM of each peptide, 50 µg/ml protein and either 0.1% DMSO or 10 µM

Chymostatin in a total volume of 300 µl. Aliquots were removed at defined time intervals, adjusted to

PBS at room temperature and aliquots were removed at defined time intervals as outlined above.

Mass spectrometry was performed with either the LTQ Orbitrap XL or an LTQ FT instrument, equipped with 10,000 psi system nanoACUITY (Waters) ultra-performance liquid chromatography (UPLC) instruments for reversed phase chromatography as above.

However, in this case an alternate EZ-Spray source (Thermo) was utilized, and reversed phase liquid chromatography was performed using an EZ-Spray C18 column (Thermo,

ES800, PepMap, 3 µm bead size, 75 µm x 15 cm). The LC was operated at 600 nL/min flow rate for loading and 300 nL/min for peptide separation over a linear gradient from 2% to 30% acetonitrile in 0.1% formic acid. For the MSP-MS assays with 64 peptides a 65 min gradient was used, while a 95 min gradient was used for the assays with 114 peptides.

The LTQ FT and LTQ Orbitrap XL mass spectrometers were operated using identical acquisition parameters as reported (1).

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Substrate profiling using a fluorescent combinatorial peptide library

The complete diverse Positional Scanning Synthetic Combinatorial Library (PS-

SCL) consists of four sub-libraries of fluorogenic substrates with the general structure

acetyl-P4-P3-P2-P1-amino-4-carbamoyl-methylcoumarin. One position is fixed in each sublibrary, while the remaining positions contain an equimolar mixture of all amino acids

(7). Recombinant Destructin-1 (15 nM) was assayed with the library in D-PBS, 0.02%

Triton X-100 for 1 hour at room temperature. All reactions were run in triplicate in 96-well

round bottom plates in a Biotek Synergy H4 using a λex 380 nm and λem 460 nm. Initial

velocities in relative fluorescent units per second were calculated using Gen5 software

(Biotek) and converted to nM of released amino-4-carbamoyl-methylcoumarin per sec.

Molecular Modeling of the Destructin-1 Structure

To generate an atomic model for Destructin-1, homologous structures were

identified (PDB entries 3whi, 2p4e, 2pwa, 2b6n, 1sh7, 3wiu, and 1r6v) and alignments were built with the hidden Markov model comparison tool HHpred (8), and applied the restraint-based comparative modeling program MODELLER-v9.10 (9). Structures were visualized using UCSF Chimera software v 1.9 (11). To model the bound substrate peptides, the Destructin-1 model was superposed with an additional structure of proteinase K (1p7w) to which was bound an inhibitor with the sequence PAPFAA, using the Matchmaker tool within Chimera, at 0.5 Å rmsd. The correlate inhibitor sidechains were mutated to either the native Destructin-1 sequence (VQANAL) or the optimal substrate sequence (IRnQKI where n = nLeu) with a rotamer library (10) accessed through

UCSF Chimera (11). These modeled structures are shown in Figure S4.

184

Collagen, elastin and keratin degradation assays

Azo dye-impregnated collagen (Sigma A4341) was incubated with conditioned media from

P. destructans (31 µg/ml) or recombinant Destructin-1 (31.2 µg/ml) in the presence of 500

µM chymostatin or 0.01% DMSO. For each reaction 3 mg of azo-collagen was washed 3

times in 50 mM Tris-HCl, pH 9 and assayed in the same buffer at 25°C in the dark under

constant shaking (250 rpm). At each indicated time point, the reaction tube was

centrifuged and 1.5 µl of the solution was removed and the absorbance read at 520 nm in

a Nanodrop (Thermo Scientific). Reactions were vortexed lightly and returned to

incubation. In addition, 3 mg of keratin-azure (Sigma K8500) and elastin-orcein (Sigma

E1500) were washed and incubated with recombinant Destructin-1 as outlined above and

absorbance was measured at 440 nm and 570 nm, respectively. Type I collagen from rat

tail (a kind gift from Chris Overall at the University of British Columbia) was solubilized in

1% acetic acid to a final concentration of 10 mg/ml. An assay containing 800 µg/ml Type

I collagen and 80 µg/ml recombinant Destructin-1 in 2X PBS was set up and incubated at room temperature. At defined time intervals, an aliquot was removed, combined with SDS gel loading buffer and flash frozen. Each aliquot was run on a 4-20% Bis-Tris gel in MOPS buffer and coomassie stained.

Acknowledgements

This research was funded in part by the UCSF Program for Breakthrough Biomedical

Research, and by the Sandler Foundation (to C.S.C.). Mass spectrometry analysis was

performed in the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A.L.

Burlingame, Director) supported by the Biomedical Technology Research Centers

program of the NIH National Institute of General Medical Sciences, NIH NIGMS

8P41GM103481. Work was also supported by the NIH T32GM007601 (to C.B.) and

185

P50GM082250 (to C.S.C). The authors also thank Jeremy A. Horst for structural modeling assistance with the Destructin-1 homology model.

186

Figures

Figure 1. Analysis of the secretome of P. destructans. (A) Composition of enzymatic activities present in conditioned medium from P. destructans. (B) Phylogenetic relationship between hydrolytic activities secreted by P. destructans. Note that secreted activities include three families of peptidases.

187

Figure 2. Peptidase substrate specificity from P. destructans conditioned medium. (A) Cleavage of azo-collagen by conditioned medium from P. destructans. (B) Outline of the MSP-MS assay used to examine peptidase activities in the secretome of P. destructans. Conditioned media was incubated with a mixture of 124 peptides and sampled at subsequent time points by LC-MS/MS peptide sequencing. (C) Cleavage sites are shown for three representative peptides in the MSP-MS assay. Incubation time at which cleavage events were first observed is indicated in minutes. (D) iceLogo generated from the pattern of cleavage events at 60 min shows the specificity of peptidase activity. Amino acids that are most frequently observed at each position are shown above the axis, and amino acids least frequently observed are shown below the axis.

188

Figure 3. Purification of a serine S8 peptidase, Destructin-1, from P. destructans conditioned medium. (A) Analysis of relative cleavage rates by P. destructans conditioned media on 15 different IQ substrates. Conditioned medium was purified using a 3-step process using (B) DEAE sepharose, (C) Phenyl sepharose, and (D) gel filtration. Peptidase activity was monitored using cleavage of IQ8 (red line) and IQ12 (blue line) substrates. Yellow line indicates total protein by absorbance at 280 nm and the grey box shows the fractions that were pooled for subsequent separation or characterization. Green line indicates protein standards on gel filtration column. The most purified fraction was also analyzed on a silver-stained SDS-PAGE gel (inset, part D).

189

Figure 4. Characterization of recombinant Destructin-1 activity. (A) Destructin-1 was co-incubated with Azo-collagen for 54 hours at 20°C and the release of Azo dye measured photometrically at 520 nm. (B) Cleavage and analysis of collagen degradation by Destructin-1 by SDS-PAGE. 1 and 1 bands indicate the major protein components of collagen. (C) iceLogo analysis of the recombinant Destructin-1 protein in the MSP-MS assay. (D) Comparison of kinetics of cleavage between IQ8, IQ-Pro and IQ-Opt substrates. kcat/Km values are shown for each IQ substrate. (E) PS-SCL profiling of the recombinant Destructin-1 protein to determine cleavage specificity at P1-P4 positions. (F) Homology model of the Destructin-1 substrate-binding pocket (grey ribbons and semitransparent surface) with the IQ-Opt sequence IRnQKIE shown in orange, and the catalytic triad residues Asp160, His192, and Ser345 in red.

190

Figure 5. Inhibition of Destructin-1 reveals the presence of other peptidases in the P. destructans secretome. (A) Inhibition of Destructin-1 peptidase activity using chymostatin, antipain or PMSF inhibitors. Activity assays were performed using the IQ8 substrate. (B) Cleavage of azo-collagen by Destructin-1 in the presence or absence of chymostatin. (C) Total number of Destructin-1 cleavage sites in the MSP-MS assay in the presence (red) or absence (black) of chymostatin. Cleavage sites that are only present in the presence of chymostatin are colored purple. (D) Examples of two peptides from the MSP-MS assay cleaved by recombinant Destructin-1 in the presence (red/purple arrows) or absence (black arrows) of chymostatin. The time in minutes at which cleavage events were first detected is indicated. (E) Positional analysis of peptide cleavage by Destructin-1 after 1 hour incubation in the MSP-MS assay in the presence or absence of chymostatin. Color scheme is the same as in D.

191

References

1. Blehert DS (2012) Fungal disease and the developing story of bat white-nose

syndrome. PLoS Pathog 8(7):e1002779.

2. Blehert DS, et al. (2009) Bat white-nose syndrome: an emerging fungal

pathogen? Science 323(5911):227.

3. Coleman J & Hibbard C (2013) News Release. U.S. Fish and Wildlife

(http://batcon.org/pdfs/whitenose/2013 WNS grants to states final.pdf).

4. Frick WF, et al. (2010) An emerging disease causes regional population collapse

of a common North American bat species. Science 329(5992):679-682.

5. Boyles JG, Cryan PM, McCracken GF, & Kunz TH (2012) Economic Importance

of Bats in Agriculture. Science 332:41-42.

6. Minnis AM & Lindner DL (2013) Phylogenetic evaluation of Geomyces and allies

reveals no close relatives of Pseudogymnoascus destructans, comb. nov., in bat

hibernacula of eastern North America. Fungal Biol 117(9):638-649.

7. Lorch JM, et al. (2011) Experimental infection of bats with Geomyces destructans

causes white-nose syndrome. Nature 480(7377):376-378.

8. Verant ML, Boyles JG, Waldrep W, Jr., Wibbelt G, & Blehert DS (2012)

Temperature-dependent growth of Geomyces destructans, the fungus that

causes bat white-nose syndrome. PLoS One 7(9):e46280.

9. Warnecke L, et al. (2012) Inoculation of bats with European Geomyces

destructans supports the novel pathogen hypothesis for the origin of white-nose

syndrome. Proc Natl Acad Sci U S A 109(18):6999-7003.

192

10. Meteyer CU, et al. (2009) Histopathologic criteria to confirm white-nose

syndrome in bats. Journal of veterinary diagnostic investigation : official

publication of the American Association of Veterinary Laboratory Diagnosticians,

Inc 21(4):411-414.

11. Wibbelt G, et al. (2013) Skin lesions in European hibernating bats associated

with Geomyces destructans, the etiologic agent of white-nose syndrome. PLoS

One 8(9):e74105.

12. Puechmaille SJ, et al. (2013) Pan-European Distribution of White-Nose

Syndrome Fungus (Geomyces destructans) Not Associated with Mass Mortality.

PLoS One 6:e19167.

13. Zukal J, et al. (2014) White-nose syndrome fungus: a generalist pathogen of

hibernating bats. PLoS One 9(5):e97224.

14. Yike I (2011) Fungal proteases and their pathophysiological effects.

Mycopathologia 171(5):299-323.

15. Monod M, et al. (2002) Secreted proteases from pathogenic fungi. International

journal of medical microbiology : IJMM 292(5-6):405-419.

16. Naglik J, Albrecht A, Bader O, & Hube B (2004) Candida albicans proteinases

and host/pathogen interactions. Cell Microbiol 6(10):915-926.

17. Baldo A, et al. (2012) Mechanisms of skin adherence and invasion by

dermatophytes. Mycoses 55(3):218-223.

18. Achterman RR & White TC (2012) Dermatophyte virulence factors: identifying

and analyzing genes that may contribute to chronic or acute skin infections.

International journal of microbiology 2012:358305.

193

19. Santos AL & Braga-Silva LA (2013) Aspartic protease inhibitors: effective drugs

against the human fungal pathogen Candida albicans. Mini reviews in medicinal

chemistry 13(1):155-162.

20. Park M, Do E, & Jung WH (2013) Lipolytic enzymes involved in the virulence of

human pathogenic fungi. Mycobiology 41(2):67-72.

21. Chaturvedi V, et al. (2010) Morphological and molecular characterizations of

psychrophilic fungus Geomyces destructans from New York bats with White

Nose Syndrome (WNS). PLoS One 5(5):e10783.

22. Reynolds HT & Barton HA (2014) Comparison of the white-nose syndrome agent

Pseudogymnoascus destructans to cave-dwelling relatives suggests reduced

saprotrophic enzyme activity. PLoS One 9(1):e86437.

23. Monod M & Borg-von ZM (2002) Secreted aspartic proteases as virulence factors

of Candida species. Biological chemistry 383(7-8):1087-1093.

24. Yang J, Tian B, Liang L, & Zhang KQ (2007) Extracellular enzymes and the

pathogenesis of nematophagous fungi. Applied microbiology and biotechnology

75(1):21-31.

25. Cryan PM, Meteyer CU, Boyles JG, & Blehert DS (2010) Wing pathology of

white-nose syndrome in bats suggests life-threatening disruption of physiology.

BMC Biol 8:135.

26. O'Donoghue AJ, et al. (2012) Global identification of peptidase specificity by

multiplex substrate profiling. Nature methods 9(11):1095-1100.

194

27. Colaert N, Helsens K, Martens L, Vandekerckhove J, & Gevaert K (2009)

Improved visualization of protein consensus sequences by iceLogo. Nature

methods 6(11):786-787.

28. Yang J, et al. (2007) Cloning and characterization of an extracellular serine

protease from the nematode-trapping fungus Arthrobotrys conoides. Archives of

microbiology 188(2):167-174.

29. Yang J, et al. (2007) Purification and cloning of a novel serine protease from the

nematode-trapping fungus Dactylellina varietas and its potential roles in infection

against nematodes. Applied microbiology and biotechnology 75(3):557-565.

30. Page MJ & Di Cera E (2008) Serine peptidases: classification, structure and

function. Cell Mol Life Sci 65(7-8):1220-1236.

31. Petersen TN, Brunak S, von Heijne G, & Nielsen H (2011) SignalP 4.0:

discriminating signal peptides from transmembrane regions. Nature methods

8(10):785-786.

32. Harris JL, et al. (2000) Rapid and general profiling of protease specificity by

using combinatorial fluorogenic substrate libraries. Proc Natl Acad Sci U S A

97(14):7754-7759.

33. O'Donoghue AJ, et al. (2008) Inhibition of a secreted glutamic peptidase prevents

growth of the fungus Talaromyces emersonii. J Biol Chem 283(43):29186-29195.

34. Small JL, et al. (2013) Substrate specificity of MarP, a periplasmic protease

required for resistance to acid and oxidative stress in Mycobacterium

tuberculosis. J Biol Chem 288(18):12489-12499.

195

35. Sorgo AG, et al. (2010) Mass spectrometric analysis of the secretome of Candida

albicans. Yeast 27(8):661-672.

36. Wartenberg D, et al. (2011) Secretome analysis of Aspergillus fumigatus reveals

Asp-hemolysin as a major secreted protein. International journal of medical

microbiology : IJMM 301(7):602-611.

37. Schaller M, Borelli C, Korting HC, & Hube B (2005) Hydrolytic enzymes as

virulence factors of Candida albicans. Mycoses 48(6):365-377.

38. Shoulders MD & Raines RT (2009) Collagen structure and stability. Annu Rev

Biochem 78:929-958.

39. Eckhard U, Huesgen PF, Brandstetter H, & Overall CM (2014) Proteomic

protease specificity profiling of clostridial collagenases reveals their intrinsic

nature as dedicated degraders of collagen. Journal of proteomics 100:102-114.

40. Tunlid A, Rosen S, Ek B, & Rask L (1994) Purification and characterization of an

extracellular serine protease from the nematode-trapping fungus Arthrobotrys

oligospora. Microbiology 140 ( Pt 7):1687-1695.

41. Li J, et al. (2010) New insights into the evolution of subtilisin-like serine protease

genes in Pezizomycotina. BMC Evol Biol 10:68.

42. Huang X, Zhao N, & Zhang K (2004) Extracellular enzymes serving as virulence

factors in nematophagous fungi involved in infection of the host. Res Microbiol

155(10):811-816.

43. Thekkiniath JC, Zabet-Moghaddam M, San Francisco SK, & San Francisco MJ

(2013) A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis

196

is induced by thyroid hormone and degrades antimicrobial peptides. Fungal Biol

117(6):451-461.

44. Newport G, et al. (2003) Inactivation of Kex2p diminishes the virulence of

Candida albicans. J Biol Chem 278:1713-1720.

197

Supplementary Figures and Tables

198

Figure S1. Protein alignment of Destructin-1, -2 and -3 with serine endopeptidases from other fungi. Alignment of serine peptidases Destructin-1(GMDG_06417),

Destructin-2 (GMDG_08491) and Destructin-3 (GMDG_04447) with those from Dactylella varietas (DvS8), Arthrobotrys conoides (AcAC1), and Engyodontium album (EvS8).

Genbank accession numbers are 440633958, 440637657, 440640127, DQ531603,

62467863 and 131077, respectively. The signal sequence, pro-domain and catalytic domain are indicated. Highlighted text indicates peptides identified by LC-MS/MS analysis and were accumulated from the three replicate secretome analyses (Table S2) and the enriched peptidase activity fraction (Table S3). Yellow text are peptides present in

Destructin-1. Green text are peptides that are present in serine peptidases but are distinct from Destructin-1. Alignment was performed using ClustalW. The autoproteolytic processing site, determined by N-terminal sequencing, is indicated with a bar at

VQAN/ALET. The predicted catalytic triad residues are marked with a red caret.

199

Figure S2. Identification of the cleavage sites in IQ8 and IQ12 substrates using

MALDI-TOF MS.

IQ8 and IQ12 were incubated for 1 hour in D-PBS with conditioned media from P. destructans. The reaction was quenched with 2% formic acid and desalted using a C18 tip. The eluted peptide was spotted on a 96-well MALDI-TOF plate. (A, B) IQ8 (tQASSRS) was cleaved between the Ser4 and Ser5 residues and both products are visible. (C, D)

IQ12 (PKRLSALL) is cleaved between the between Leu7 and Leu8and only the larger product, PKRLSAL is detected. A secondary, minor cleavage site is found between Leu4

and Ser5.

200

Figure S3. Determination of purity and pH Optimum for Destructin-1. (A)

Coomassie-stained SDS-PAGE gel of Destructin-1 expressed and purified from P. pastoris. Lane 2, protein after nickel chromatography. Lane 3, protein after nickel and

DEAE chromatography. (B) Analysis of Destructin-1 activity at different pH values.

Experiments were performed in phosphate-citrate (pH 4-8), Tris-HCl (pH 7.5-9) and glycine (pH 9-10.5) buffers and activity determined using the IQ12 substrate assay.

201

Figure S4. Homology model of Destructin-1. The homology model of Destructin-1 is shown with residues 120-400 modeled as a ribbon diagram and semi-transparent solvent accessible surface. Catalytic triad residues D160, H192 and S345 are shown in red.

Binding to the native sequence (VQANAL) is shown (A, C) or to the optimal substrate sequence (IRnQKI) (B,D).

202

Table S1.

GdDB Number of Average Accession No. Protein Description of best Accession Replicates Spectral for best BLASTp hit Number Observed Count BLASTp Hit GMDG_07937 3 33 P32470 chitinase 1 GMDG_08104 3 32 P84675 fungistatic metabolite GMDG_03116 3 14 P84675 fungistatic metabolite GMDG_07140 3 13 Q12599 cytochrome p450 55a3 GMDG_01515 2 7 A4QUT2 catalase-peroxidase 2 GMDG_03914 3 5 Q8J0P4 glycosidase crf1 GMDG_07779 3 5 A1DJ47 endo-1,3-beta-glucosidase GMDG_06417 3 5 A1CIA7 alkaline protease 1 GMDG_08491 3 4 P29118 alkaline proteinase GMDG_03456 3 3 EYE90739.1 six-hairpin glycosidase GMDG_08145 3 9 Q9URY4 amidase GMDG_04446 3 5 P49426 glucan 1,3-beta-glucosidase GMDG_08378 2 3 Q4WCX9 uncharacterized protein GMDG_08552 3 4 P43485 mitomycin radical oxidase GMDG_06569 3 3 Q1E3R8 endochitinase 1 GMDG_05333 3 5 D0VKF5 probable beta-glucosidase a GMDG_05452 3 3 P52719 carboxypeptidase GMDG_08595 2 2 P43077 beta-hexosaminidase GMDG_03838 3 3 XP_008087208.1 hypothetical protein GMDG_02198 3 3 Q00363 race-specific elicitor a4 GMDG_07699 2 4 P15703 glucan 1,3-beta-glucosidase GMDG_02357 2 2 P84675 fungistatic metabolite GMDG_01729 3 2 XP_001597233.1 hypothetical protein GMDG_02668 2 3 CCD53300.1 hypothetical protein GMDG_00244 1 2 Q2U325 probable beta-glucosidase g

203

Probable aspartic-type GMDG_03638 2 1 D4D8U6 endopeptidase GMDG_02429 3 2 XP_007296051.1 IgE-binding protein GMDG_00651 2 2 P17573 lipase 1 GMDG_03210 2 1 XP_006696953.1 hypothetical protein GMDG_00548 3 1 P32949 lipase 5 GMDG_04447 3 2 A1CWF3 alkaline protease 1 GMDG_07342 2 1 XP_002568012.1 hypothetical protein oxygen-dependent choline GMDG_06725 2 2 A6X2G7 dehydrogenase GMDG_07037 2 1 Q5ACV9 cell surface superoxide dismutase GMDG_05916 1 1 P14804 glucoamylase GMDG_04044 1 1 P0C7S9 1,3-beta-glucanosyltransferase GMDG_08409 1 1 EWG53341.1 murein transglycosylase GMDG_03077 1 1 P9WHH0 thioredoxin reductase GMDG_04107 1 1 XP_001273330.1 endo-chitosanase GMDG_07215 1 1 P37552 2-iminopropanoate deaminase GMDG_06096 1 1 A6RUD7 carboxypeptidase y GMDG_06446 1 1 Q9Y8D9 superoxide dismutase GMDG_07870 1 2 Q96WM9 Laccase 2 GMDG_05222 1 1 Q4WLB9 Uncharacterized protein

Table S1. Protein Identifications by LC-MS/MS in the P. destructans secretome using 3 cultures replicates. Accession numbers for the best BlastP hits are included and the predicted function of each protein is indicated. Table S1 is a summary of the information presented in Table S2.

Table S2. Protein Identifications by LC-MS/MS in the P. destructans secretome across

N=3 replicates, with GO annotations. Provided are the following MS/MS-related metrics

204 from Protein Prospector software (the protein score, the number of uniquely mapped peptides, the number of observed spectra for a given protein identified—the spectral count, the best individual peptide score, percent sequence coverage, the best discriminant score, and the best expectation value for an individual peptide in each secretome analysis). Also provided are BLAST2GO protein matches, with the number of hits identified and the expectation value for the confidence of a shared annotation with the best hit, as well as mean similarity across these hits, and mention of the associated GO annotations, as well as Enzyme Code (EC) annotations for any proteins with associated enzymatic activity.

Table S3. Peptide LC-MS/MS Data supporting the identification of proteins in the P. destructans secretome. Provided are MS/MS details in a peptide report format from

Protein Prospector, indicating the following: the number of unique peptides matched to a protein identification, the percent sequence coverage, the best discriminant score and best expectation value for the best matched peptide to that protein, mass to charge ratio (m/z) and charge (z), peptide sequence, chromatographic retention time in minutes, and the individual peptide score and expectation value for that peptide as it is matched to a specific protein in the P. destructans genome. This species was formerly named G. destructans and GdDB accession numbers are provided.

Table S4. Peptide LC-MS/MS Data supporting P. destructans protein identification in peptidase-enriched chromatographic fractions. The peptide report format provided here contains the same information from Protein Prospector as summarized for Table S2.

205

Table S5. Composition of the 15 IQ peptide substrates used in Figure 3A.

Table S6. Pearson chi-squared test to compare the P1 to P4 specificity profile of recombinant Destructin-1 using MSP-MS and PS-PSL

206

Supplementary References

1. O'Donoghue AJ, et al. (2012) Global identification of peptidase specificity by

multiplex substrate profiling. Nature methods 9(11):1095-1100.

2. Olsen JV, et al. (2005) Parts per million mass accuracy on an Orbitrap mass

spectrometer via lock mass injection into a C-trap. Mol Cell Proteomics 4(12):2010-

2021.

3. Chalkley RJ, Baker PR, Medzihradszky KF, Lynn AJ, & Burlingame AL (2008) In-

depth analysis of tandem mass spectrometry data from disparate instrument types.

Mol Cell Proteomics 7(12):2386-2398.

4. Choi H, Fermin D, & Nesvizhskii AI (2008) Significance analysis of spectral count

data in label-free shotgun proteomics. Mol Cell Proteomics 7(12):2373-2385.

5. Conesa A, et al. (2005) Blast2GO: a universal tool for annotation, visualization and

analysis in functional genomics research. Bioinformatics 21(18):3674-3676.

6. Quan J & Tian J (2009) Circular polymerase extension cloning of complex gene

libraries and pathways. PLoS One 4(7):e6441.

7. Harris JL, et al. (2000) Rapid and general profiling of protease specificity by using

combinatorial fluorogenic substrate libraries. Proc Natl Acad Sci U S A

97(14):7754-7759.

8. Soding J, Biegert A, & Lupas AN (2005) The HHpred interactive server for protein

homology detection and structure prediction. Nucleic Acids Res 33(Web Server

issue):W244-248.

207

9. Sali A & Blundell TL (1993) Comparative protein modelling by satisfaction of spatial

restraints. J Mol Biol 234(3):779-815.

10. Dunbrack RL, Jr. (2002) Rotamer libraries in the 21st century. Current opinion in

structural biology 12(4):431-440.

11. Yang Z, et al. (2012) UCSF Chimera, MODELLER, and IMP: an integrated

modeling system. Journal of structural biology 179(3):269-278.

208

APPENDIX B: Supplementary proteomic Tables for Chapter

209

Replicate 1 Replicate 2 Replicate 3 Acc # Num Unique Peptide Count % Cov Best Disc Score Best Expect Val Num Unique Peptide Count % Cov Best Disc Score Best Expect Val Num Unique Peptide Count % Cov Best Disc Score Best Expect Val Protein MW Protein Name

GMDG_08104T0 33 217 75.8 18.1 6.50E-33 17 40 45 18.07 7.40E-33 17 29 35.3 16.63 3.50E-30 46124.2 GMDG_08104 GMDG_07937T0 31 82 56.3 11.79 3.20E-21 27 41 46.2 10.83 1.90E-19 30 93 51.3 11.22 3.60E-20 47553.9 GMDG_07937 GMDG_02198T0 19 27 47.1 8.99 4.90E-16 3 3 8.5 4.32 6.30E-10 22 34 42.1 11.03 8.00E-20 52242.6 GMDG_02198 GMDG_03130T0 18 32 43.4 12.17 6.30E-22 8 11 24.2 9.85 1.20E-17 14 25 30.5 10.06 5.20E-18 56701.1 GMDG_03130 GMDG_01515T0 20 23 34.1 9.31 1.30E-16 8 9 10.9 6.99 2.40E-12 25 29 33.7 10.1 4.30E-18 86802.1 GMDG_01515 GMDG_05333T0 9 9 9.1 8.41 5.70E-15 6 6 7.2 5.14 6.50E-09 21 24 24.5 9.89 1.00E-17 89645.4 GMDG_05333 GMDG_03210T0 14 35 45.8 11.53 9.60E-21 11 14 34 9.55 4.50E-17 11 55 28.1 10.23 2.50E-18 33280.5 GMDG_03210 GMDG_07140T0 21 45 27.6 8.39 6.10E-15 17 25 20.6 8.38 6.60E-15 18 32 24.4 8.51 3.70E-15 79347.1 GMDG_07140 GMDG_03116T0 17 19 17.5 10.4 1.20E-18 16 18 17.2 13.62 1.30E-24 14 14 15.7 13.22 7.10E-24 123890.9 GMDG_03116 GMDG_02357T0 16 20 25 13.98 2.80E-25 9 9 13.3 6.76 6.40E-12 12 12 16.1 6.62 1.20E-11 81522.8 GMDG_02357 GMDG_03442T0 13 16 40.9 9.88 1.10E-17 15 24 34.9 7.43 3.80E-13 47548.7 GMDG_03442 GMDG_04989T0 6 7 10.4 6.71 8.10E-12 1 1 1.8 4.35 2.80E-08 14 16 21.5 8.09 2.30E-14 71663.6 GMDG_04989 GMDG_08145T0 10 10 19 6.35 3.80E-11 5 5 8.6 4.66 6.00E-09 17 22 19.2 6.63 1.20E-11 59077.9 GMDG_08145 GMDG_03914T0 8 8 22.9 9.55 4.40E-17 11 12 38 8.23 1.20E-14 13 20 36.1 8.58 2.80E-15 39063.9 GMDG_03914 GMDG_08552T0 13 16 34.2 8.14 1.80E-14 9 9 21.5 6 1.70E-10 12 15 25.4 6.48 2.20E-11 55559 GMDG_08552 GMDG_07699T0 1 1 3.9 4.08 7.30E-09 2 2 7.2 4.46 2.00E-08 10 14 32.9 12.06 1.00E-21 32716.1 GMDG_07699 GMDG_03838T0 9 11 47.3 8.87 8.00E-16 8 8 41.8 8.13 1.90E-14 8 11 41.8 9.48 6.10E-17 19565.1 GMDG_03838 GMDG_02454T0 8 12 24.7 9.01 4.40E-16 5 6 18.1 7.55 2.20E-13 10 28 26.9 7.99 3.50E-14 33465.5 GMDG_02454 GMDG_04457T0 4 4 12.8 8.4 6.10E-15 8 8 22.2 14.04 2.20E-25 6 8 17.2 10.9 1.40E-19 56275.2 GMDG_04457 GMDG_01891T0 8 8 18.6 9.06 3.60E-16 7 7 16.7 5.11 7.60E-09 10 10 25.7 8.47 4.50E-15 51458.7 GMDG_01891 GMDG_05000T0 7 11 32.8 10.58 5.40E-19 3 5 11.1 10.49 8.00E-19 8 13 23.7 9.89 1.00E-17 41521.7 GMDG_05000 GMDG_04446T0 5 5 7.1 5.85 3.20E-10 8 9 16 8.51 3.70E-15 83220.4 GMDG_04446 GMDG_00092T0 6 9 23.8 7.38 4.70E-13 6 8 22.4 8.37 6.70E-15 9 13 31.1 7.83 6.80E-14 36087 GMDG_00092 GMDG_02498T0 2 2 2.1 3.11 1.80E-06 10 10 11.8 6.56 1.50E-11 88592.3 GMDG_02498 GMDG_07779T0 8 12 15.5 7.74 1.00E-13 7 9 14.2 6.26 5.60E-11 9 14 18 8.36 7.00E-15 46656.4 GMDG_07779 GMDG_00637T0 5 6 25 12.33 3.20E-22 2 2 16.5 6.22 6.40E-11 2 2 16.5 12.4 2.30E-22 21925.2 GMDG_00637 GMDG_00633T0 1 1 2.1 2.54 1.20E-06 2 2 5.4 4.48 1.80E-09 6 7 20 14.58 2.10E-26 42385.9 GMDG_00633 GMDG_07208T0 4 5 10.1 8.7 1.70E-15 2 2 5.2 7.62 1.70E-13 7 9 15.7 8.8 1.10E-15 62035.5 GMDG_07208 GMDG_03077T0 3 3 7.8 6.97 2.70E-12 8 8 20.7 6.1 1.10E-10 9 9 24.6 6.88 4.00E-12 42113.9 GMDG_03077 GMDG_05222T0 4 8 16.8 14.41 4.50E-26 3 4 16.8 14.17 1.30E-25 3 3 16.8 11.72 4.20E-21 26017.4 GMDG_05222 GMDG_03526T0 7 13 10.4 7.28 7.30E-13 7 12 10.4 6.98 2.50E-12 7 17 10.4 7.21 9.50E-13 68168.5 GMDG_03526 GMDG_03068T0 7 8 79.5 8.69 1.70E-15 3 3 40.9 4.24 3.30E-09 2 2 30.7 7.97 3.70E-14 8879.7 GMDG_03068 GMDG_04818T0 6 6 20.7 9.08 3.40E-16 6 6 20.9 9.59 3.70E-17 43613.4 GMDG_04818 GMDG_07491T0 6 7 12.4 6.46 2.30E-11 5 5 9.8 6.78 3.70E-12 8 9 16.5 5.66 7.10E-10 59236 GMDG_07491 GMDG_05452T0 4 5 6.3 5.24 4.30E-09 3 3 6.3 5.44 3.60E-10 7 7 13.5 8.44 5.00E-15 59888.6 PdCP1 GMDG_07480T0 2 2 4.3 6.44 2.20E-11 6 6 14 8.82 9.80E-16 54412.5 GMDG_07480 GMDG_03975T0 5 7 44.4 6.75 6.90E-12 2 2 18.2 4.49 1.00E-07 6 13 52.5 7.6 1.90E-13 10991.7 GMDG_03975 GMDG_08350T0 1 1 2.8 5.9 2.60E-10 6 6 14.2 6.32 4.30E-11 1 1 2.8 6.03 1.50E-10 54444.6 GMDG_08350 GMDG_00651T0 5 5 10 7.31 6.40E-13 6 6 11.7 6.17 8.10E-11 6 6 11.7 5.79 4.10E-10 63126.5 GMDG_00651 GMDG_06611T0 4 9 26.2 10 6.50E-18 3 3 12.2 6.95 2.90E-12 3 4 12.2 7.13 1.30E-12 22702.6 GMDG_06611 GMDG_08378T0 5 20 14.9 6.49 2.10E-11 5 10 14.9 6.53 1.80E-11 5 42 14.9 7.06 1.80E-12 21902.1 GMDG_08378 GMDG_05916T0 1 1 2.8 5.69 3.30E-11 5 5 8 7.06 1.80E-12 72541.3 GMDG_05916 GMDG_00696T0 4 8 16.2 7.78 8.30E-14 3 9 16.2 7.22 9.20E-13 4 11 16.2 7.54 2.30E-13 19164.9 GMDG_00696 GMDG_06522T0 5 5 28.2 6.85 4.40E-12 5 5 26.8 8.09 2.20E-14 22574 GMDG_06522 GMDG_03019T0 3 4 16.6 9.77 1.70E-17 3 3 16.6 6.21 6.80E-11 4 5 16.6 9.31 1.20E-16 18325.4 GMDG_03019 GMDG_02704T0 4 5 11.8 9.1 3.10E-16 4 4 11.8 6.5 1.20E-11 4 4 6.8 7.03 2.10E-12 41010 GMDG_02704 GMDG_03955T0 2 3 46.9 7.13 1.30E-12 3 6 72.8 10.22 2.60E-18 1 1 19.8 6.35 3.70E-11 8735.8 GMDG_03955 GMDG_00646T0 2 2 4.1 4.9 2.70E-09 5 5 8.2 6.4 3.00E-11 63827.7 GMDG_00646 GMDG_05533T0 2 3 6.3 5.81 3.80E-10 5 6 16.3 6.28 5.10E-11 5 6 16.9 6.54 1.70E-11 34108.6 GMDG_05533 GMDG_05358T0 4 5 20.3 4.53 7.40E-08 5 5 25.2 5.7 6.10E-10 24036.3 GMDG_05358 GMDG_07093T0 3 4 11.6 7.5 2.70E-13 1 1 3 7.45 3.50E-13 1 1 3 7.15 3.60E-13 37074.6 GMDG_07093 GMDG_00903T0 1 1 4.2 9.27 1.50E-16 4 4 11.2 9.54 4.70E-17 2 2 7.6 8.3 9.20E-15 34479.2 GMDG_00903 GMDG_01066T0 2 3 6.4 5.6 9.40E-10 3 4 10.8 8.67 1.90E-15 41359.4 GMDG_01066 GMDG_07602T0 2 2 4.9 5.96 2.60E-11 3 3 11.1 9.02 4.30E-16 49592.6 GMDG_07602 GMDG_01434T0 2 2 25.9 9.83 1.40E-17 3 3 42.6 9.75 1.90E-17 2 2 25.9 7.05 1.90E-12 11837.6 GMDG_01434 GMDG_07064T0 4 5 17.5 7.15 1.20E-12 2 2 9.3 3.13 8.20E-06 18167.7 GMDG_07064 GMDG_08299T0 3 3 8.3 8.79 1.10E-15 3 3 6.5 5.52 1.30E-09 2 2 2.9 5.39 2.20E-09 46117.8 GMDG_08299 GMDG_00372T0 3 4 18.6 7.15 1.20E-12 1 1 7.8 6.94 3.00E-12 26825.7 GMDG_00372 GMDG_05440T0 1 1 3.4 9.07 3.50E-16 2 3 7.6 9.24 1.70E-16 51025.5 GMDG_05440 GMDG_07037T0 3 3 14 5.67 6.80E-10 4 4 16.9 4.98 1.30E-08 23680.8 GMDG_07037 GMDG_08491T0 3 3 7.5 5.35 2.60E-09 4 4 11.2 5.57 1.10E-09 40739.7 GMDG_08491 GMDG_04925T0 3 3 13 6.88 2.00E-12 2 2 11 5.09 2.00E-09 3 3 17.5 4.75 3.40E-08 16625.8 GMDG_04925 GMDG_06417T0 3 3 7.5 5.38 2.40E-09 4 4 9.2 6 1.70E-10 40489.2 GMDG_06417 GMDG_05256T0 3 3 49.3 7.93 4.40E-14 1 2 13.7 5 6.50E-10 3 4 49.3 6.1 1.10E-10 7726.5 GMDG_05256 GMDG_03638T0 3 4 6.6 7.55 2.20E-13 1 1 3.4 4.42 4.50E-08 2 3 6.6 6.69 9.00E-12 55764.3 GMDG_03638 GMDG_03414T0 3 4 63.9 5.81 3.70E-10 1 1 18.1 6.18 7.70E-11 3 3 58.3 6.23 6.40E-11 7428.1 GMDG_03414 GMDG_01295T0 1 1 6.2 5.86 2.90E-11 2 2 12.9 9 4.70E-16 26174.7 GMDG_01295 GMDG_08409T0 4 4 7.1 3.92 9.10E-08 2 2 2.9 3.52 1.90E-07 61250.6 GMDG_08409 GMDG_06268T0 1 1 7.1 6.19 7.40E-11 3 3 17.8 7.09 1.60E-12 3 3 17.8 6.25 5.70E-11 21504.5 GMDG_06268 GMDG_01608T0 3 3 33 4.81 2.70E-08 1 1 9.6 4.03 1.60E-07 12552.1 GMDG_01608 GMDG_01630T0 1 1 2.3 5.54 1.20E-09 2 2 3.8 8.03 2.90E-14 57171.1 GMDG_01630 GMDG_00384T0 3 3 8.6 5.07 8.70E-09 4 4 12 3.63 1.60E-06 3 3 8.9 6.55 1.60E-11 44387.3 GMDG_00384 GMDG_07510T0 2 4 10.9 8.45 4.80E-15 1 2 7 7.38 4.60E-13 2 3 6.3 3.91 4.60E-10 28439.1 GMDG_07510 GMDG_02429T0 2 3 15.3 6.61 1.30E-11 2 5 15.3 6.82 5.00E-12 2 5 15.3 6.85 4.40E-12 20995.5 GMDG_02429 GMDG_00224T0 1 1 1.7 1.87 6.00E-08 3 3 4.2 5.55 3.30E-11 1 1 1.7 5.32 1.20E-10 64656.1 GMDG_00224 GMDG_04810T0 2 4 11 8.32 8.60E-15 2 2 11 3.63 1.30E-06 1 25279.4 GMDG_04810 GMDG_04626T0 2 2 8.1 7.32 5.90E-13 2 2 7.5 7.55 2.30E-13 1 1 2.7 3.23 3.10E-07 31426.8 GMDG_04626 GMDG_05832T0 2 2 8.1 4.57 7.50E-08 3 3 8.1 5.15 1.30E-09 2 2 8.1 4.29 1.80E-08 25643.4 GMDG_05832 GMDG_05500T0 2 2 6.6 5.23 4.40E-09 2 2 7.9 4.68 1.70E-08 35297.5 GMDG_05500 GMDG_06714T0 2 2 14 5.68 6.60E-10 2 2 14 5.2 5.00E-09 16944.1 GMDG_06714 GMDG_08270T0 2 2 3.6 3.89 7.20E-08 2 2 3.6 4.55 8.20E-09 2 2 3.6 4.08 6.60E-08 60003.2 GMDG_08270 GMDG_04735T0 2 2 2.7 4.2 3.50E-07 3 3 5.4 2.74 1.40E-07 1 44869.5 GMDG_04735 GMDG_02668T0 1 1 8.5 5.97 1.90E-10 1 1 8.5 6.32 4.20E-11 19087.6 GMDG_02668 GMDG_01086T0 1 1 11.2 6.12 1.00E-10 1 1 11.2 3.98 2.10E-09 13026.6 GMDG_01086 GMDG_01408T0 1 1 1.2 4.49 1.10E-07 2 2 2.6 3.63 2.70E-08 93325 GMDG_01408 GMDG_00087T0 1 2 11 5.43 1.90E-09 1 2 11 5.88 2.70E-10 1 2 11 6.06 1.30E-10 10501.2 GMDG_00087 GMDG_06420T0 1 1 6 5.78 4.20E-10 1 1 2.9 6.02 4.60E-11 52913.7 GMDG_06420 GMDG_00797T0 1 1 3.3 5.6 8.10E-10 2 2 4.9 3.37 2.10E-07 38184.1 GMDG_00797 GMDG_07199T0 1 1 1.5 4.03 8.70E-08 1 1 1.5 5.05 5.40E-09 1 1 1.5 5.87 2.90E-10 87529.3 GMDG_07199 GMDG_06638T0 1 2 2.4 3.28 1.40E-08 2 2 3.2 2.81 8.00E-06 1 57455.4 GMDG_06638 GMDG_08205T0 1 1 2 2.7 2.70E-07 1 1 2 4.44 5.70E-08 61749.3 GMDG_08205 GMDG_06914T0 1 1 7.3 2.46 2.90E-06 1 1 5.1 2.51 6.70E-06 1 18595.5 GMDG_06914 Supplementary Table 1. Proteins identified in P. destructans secretome by LC-MS/MS

210

VE01_02776-T1 49 58 31.2 12.21 5.20E-22 54 69 30 11.82 2.80E-21 46 52 24.2 12.51 1.50E-22 215848.5 VE01_02776-T1 VE01_05992-T1 30 42 42.1 10.81 2.10E-19 31 40 43.3 10.37 1.30E-18 30 42 35.9 10.32 1.70E-18 86864.2 VE01_05992-T1 VE01_05095-T1 20 22 28.3 8.79 1.10E-15 31 44 35.7 8.7 1.70E-15 15 16 18.5 7.18 1.10E-12 102361.7 VE01_05095-T1 VE01_05425-T1 17 33 48.3 13.57 1.60E-24 14 28 42.4 13.6 1.40E-24 11 24 36.2 13.22 7.20E-24 43677.3 VE01_05425-T1 VE01_05195-T1 14 24 47.6 10.83 1.90E-19 20 26 46.6 11.03 8.10E-20 17 21 42.9 11.49 1.10E-20 49665.5 VE01_05195-T1 VE01_04855-T1 22 23 31.5 10 6.60E-18 12 12 14.9 8.08 2.40E-14 8 8 9.4 6.89 3.80E-12 98605.5 VE01_04855-T1 VE01_02280-T1 13 14 18 8.59 2.60E-15 21 25 33.2 11.11 5.60E-20 13 14 17.2 9.35 1.10E-16 89758.6 VE01_02280-T1 VE01_06555-T1 20 24 39.9 12.4 2.40E-22 11 11 16.8 6.61 1.20E-11 4 4 5.4 2.64 5.70E-06 65042.1 VE01_06555-T1 VE01_05351-T1 15 23 50.4 11.36 2.00E-20 12 15 36.1 10.25 2.30E-18 10 12 27.8 10.48 8.30E-19 42975.3 VE01_05351-T1 VE01_03133-T1 18 24 69.3 11.37 1.90E-20 11 12 34.5 9.27 1.50E-16 10 13 24.9 8.96 5.40E-16 37684 VE01_03133-T1 VE01_07389-T1 12 16 59.4 9.87 1.20E-17 17 21 73.5 9.01 4.40E-16 6 6 22 7.18 1.10E-12 32467.1 VE01_07389-T1 VE01_08417-T1 12 31 37.8 10.98 1.00E-19 14 47 39 11.3 2.60E-20 12 37 37.8 13.14 1.00E-23 34138.9 VE01_08417-T1 VE01_06376-T1 15 16 17.1 10.32 1.70E-18 16 17 20.9 10.52 7.20E-19 11 13 13.1 12.7 6.50E-23 117853.9 VE01_06376-T1 VE01_07963-T1 16 17 46.5 10.81 2.10E-19 8 9 23.5 10.61 4.90E-19 5 6 13.2 11.05 7.30E-20 47203.5 VE01_07963-T1 VE01_00201-T1 14 20 45.7 9.8 1.60E-17 10 13 37.2 10.29 1.90E-18 4 4 14.9 8.27 1.00E-14 46936.9 VE01_00201-T1 VE01_06806-T1 15 16 32.7 13.08 1.30E-23 10 11 20.4 9.66 2.80E-17 7 7 10.3 5.69 6.20E-10 72223.5 VE01_06806-T1 VE01_02953-T1 3 3 7.8 6.36 3.70E-11 13 16 34.5 14.92 5.20E-27 1 47693.4 VE01_02953-T1 VE01_06488-T1 12 14 36.9 13.57 1.60E-24 5 6 13 12.28 3.80E-22 3 3 5.2 4.35 1.90E-07 57537.9 VE01_06488-T1 VE01_06819-T1 15 18 28.9 9.16 2.30E-16 14 16 25.6 9.76 1.80E-17 8 9 11.1 5.95 2.10E-10 72489.1 VE01_06819-T1 VE01_05479-T1 10 15 27 11.93 1.70E-21 11 15 27.9 11.77 3.40E-21 7 9 19.5 11.16 4.60E-20 57360.9 VE01_05479-T1 VE01_06761-T1 8 13 56.4 9.83 1.30E-17 10 35 57.1 13.93 3.40E-25 10 92 57.1 10.79 2.20E-19 13937 VE01_06761-T1 VE01_05856-T1 10 11 23.5 12.38 2.60E-22 8 10 17.7 9.29 1.30E-16 14 20 27.5 8.94 6.00E-16 69546.7 VE01_05856-T1 VE01_05199-T1 11 15 60.6 14.73 1.10E-26 9 10 40.2 13.92 3.60E-25 9 9 40.2 12.37 2.60E-22 25424.5 VE01_05199-T1 VE01_00443-T1 12 19 38.5 8.95 5.60E-16 10 14 34.5 9.18 2.10E-16 11 14 38.3 8.61 2.50E-15 42648.1 VE01_00443-T1 VE01_06509-T1 2 2 6.9 5.06 4.60E-09 8 13 30.6 6.34 3.90E-11 14 25 39.5 12.58 1.10E-22 26799.6 VE01_06509-T1 VE01_06516-T1 9 12 24.4 8.9 7.00E-16 13 15 29.7 8.97 5.20E-16 10 10 20.6 9.04 3.80E-16 67391 VE01_06516-T1 VE01_00429-T1 10 15 30.5 10.36 1.40E-18 9 10 30.1 14.84 7.00E-27 6 7 20.3 8.48 4.30E-15 46672.8 VE01_00429-T1 VE01_01013-T1 11 12 28.1 12.64 8.60E-23 4 4 10.1 7.08 1.70E-12 5 6 10.8 7.57 2.10E-13 60038.4 VE01_01013-T1 VE01_07557-T1 10 18 26.4 11.58 7.70E-21 9 17 19.4 9.37 9.70E-17 7 15 15.5 7.99 3.50E-14 45650 VE01_07557-T1 VE01_03196-T1 10 13 41.8 10.83 1.90E-19 10 12 43.8 10.13 3.70E-18 5 5 12.8 5.42 2.00E-09 40341.1 VE01_03196-T1 VE01_07152-T1 12 14 25.3 7.87 5.80E-14 1 1 1.7 6.86 7.70E-13 1 1 1.7 3.3 1.20E-07 76674.9 VE01_07152-T1 VE01_06546-T1 11 15 21 8.49 4.20E-15 8 14 17.3 8.46 4.70E-15 6 7 11.5 6.4 3.00E-11 70623.5 VE01_06546-T1 VE01_06364-T1 9 11 26.4 14.92 5.00E-27 9 10 21.8 14.47 3.40E-26 6 7 15.6 9.52 5.00E-17 56686.9 VE01_06364-T1 VE01_02747-T1 12 18 39.6 9.19 2.10E-16 13 18 36.8 8.31 8.80E-15 10 13 25.6 6.55 1.60E-11 41530.7 VE01_02747-T1 VE01_00132-T1 11 11 28.3 11.38 1.80E-20 7 10 19 11.02 8.30E-20 4 5 6.9 5.66 7.20E-10 63008.3 VE01_00132-T1 VE01_02196-T1 10 11 45.5 11.63 6.30E-21 11 13 43.6 11.99 1.30E-21 9 10 29.4 11.4 1.70E-20 32725.1 VE01_02196-T1 VE01_06978-T1 3 3 19.4 8.31 8.70E-15 11 12 74.8 9.2 2.00E-16 3 3 11.2 3.08 3.00E-07 26491.4 VE01_06978-T1 VE01_08061-T1 11 17 33.4 9.28 1.40E-16 11 17 32.1 10.28 2.00E-18 12 18 36 9.12 2.80E-16 40292.6 VE01_08061-T1 VE01_02707-T1 9 13 76.8 9.66 2.80E-17 3 3 18.8 7.06 1.90E-12 21906.2 VE01_02707-T1 VE01_07986-T1 3 3 7.9 9.35 1.00E-16 7 7 20.7 13.17 8.70E-24 10 10 25.9 8.63 2.30E-15 53249.7 VE01_07986-T1 VE01_04324-T1 11 11 25.3 8.62 2.30E-15 4 4 6.5 4.88 2.00E-08 2 2 2.7 3.85 1.00E-08 59882.3 VE01_04324-T1 VE01_02284-T1 9 9 26.3 9.94 8.60E-18 4 4 9.1 6.06 1.30E-10 3 3 6.1 7.05 1.90E-12 70530.2 VE01_02284-T1 VE01_05202-T1 9 10 27.3 10.79 2.20E-19 3 3 7.4 7.83 7.00E-14 2 2 4.1 6.29 4.80E-11 63723.7 VE01_05202-T1 VE01_07751-T1 5 5 19.1 5.75 4.90E-10 8 8 28.1 7.14 1.30E-12 11 12 51.1 7.62 1.70E-13 25259.7 VE01_07751-T1 VE01_03388-T1 6 22 52.9 9.47 6.40E-17 6 26 52.9 9.79 1.60E-17 8 62 64.7 10.93 1.20E-19 11255.7 VE01_03388-T1 VE01_02778-T1 8 10 18.2 7.5 2.80E-13 10 13 22.4 10.66 4.00E-19 9 14 22.4 10.35 1.50E-18 48054.7 VE01_02778-T1 VE01_07492-T1 8 12 53.6 13.72 8.30E-25 3 3 17.5 5.74 5.10E-10 3 3 9.5 8.13 1.90E-14 26029.6 VE01_07492-T1 VE01_09050-T1 10 14 37.2 10.07 4.80E-18 8 9 21 7.64 1.60E-13 10 11 23.2 9.36 1.00E-16 42099.8 VE01_09050-T1 VE01_06213-T1 5 13 19.8 11.59 7.40E-21 6 12 26.4 10.73 2.90E-19 3 7 12.9 10.54 6.50E-19 51295.6 VE01_06213-T1 VE01_05385-T1 11 16 30.4 9.07 3.50E-16 7 9 20.3 6.36 3.60E-11 9 10 23.4 6.58 1.40E-11 42395.3 VE01_05385-T1 VE01_02450-T1 8 10 58.1 8.87 8.20E-16 6 22 37.5 7.76 9.30E-14 6 17 37.5 7.83 6.90E-14 14626.8 VE01_02450-T1 VE01_04762-T1 6 7 25.8 9.4 8.40E-17 6 6 32.9 12.55 1.20E-22 5 5 27.4 9.21 1.90E-16 32696.2 VE01_04762-T1 VE01_04878-T1 6 6 31.4 11.1 5.90E-20 9 11 45 9.64 3.10E-17 4 4 16.7 8.12 2.00E-14 27970 VE01_04878-T1 VE01_01811-T1 8 8 12.4 8.84 9.20E-16 4 4 5.2 6.45 2.40E-11 3 3 3.8 4.85 8.50E-09 94181.6 VE01_01811-T1 VE01_00639-T1 8 9 15.3 9.75 1.90E-17 10 12 17.8 7.97 3.80E-14 10 11 17.8 8.68 1.80E-15 46886.6 VE01_00639-T1 VE01_03592-T1 4 4 26.9 11.36 2.00E-20 7 12 34 11.43 1.40E-20 5 11 27.4 10.41 1.10E-18 23508.3 VE01_03592-T1 VE01_04998-T1 6 12 24.3 11.55 8.70E-21 5 7 23.9 11.28 2.80E-20 6 10 26.8 11.88 2.20E-21 27956.4 VE01_04998-T1 VE01_03617-T1 6 9 36.3 10.55 6.30E-19 6 11 28.2 8.86 8.50E-16 6 11 28.2 8.52 3.60E-15 26265.6 VE01_03617-T1 VE01_08610-T1 7 7 18.2 8.12 1.90E-14 6 6 13.1 7.68 1.30E-13 4 4 6.7 6.77 6.30E-12 68006.2 VE01_08610-T1 VE01_02047-T1 6 9 20.4 8.13 1.90E-14 3 3 8.8 7.05 1.90E-12 2 2 5.9 6.31 4.50E-11 47350.2 VE01_02047-T1 VE01_08982-T1 3 3 6.9 6.31 4.40E-11 7 7 14.9 6.84 4.70E-12 9 9 17.7 5.64 7.60E-10 64986.1 VE01_08982-T1 VE01_06762-T1 5 7 23.5 9.02 4.30E-16 2 2 13.2 5.68 6.50E-10 2 3 7.5 9.01 4.50E-16 43151.7 VE01_06762-T1 VE01_02044-T1 3 3 7.3 5.14 5.00E-10 1 1 2.1 2.81 4.40E-06 7 7 22.2 8.5 4.00E-15 42714.7 VE01_02044-T1 VE01_00283-T1 4 6 46.7 17.73 3.10E-32 4 4 46.7 15.01 3.40E-27 4 8 46.7 16.01 4.80E-29 12792.7 VE01_00283-T1 VE01_00473-T1 4 7 26 11.94 1.70E-21 4 6 26 10.29 1.90E-18 4 5 26 9.61 3.40E-17 25545 VE01_00473-T1 VE01_05143-T1 6 6 19.8 7.24 8.40E-13 4 4 10.1 7.28 7.00E-13 5 5 12.8 7.44 3.50E-13 43291.7 VE01_05143-T1 VE01_06727-T1 3 3 8.6 5.38 2.30E-09 2 2 5.9 7.6 1.80E-13 7 7 16.5 8.81 1.00E-15 51768 VE01_06727-T1 VE01_08881-T1 4 4 23.8 5.93 2.30E-10 6 6 43.1 8.1 2.20E-14 5 5 31.5 5.82 3.50E-10 19496.1 VE01_08881-T1 VE01_08352-T1 5 6 34 9.36 1.00E-16 4 7 26.7 9.93 8.70E-18 5 8 34 9.19 2.10E-16 19882.4 VE01_08352-T1 VE01_00133-T1 3 3 9.2 8.39 6.20E-15 4 4 6.5 3.38 1.70E-07 9 9 15.8 6.17 2.00E-11 56209 VE01_00133-T1 VE01_05264-T1 5 5 46 11.45 1.30E-20 1 1 6.9 5.63 8.00E-10 2 2 12.2 5.22 4.50E-09 20639.7 VE01_05264-T1 VE01_08822-T1 4 6 12.5 7.89 5.40E-14 6 8 13 6.91 3.40E-12 55922.8 VE01_08822-T1 VE01_07612-T1 3 7 58.5 16.64 3.30E-30 4 12 59.6 18.87 2.40E-34 3 15 58.5 13.64 1.20E-24 9934.2 VE01_07612-T1 VE01_05790-T1 1 1 9.2 6.28 5.10E-11 5 6 43.9 11.94 1.70E-21 1 1 7.5 3.19 2.10E-07 16539.6 VE01_05790-T1 VE01_05850-T1 4 5 17.7 10.05 5.30E-18 1 1 2 3.45 7.40E-06 52271.7 VE01_05850-T1 VE01_00252-T1 1 1 2.8 2.84 2.10E-07 5 7 16.5 9.35 1.00E-16 41932.5 VE01_00252-T1 VE01_04401-T1 5 5 40.7 9.28 1.40E-16 2 2 10.2 5.22 4.60E-09 29784.4 VE01_04401-T1 VE01_02403-T1 4 4 22.5 8.69 1.80E-15 2 2 8.8 6.51 1.90E-11 2 2 8.8 7.02 2.10E-12 31390.8 VE01_02403-T1 VE01_00710-T1 3 3 11.4 7.75 9.80E-14 6 6 15.7 8.02 3.00E-14 42734 VE01_00710-T1 VE01_04945-T1 4 4 18.3 12.22 5.10E-22 2 2 7.9 5.95 2.90E-11 2 2 7.9 6.3 4.10E-11 25808.9 VE01_04945-T1 VE01_05051-T1 4 6 76.9 10.07 4.90E-18 2 2 13.8 6.53 1.70E-11 2 13002.7 VE01_05051-T1 VE01_07469-T1 4 7 27.8 9.13 2.70E-16 3 6 12.1 10.21 2.70E-18 3 4 12.1 8.84 9.10E-16 34193.6 VE01_07469-T1 VE01_07713-T1 6 7 10.5 9.53 4.90E-17 4 4 5.1 5.62 8.50E-10 2 2 2.4 4.5 2.30E-08 91323.5 VE01_07713-T1 VE01_02716-T1 4 4 27.8 7.87 5.70E-14 4 7 27.8 8.99 4.80E-16 2 3 8.1 6.69 8.80E-12 23507.8 VE01_02716-T1 VE01_07926-T1 3 3 7 7.2 1.00E-12 5 5 9.4 6.63 1.10E-11 6 6 10.6 6.21 7.00E-11 73699.1 VE01_07926-T1 VE01_04568-T1 2 2 57.1 14.08 1.80E-25 2 2 57.1 15.45 5.30E-28 2 3 57.1 17.88 1.70E-32 7523.8 VE01_04568-T1 VE01_01732-T1 5 9 21.2 6.99 2.40E-12 7 10 24.7 6.6 1.30E-11 7 12 24.7 7.44 3.60E-13 27725.8 VE01_01732-T1 VE01_03108-T1 1 1 10.9 5.97 3.20E-11 4 6 33.7 9.16 2.30E-16 4 9 33.7 9.12 2.70E-16 9756.9 VE01_03108-T1 VE01_06755-T1 4 4 66.4 8.68 1.80E-15 3 3 41.6 10.48 8.60E-19 1 1 8 4.49 1.10E-07 14508.5 VE01_06755-T1 VE01_05938-T1 3 3 21.5 11.49 1.10E-20 3 4 21.5 11.51 1.10E-20 3 5 21.5 11.01 8.70E-20 20605 VE01_05938-T1 VE01_03033-T1 3 3 9 11.11 5.70E-20 6 6 10.6 6.31 4.50E-11 68433.8 VE01_03033-T1 VE01_03504-T1 1 1 0.5 3.77 1.20E-08 5 5 2.9 7.35 5.30E-13 197006.1 VE01_03504-T1

211

VE01_01925-T1 6 6 19 7.56 2.10E-13 2 2 5.4 2.21 1.00E-06 34983.8 VE01_01925-T1 VE01_05800-T1 4 5 15.4 9.94 8.50E-18 2 2 4.2 2.17 8.20E-06 4 6 15.4 9.46 6.70E-17 36620.9 VE01_05800-T1 VE01_05126-T1 1 1 3.6 8.68 1.80E-15 3 3 9.3 12.98 2.00E-23 3 6 9.3 12.54 1.30E-22 40586.5 VE01_05126-T1 VE01_02502-T1 2 3 46.2 19.8 4.60E-36 1 1 25.5 13.39 3.40E-24 15150.6 VE01_02502-T1 VE01_06973-T1 1 1 5.3 4.92 1.60E-08 5 5 27.4 7.38 4.60E-13 3 3 15.9 8.12 2.00E-14 22684.1 VE01_06973-T1 VE01_06458-T1 4 5 8.4 7.56 2.20E-13 3 5 6.1 8.19 1.50E-14 1 1 2.4 7.99 3.40E-14 67431.9 VE01_06458-T1 VE01_02780-T1 4 4 26.6 10.44 9.80E-19 5 5 32 9.02 4.40E-16 1 25251.8 VE01_02780-T1 VE01_02701-T1 1 1 8.8 9.15 2.50E-16 4 5 18.8 8.1 2.20E-14 34135.8 VE01_02701-T1 VE01_03427-T1 4 4 19.6 7.22 9.20E-13 3 4 13.1 6.04 1.40E-10 1 1 3.3 5.65 7.40E-10 43762.9 VE01_03427-T1 VE01_05459-T1 1 1 10.5 5.36 2.50E-09 2 2 21.1 5.54 1.20E-09 4 4 50 6.9 3.60E-12 12468.1 VE01_05459-T1 VE01_01861-T1 3 3 8 6.79 5.70E-12 1 1 2.6 7.59 1.90E-13 3 3 8.5 7.72 1.10E-13 56039.5 VE01_01861-T1 VE01_04982-T1 4 5 27.6 6.66 1.00E-11 3 9 26.7 7.1 1.50E-12 3 10 26.7 7.75 9.70E-14 11952.8 VE01_04982-T1 VE01_02715-T1 4 5 15 6.94 3.10E-12 4 6 15 5.14 6.50E-09 4 8 15 5.58 1.00E-09 22004.9 VE01_02715-T1 VE01_08809-T1 4 4 9.7 7.47 3.20E-13 3 3 4.9 7.2 1.00E-12 4 4 5.4 4.12 1.90E-07 71664.8 VE01_08809-T1 VE01_01092-T1 4 4 15.1 6.55 1.60E-11 1 1 3.4 3.04 4.20E-07 33177.3 VE01_01092-T1 VE01_09038-T1 3 3 16.3 7.26 7.80E-13 4 4 17.9 6.3 4.70E-11 2 2 9.2 3.97 3.00E-07 21507.5 VE01_09038-T1 VE01_05132-T1 3 3 15.9 7.33 5.80E-13 2 3 6.1 4.74 3.60E-08 3 4 10.5 8.24 1.20E-14 41149 VE01_05132-T1 VE01_03624-T1 1 1 1.6 2.49 5.90E-07 1 1 1.6 2.42 7.50E-07 4 4 12.5 6.81 5.30E-12 39099.4 VE01_03624-T1 VE01_07730-T1 3 5 6 8.8 1.10E-15 2 4 4.2 8.89 7.50E-16 3 4 5.5 7.09 1.20E-12 63019.2 VE01_07730-T1 VE01_01168-T1 3 3 12.6 8.06 2.60E-14 1 1 2 3.12 5.50E-07 47801.8 VE01_01168-T1 VE01_08297-T1 3 3 13.5 7.72 1.10E-13 4 4 15.5 7.57 2.00E-13 4 5 15.5 6.53 1.70E-11 41068 VE01_08297-T1 VE01_08127-T1 1 1 5.2 5.21 4.80E-09 3 3 22.9 8.54 3.30E-15 1 1 5.2 5.32 3.00E-09 23243 VE01_08127-T1 VE01_00761-T1 2 3 14.4 6.97 2.70E-12 4 5 18.7 7.01 2.20E-12 3 3 14.4 5.63 8.10E-10 20140.9 VE01_00761-T1 VE01_00516-T1 4 4 20.1 4.9 1.80E-08 2 2 9.1 3.87 2.50E-07 22701.6 VE01_00516-T1 VE01_04418-T1 2 2 4.8 5.25 4.00E-09 1 1 1.3 4.17 1.70E-08 4 4 7.2 5.24 7.70E-10 64879.5 VE01_04418-T1 VE01_04925-T1 4 4 9.7 4.87 2.10E-08 1 1 2.4 4.27 2.60E-07 56720.7 VE01_04925-T1 VE01_08741-T1 2 4 7 8.71 1.60E-15 2 5 7 9.01 4.40E-16 2 4 7 8.3 9.10E-15 26072.1 VE01_08741-T1 VE01_08580-T1 2 2 14.5 6.49 2.10E-11 2 2 19.5 10.32 1.70E-18 1 1 10.9 9.64 3.00E-17 21701.9 VE01_08580-T1 VE01_07214-T1 2 2 17.5 8.75 1.30E-15 1 1 6.9 9.59 3.80E-17 1 1 6.9 8.94 6.00E-16 17996 VE01_07214-T1 VE01_08424-T1 4 4 11.1 4.59 6.90E-08 4 4 15.5 6.07 1.20E-10 3 3 8.5 5.1 7.60E-09 35692.8 VE01_08424-T1 VE01_00003-T1 2 2 12 9.66 2.80E-17 1 1 5.8 5.36 1.40E-09 27525.9 VE01_00003-T1 VE01_00679-T1 4 4 8.8 5.73 2.20E-10 1 1 1.6 0.8 8.30E-06 61307.6 VE01_00679-T1 VE01_02884-T1 3 3 8.5 6.47 2.20E-11 4 4 8.5 5.13 6.90E-09 4 4 7.7 4.66 5.00E-08 53819 VE01_02884-T1 VE01_03503-T1 3 3 3.9 6.67 9.40E-12 2 2 2.3 4.46 2.90E-08 3 3 4 4.18 5.50E-08 91526.8 VE01_03503-T1 VE01_01505-T1 2 2 12.5 11.36 2.00E-20 2 2 3.6 3.07 2.40E-07 46263.6 VE01_01505-T1 VE01_07248-T1 1 2 13.1 7.54 2.30E-13 3 3 30.8 7.04 2.00E-12 1 1 9.3 3.93 1.20E-06 11903.8 VE01_07248-T1 VE01_08339-T1 3 3 7.8 5.82 3.50E-10 2 2 5 5.19 1.70E-09 43262 VE01_08339-T1 VE01_00423-T1 2 2 34.3 8.07 2.50E-14 2 3 34.3 5.3 3.30E-09 1 2 11.1 5.39 2.30E-09 10604.3 VE01_00423-T1 VE01_06361-T1 2 2 9.6 8.44 5.10E-15 1 1 4.5 7.31 6.10E-13 38769.3 VE01_06361-T1 VE01_00419-T1 3 3 8.1 4.74 3.60E-08 3 3 8.1 5.35 2.70E-09 1 1 2.9 3.86 8.00E-07 36314.3 VE01_00419-T1 VE01_01908-T1 3 4 12 6.96 2.70E-12 3 3 12 6.27 5.30E-11 3 3 12 6.2 7.10E-11 23862.5 VE01_01908-T1 VE01_07731-T1 1 1 4.4 6.33 4.10E-11 2 2 9.1 8.05 2.70E-14 1 1 3.8 5.01 1.10E-08 34685.6 VE01_07731-T1 VE01_00397-T1 2 2 16.5 8.18 1.50E-14 2 2 13.4 6.06 1.10E-11 25435.4 VE01_00397-T1 VE01_05243-T1 3 3 21.9 4.75 3.40E-08 2 2 13.5 2.36 9.40E-07 16799.1 VE01_05243-T1 VE01_09010-T1 2 4 14.9 6.6 1.30E-11 2 3 14.9 6.38 3.30E-11 2 2 14.9 4.72 1.10E-08 20950.6 VE01_09010-T1 VE01_08262-T1 4 4 14.5 4.15 7.20E-08 2 2 6.9 2.97 1.20E-06 29374.1 VE01_08262-T1 VE01_06422-T1 3 3 15.4 5.07 5.40E-09 3 3 15.4 5.48 6.00E-10 1 17914 VE01_06422-T1 VE01_08806-T1 1 1 17.8 11.59 7.30E-21 1 1 17.8 10.73 2.90E-19 1 1 17.8 8.6 2.60E-15 15303.6 VE01_08806-T1 VE01_01585-T1 2 2 10.9 7.59 1.90E-13 1 1 8 7.54 2.30E-13 40550.8 VE01_01585-T1 VE01_03332-T1 2 2 6.3 6.43 2.60E-11 1 1 2.4 6.65 1.10E-11 56091.3 VE01_03332-T1 VE01_05359-T1 1 1 6.1 4.78 5.60E-09 2 2 6.1 6.53 1.70E-11 2 2 6.1 6.06 1.30E-10 19759.5 VE01_05359-T1 VE01_08977-T1 2 2 18.3 6.31 4.50E-11 2 2 18.3 5.6 9.20E-10 1 1 8.3 3.98 1.60E-07 12942.9 VE01_08977-T1 VE01_04627-T1 2 2 4.3 6.03 1.50E-10 2 2 4.3 6.47 2.30E-11 48649.4 VE01_04627-T1 VE01_01353-T1 1 3 3 18 4.33 4.30E-08 2 2 16.2 4.14 6.80E-08 11846.4 VE01_01353-T1 VE01_02923-T1 2 2 9 5.63 8.00E-10 1 1 4.5 3.24 8.60E-08 1 1 4.5 5.03 1.10E-08 23739.5 VE01_02923-T1 VE01_00498-T1 2 3 7.3 6.3 4.70E-11 1 1 4.2 4.56 2.10E-09 1 6 4.2 4.55 6.70E-10 27099.7 VE01_00498-T1 VE01_01196-T1 1 1 8.2 2.99 3.60E-06 2 3 24.5 7.01 2.20E-12 1 11772.8 VE01_01196-T1 VE01_08337-T1 3 3 21 2.72 8.40E-07 3 3 21 4.34 5.70E-09 13334.2 VE01_08337-T1 VE01_05361-T1 1 1 6.3 9.55 4.40E-17 1 1 6.3 9.39 8.70E-17 40229 VE01_05361-T1 VE01_07945-T1 1 1 3.3 4.67 4.90E-08 2 2 6 4.9 1.80E-08 40158.1 VE01_07945-T1 VE01_01840-T1 2 2 21.9 5.54 6.30E-11 1 1 5.9 4.04 1.40E-07 1 1 5.9 3.22 5.50E-07 22886.8 VE01_01840-T1 VE01_04636-T1 1 1 6.4 6.24 6.00E-11 1 1 8.8 8.89 7.30E-16 16181.7 VE01_04636-T1 VE01_06014-T1 1 1 4.9 4 8.40E-07 1 1 8.7 8.87 8.00E-16 19593.3 VE01_06014-T1 VE01_07701-T1 1 1 21.5 8.4 6.10E-15 1 1 21.5 6.36 3.70E-11 1 2 21.5 8.86 8.50E-16 6829.2 VE01_07701-T1 VE01_02532-T1 2 2 4.6 5.67 6.80E-10 1 1 3.2 5.65 7.40E-10 47198.6 VE01_02532-T1 VE01_05383-T1 1 1 10.3 8.74 1.40E-15 1 1 10.3 7.17 1.10E-12 16775.7 VE01_05383-T1 VE01_00676-T1 1 1 15.7 7.7 1.20E-13 1 2 15.7 8.38 6.50E-15 1 1 15.7 7.98 3.60E-14 11410.2 VE01_00676-T1 VE01_08259-T1 1 1 2.3 5.35 2.20E-09 2 2 3.6 6 1.70E-10 56417.1 VE01_08259-T1 VE01_05013-T1 2 2 17.6 4.17 4.00E-07 1 1 10.2 3.99 8.80E-07 12100.8 VE01_05013-T1 VE01_05710-T1 1 1 1.8 8 3.30E-14 1 1 0.8 2.17 1.40E-06 1 1 0.8 3.02 1.30E-07 110358.4 VE01_05710-T1 VE01_00313-T1 1 1 1.6 4.55 5.20E-08 2 2 3.7 5.37 2.40E-09 72662.3 VE01_00313-T1 VE01_04077-T1 1 1 9.3 5.82 3.60E-10 1 1 6 7.63 1.60E-13 34066.1 VE01_04077-T1 VE01_03523-T1 1 1 10.8 4.41 1.40E-07 2 2 16.2 4.02 2.60E-07 11956.8 VE01_03523-T1 VE01_03554-T1 1 1 4.5 6.46 2.40E-11 1 1 4.5 6.4 3.00E-11 1 1 4.5 5.68 6.60E-10 23379.2 VE01_03554-T1 VE01_05784-T1 1 1 2.9 3.5 3.40E-07 2 2 4.4 4.43 9.80E-09 41868.8 VE01_05784-T1 VE01_06449-T1 1 1 1.8 6.11 1.10E-10 1 1 1.8 6.08 1.20E-10 71877 VE01_06449-T1 VE01_08855-T1 1 1 2.8 5.88 2.80E-10 1 1 2.8 5.6 9.30E-10 56051.4 VE01_08855-T1 VE01_06215-T1 1 1 7.9 5.48 1.30E-09 1 1 7.9 5.04 2.90E-09 1 1 7.9 5.2 5.00E-09 13426.2 VE01_06215-T1 VE01_03394-T1 1 1 6.6 5.29 3.50E-09 1 1 6.6 2.94 2.40E-07 24546.7 VE01_03394-T1 VE01_07383-T1 1 1 2.4 4.46 9.80E-08 1 1 2.4 3.32 2.80E-07 46479.7 VE01_07383-T1 VE01_09066-T1 1 1 4.2 4.16 7.90E-08 1 1 1 4.2 4.45 7.70E-09 28706.8 VE01_09066-T1 VE01_03121-T1 1 1 13.8 3.28 8.10E-09 1 1 6.9 3.11 1.00E-06 17292.9 VE01_03121-T1 VE01_09076-T1 1 1 8.8 3.02 8.80E-08 1 1 8.8 2.93 6.30E-07 11094 VE01_09076-T1 VE01_07429-T1 4 4 13.5 3.15 1.30E-06 2 2 6.6 2.97 1.20E-06 30408.2 VE01_07429-T1 VE01_07752-T1 5 5 15.1 4.35 1.90E-07 3 3 6.9 5.02 1.10E-08 11 12 38.2 8.57 3.00E-15 26695.9 VE01_07752-T1 VE01_06111-T1 7 8 20.6 7.86 6.10E-14 5 5 17.8 7.21 9.40E-13 6 7 17.8 6.86 4.20E-12 36356.4 VE01_06111-T1

Supplementary Table 2. Proteins identified in P. verrucosus secretome by LC-MS/MS

212

VE02_07717-T1 8 34 26.7 14.59 2.10E-26 4 9 9 7.83 6.70E-14 2 2 9.4 8.07 2.40E-14 27994.7 VE02_07717-T1 VE02_00285-T1 16 29 34.6 13.66 1.10E-24 9 16 17 9.76 1.90E-17 6 10 13.1 8.97 5.30E-16 46559.7 VE02_00285-T1 VE02_08312-T1 7 10 67.1 12.81 4.10E-23 3 4 14.7 12.51 1.40E-22 1 2 6.6 12.94 2.30E-23 25558 VE02_08312-T1 VE02_03721-T1 3 4 12.5 12.61 9.50E-23 1 1 6.1 6.06 6.60E-13 27400.4 VE02_03721-T1 VE02_01241-T1 11 14 25.7 12.47 1.80E-22 5 6 10.8 8.75 1.30E-15 2 2 4.2 5.37 2.50E-09 58743.7 VE02_01241-T1 VE02_01083-T1 2 4 17.5 11.59 7.30E-21 2 2 17.5 7.04 2.00E-12 1 1 12 6.95 5.60E-13 17248.5 VE02_01083-T1 VE02_00413-T1 17 37 34.3 11.53 9.50E-21 13 19 21.9 8.81 1.00E-15 9 11 15.2 7.29 6.90E-13 56536.2 VE02_00413-T1 VE02_01208-T1 19 41 56.9 11.49 1.10E-20 13 26 31.5 11.27 2.90E-20 11 13 27 10.3 1.80E-18 43996.9 VE02_01208-T1 VE02_07542-T1 11 13 22.4 11.29 2.70E-20 14 18 23.8 9.81 1.50E-17 5 6 9 8.23 1.30E-14 69485.6 VE02_07542-T1 VE02_01417-T1 15 44 41.5 11.1 6.00E-20 11 28 16.8 7.92 4.60E-14 10 18 16.6 8.2 1.40E-14 45356.7 VE02_01417-T1 VE02_04065-T1 3 3 8.9 11.09 6.20E-20 2 2 5.2 6.34 3.90E-11 1 1 2.3 5.93 2.30E-10 69469.3 VE02_04065-T1 VE02_00100-T1 4 10 20 11.07 6.80E-20 2 5 9.2 10.48 8.50E-19 51106.8 VE02_00100-T1 VE02_07224-T1 9 14 33.9 10.93 1.20E-19 2 3 3.7 3.47 2.00E-07 42418.7 VE02_07224-T1 VE02_04705-T1 5 16 39.3 10.72 3.00E-19 3 10 27.7 10.44 9.90E-19 2 3 22 8.63 2.30E-15 19650.2 VE02_04705-T1 VE02_00402-T1 18 20 21.1 10.52 7.10E-19 18 22 21.8 14.2 1.10E-25 11 12 13.5 13.91 3.80E-25 119779.9 VE02_00402-T1 VE02_03887-T1 14 60 47.4 10.39 1.20E-18 12 46 29.4 11.86 2.30E-21 6 9 18.3 7.6 1.80E-13 34167.9 VE02_03887-T1 VE02_03433-T1 6 7 41.6 10.36 1.40E-18 2 2 16 5.81 3.80E-10 2 2 11.7 6.29 4.80E-11 23443.1 VE02_03433-T1 VE02_07523-T1 4 4 24.8 10.35 1.50E-18 1 1 3.3 2.66 4.50E-06 25451.3 VE02_07523-T1 VE02_06038-T1 11 13 44.9 10.34 1.60E-18 7 8 25.4 13.07 1.40E-23 2 2 9.2 7.3 6.40E-13 32648.2 VE02_06038-T1 VE02_08921-T1 1 2 14.7 10 6.50E-18 1 1 14.7 7.35 4.70E-14 1 1 14.7 6.26 7.00E-13 11333.2 VE02_08921-T1 VE02_05466-T1 9 9 11.5 9.8 1.50E-17 3 3 1.9 3.07 4.30E-07 111450.4 VE02_05466-T1 VE02_02241-T1 7 11 58.1 9.75 1.90E-17 5 11 25.7 6.6 1.30E-11 3 4 30.9 9.44 7.20E-17 14606.8 VE02_02241-T1 VE02_02877-T1 1 1 3.6 9.65 2.80E-17 1 1 3.1 4.2 3.60E-07 1 1 3.1 3.9 1.30E-06 40553.5 VE02_02877-T1 VE02_05228-T1 7 9 40.9 9.57 4.10E-17 7 7 39.7 7.57 2.00E-13 5 5 35 7.43 3.80E-13 26921.8 VE02_05228-T1 VE02_08573-T1 2 3 10.4 9.52 5.10E-17 2 2 11.9 8.58 2.80E-15 34500.8 VE02_08573-T1 VE02_07822-T1 12 12 36.3 9.42 7.60E-17 6 6 11.1 8.71 1.60E-15 1 1 2.1 3.78 2.20E-06 49558.4 VE02_07822-T1 VE02_02185-T1 5 11 16 9.4 8.60E-17 6 9 16 8.67 1.90E-15 4 4 14 6.33 1.40E-11 40831.9 VE02_02185-T1 VE02_08171-T1 8 10 53.3 9.3 1.30E-16 4 4 20.8 7.5 2.80E-13 5 5 21.5 9.81 1.50E-17 29591.2 VE02_08171-T1 VE02_04513-T1 5 18 21.6 9.03 4.00E-16 4 6 15.4 7.03 2.10E-12 1 2 4.8 4.82 1.30E-08 22581.3 VE02_04513-T1 VE02_06960-T1 7 7 44.2 9.03 4.10E-16 6 6 35.4 7.99 3.40E-14 19470.1 VE02_06960-T1 VE02_07495-T1 1 1 17.5 9.03 4.10E-16 1 1 8.3 2.67 6.00E-07 12846.7 VE02_07495-T1 VE02_02323-T1 3 4 13.1 8.96 5.50E-16 4 5 15.3 10.56 6.10E-19 2 2 7.5 2.54 3.10E-07 36839.2 VE02_02323-T1 VE02_07304-T1 34 42 38.4 8.95 5.80E-16 20 23 24.6 9.5 5.50E-17 5 5 6 6.45 2.40E-11 98557.7 VE02_07304-T1 VE02_02270-T1 7 12 18.2 8.89 7.40E-16 5 6 10.8 7.58 2.00E-13 1 2 2.5 6.17 8.10E-11 63838.9 VE02_02270-T1 VE02_00625-T1 5 7 11.7 8.88 7.80E-16 3 4 9 5.21 1.10E-10 2 2 4.5 5.25 1.30E-10 43216.2 VE02_00625-T1 VE02_04841-T1 4 7 10.3 8.61 2.50E-15 1 1 2.4 6.21 6.70E-11 67073.6 VE02_04841-T1 VE02_05425-T1 12 15 26.5 8.58 2.80E-15 8 10 21 7.19 1.00E-12 6 6 11.3 6.62 1.20E-11 47065.9 VE02_05425-T1 VE02_06229-T1 4 5 27.6 8.47 4.40E-15 2 2 20.7 7.29 6.90E-13 4 4 25 8.67 1.90E-15 11939.7 VE02_06229-T1 VE02_02686-T1 5 5 72.9 8.38 6.50E-15 2 2 37.4 7.21 9.50E-13 11937.7 VE02_02686-T1 VE02_02081-T1 1 1 17.5 8.36 7.00E-15 1 1 7.2 3.37 1.30E-06 17371.9 VE02_02081-T1 VE02_04151-T1 16 30 40.3 8.26 1.10E-14 19 29 43.5 8.91 6.90E-16 14 14 31.8 7.57 2.10E-13 39299.8 VE02_04151-T1 VE02_07280-T1 2 2 20.8 8.2 1.40E-14 1 1 10.1 5.96 2.00E-12 15919.8 VE02_07280-T1 VE02_03940-T1 1 1 2.7 8.2 1.40E-14 2 2 3.3 2.48 6.60E-07 2 2 3.6 2.95 3.20E-07 71532.6 VE02_03940-T1 VE02_01490-T1 3 4 9.3 8.08 2.30E-14 4 4 8.4 7.92 4.70E-14 1 1 1.6 2.89 5.60E-07 57661.6 VE02_01490-T1 VE02_00279-T1 2 2 34.3 7.94 4.30E-14 1 1 11.1 5.06 9.20E-09 10602.4 VE02_00279-T1 VE02_04099-T1 4 4 14.8 7.81 7.30E-14 3 3 9 4.5 1.00E-07 40184.9 VE02_04099-T1 VE02_04801-T1 5 5 13.4 7.73 1.00E-13 3 3 7.7 5.93 2.20E-10 1 2 3.2 5.02 6.20E-09 47066.3 VE02_04801-T1 VE02_02402-T1 2 2 17.9 7.56 2.10E-13 3 4 32.1 6.66 1.00E-11 3 3 32.1 6.55 1.60E-11 18748.7 VE02_02402-T1 VE02_08838-T1 4 4 20.2 7.56 2.20E-13 2 2 9.2 7.77 9.00E-14 30243.4 VE02_08838-T1 VE02_07376-T1 1 1 9.9 7.48 3.10E-13 1 2 10.5 6.02 1.50E-10 16736.9 VE02_07376-T1 VE02_05497-T1 5 7 26.5 7.34 5.40E-13 3 4 18.9 4.96 1.40E-08 19612.2 VE02_05497-T1 VE02_07142-T1 6 6 11.1 7.32 6.00E-13 11 11 20.7 8.69 1.70E-15 4 4 6.7 4.86 3.40E-09 68556.9 VE02_07142-T1 VE02_04269-T1 19 22 25 7.28 7.30E-13 14 15 20.2 8.62 2.30E-15 6 6 8.5 6.26 5.50E-11 81384.3 VE02_04269-T1 VE02_01935-T1 2 4 14.9 7.27 7.40E-13 1 3 5.9 6.3 4.70E-11 1 3 5.9 6.03 1.40E-10 21039.7 VE02_01935-T1 VE02_06498-T1 2 2 12.8 7.21 9.60E-13 3 3 14.5 8.45 4.80E-15 26460.4 VE02_06498-T1 VE02_08638-T1 2 2 10.5 7.13 1.30E-12 2 2 8.9 4.73 3.80E-08 25738.2 VE02_08638-T1 VE02_05773-T1 5 7 42.3 7.04 1.90E-12 2 2 14.1 4.74 2.90E-08 1 1 6.4 2.95 8.60E-06 16681.7 VE02_05773-T1 VE02_00693-T1 4 4 38.7 7.04 2.00E-12 6 6 42.3 5.12 7.20E-09 11845.3 VE02_00693-T1 VE02_07216-T1 2 2 22.4 6.71 8.10E-12 1 1 5 3.67 5.30E-07 18519.5 VE02_07216-T1 VE02_00758-T1 4 4 13.6 6.45 2.50E-11 2 2 3 3.03 4.80E-07 40423.4 VE02_00758-T1 VE02_00871-T1 4 4 12.1 6.41 2.90E-11 2 3 4 5.13 7.50E-10 2 2 4.2 4.26 1.00E-08 52432.5 VE02_00871-T1 VE02_08168-T1 1 1 7.1 6.33 4.00E-11 2 2 13.3 5.4 8.50E-10 1 1 4.6 3.38 3.70E-06 21453.4 VE02_08168-T1 VE02_01698-T1 2 5 11.7 6.32 4.20E-11 2 3 11.7 7 2.30E-12 3 4 11.7 9.57 4.00E-17 21334.9 VE02_01698-T1 VE02_01018-T1 1 1 3.7 6.13 9.50E-11 5 5 14.8 6.82 5.00E-12 47665.4 VE02_01018-T1 VE02_01744-T1 1 1 3.4 6.11 1.00E-10 5 5 15.1 6.81 5.30E-12 44364.2 VE02_01744-T1 VE02_04837-T1 4 4 7.2 6.06 1.30E-10 4 4 6.9 5.58 1.00E-09 2 3 4.4 3.7 2.90E-08 68173.5 VE02_04837-T1 VE02_01345-T1 4 7 10.7 6.06 1.30E-10 1 1 6 4.33 2.40E-09 22208.3 VE02_01345-T1 VE02_05113-T1 1 1 2.8 5.89 2.70E-10 3 3 8.4 9.1 3.10E-16 56051.5 VE02_05113-T1 VE02_07527-T1 1 1 18.3 5.88 2.80E-10 1 1 18.3 4.93 6.60E-09 7460.1 VE02_07527-T1 VE02_07056-T1 1 1 6.1 5.84 3.30E-10 2 2 17.3 10.16 3.30E-18 21118.4 VE02_07056-T1 VE02_06386-T1 1 1 5.2 5.75 4.90E-10 1 1 5.2 6.25 5.70E-11 29969.1 VE02_06386-T1 VE02_05159-T1 1 1 8 5.45 1.70E-09 1 1 8 5.61 8.70E-10 1 1 8 4.24 4.10E-10 15544.5 VE02_05159-T1 VE02_04174-T1 2 2 7.3 5.36 2.60E-09 1 1 4.2 5.92 2.30E-10 35365.1 VE02_04174-T1 VE02_01260-T1 4 4 43.9 5.33 3.00E-09 2 3 12.3 3.75 4.30E-07 1 1 12.3 5.27 3.40E-09 12554.2 VE02_01260-T1 VE02_02941-T1 2 2 5 5.26 3.80E-09 2 2 5.7 4.44 4.30E-10 47584.2 VE02_02941-T1 VE02_07976-T1 1 1 5.9 5.2 5.10E-09 1 1 5.9 5.29 3.40E-09 1 1 5.9 3.14 4.50E-07 23002 VE02_07976-T1 VE02_02851-T1 1 1 2.9 5.17 5.60E-09 3 3 12 6.5 2.00E-11 41251 VE02_02851-T1 VE02_08042-T1 1 1 17.2 5.1 7.60E-09 1 1 17.2 2.03 3.40E-06 7165.3 VE02_08042-T1 VE02_05149-T1 1 1 3.2 4.17 1.30E-08 1 1 3.2 4.98 5.80E-09 1 1 3.2 4.97 1.40E-08 58324.7 VE02_05149-T1 VE02_00111-T1 3 3 7.8 4.94 1.50E-08 4 5 8.2 8.5 4.00E-15 3 3 7.8 6.7 8.40E-12 52768.3 VE02_00111-T1 VE02_01358-T1 2 2 6.4 4.68 1.60E-08 2 2 6.4 4.75 7.20E-09 34206.8 VE02_01358-T1 VE02_00001-T1 3 3 9.4 4.78 3.00E-08 2 2 6.5 5.03 1.00E-08 2 2 6.5 5.04 9.90E-09 36732.7 VE02_00001-T1 VE02_00004-T1 1 2 2.8 4.09 1.70E-07 3 3 12.3 4.89 1.50E-08 35034.5 VE02_00004-T1 VE02_05105-T1 1 1 3.4 3.95 1.00E-06 5 5 15.4 6.32 4.20E-11 33177.3 VE02_05105-T1 VE02_04325-T1 1 1 2.2 3.96 1.00E-06 2 2 4.5 2.93 4.80E-07 53679.6 VE02_04325-T1 VE02_05742-T1 1 1 0.6 1.83 1.90E-06 1 1 0.6 3.62 3.70E-08 2 2 1.7 7.15 7.40E-13 193445.6 VE02_05742-T1 VE02_00476-T1 2 2 16.1 4 1.80E-07 1 1 7.1 3.63 2.00E-07 11955 VE02_00476-T1 VE02_00509-T1 2 2 4.8 6.86 4.30E-12 2 3 4.8 6.59 1.30E-11 67411 VE02_00509-T1 VE02_05772-T1 1 1 3.1 5.94 2.20E-10 1 1 3.1 5.13 1.20E-09 42154.9 VE02_05772-T1 VE02_08089-T1 2 2 2.8 5.44 1.80E-09 2 2 3.4 3.82 1.20E-07 73694.6 VE02_08089-T1 VE02_02804-T1 3 3 12 5.42 2.00E-09 1 1 6 5.82 2.50E-10 33625.5 VE02_02804-T1 VE02_00826-T1 2 2 4.7 2.45 1.70E-07 4 4 8.3 5.88 2.70E-10 59780.2 VE02_00826-T1 VE02_08208-T1 2 2 6 2.84 8.50E-07 6 8 13.7 6.05 1.40E-10 50966.8 VE02_08208-T1 VE02_06828-T1 1 1 3.7 1.04 9.90E-06 6 7 13.4 7.54 2.30E-13 55460.6 VE02_06828-T1 Supplementary Table 3. Proteins identified in P. sp. 03VT05 secretome by LC-MS/MS 213

VF21_06666-T1 27 38 36.4 17.31 1.90E-31 17 20 19.9 12.66 7.70E-23 16 18 19.8 10.82 2.00E-19 117414 VF21_06666-T1 VF21_04065-T1 18 37 48 15.21 1.50E-27 9 15 18 9.97 7.60E-18 9 21 12.5 7.76 9.30E-14 45298.6 VF21_04065-T1 VF21_02391-T1 16 21 70.2 15.95 6.10E-29 9 13 35.7 10.52 7.20E-19 8 12 28.9 8.4 6.10E-15 32421 VF21_02391-T1 VF21_07390-T1 15 35 49.9 12.63 8.90E-23 11 18 35.9 10.22 2.60E-18 9 11 27.9 10.34 1.50E-18 44055.8 VF21_07390-T1 VF21_09197-T1 14 22 74.5 11.07 6.90E-20 14 24 65 10.49 8.00E-19 7 7 27.7 10.38 1.30E-18 29648.2 VF21_09197-T1 VF21_08415-T1 14 18 32.2 11.92 1.80E-21 8 12 17.6 10.45 9.60E-19 7 8 12 7.77 8.60E-14 56601 VF21_08415-T1 VF21_04730-T1 16 30 44.7 10.84 1.80E-19 10 13 33.5 10.15 3.40E-18 14 17 23.3 10.32 1.70E-18 49635.3 VF21_04730-T1 VF21_05173-T1 15 19 26.4 11.57 7.90E-21 10 14 22.4 11.96 1.50E-21 10 11 18 12.21 5.30E-22 58411.2 VF21_05173-T1 VF21_09646-T1 16 61 61.3 12.01 1.30E-21 14 55 39 10.73 2.80E-19 10 36 36.5 9.16 2.40E-16 34170.8 VF21_09646-T1 VF21_01931-T1 19 21 27 10.66 4.00E-19 8 8 9.5 10.2 2.80E-18 10 15 11.5 9.91 9.60E-18 110016.9 VF21_01931-T1 VF21_09012-T1 14 17 26.6 9.58 3.90E-17 6 6 9.3 7.8 7.90E-14 13 15 23.8 9.5 5.50E-17 69469.6 VF21_09012-T1 VF21_08412-T1 6 7 29.7 10.73 2.90E-19 12 22 44.1 10.47 8.70E-19 4 7 12.6 8.86 8.40E-16 39107.8 VF21_08412-T1 VF21_06874-T1 15 23 72.7 10.7 3.30E-19 13 22 59.1 9.31 1.20E-16 15 21 59.1 8.51 3.80E-15 26316.6 VF21_06874-T1 VF21_06240-T1 14 19 27.7 8.7 1.70E-15 5 5 11.5 6.99 2.40E-12 12 14 19.9 7.72 1.10E-13 70594.8 VF21_06240-T1 VF21_01430-T1 13 21 36.9 14.34 5.90E-26 12 15 25.5 9.09 3.10E-16 9 11 21.1 10.5 7.70E-19 57552.5 VF21_01430-T1 VF21_06763-T1 12 15 43 11.09 6.20E-20 1 1 1.7 3.54 2.10E-07 10 14 28 9.59 3.70E-17 42460.8 VF21_06763-T1 VF21_04669-T1 7 11 68.4 9.46 6.60E-17 12 24 70.2 9.75 1.90E-17 2 3 20.5 5.12 7.10E-09 18659.4 VF21_04669-T1 VF21_08575-T1 12 15 44.2 11.27 2.90E-20 10 11 40.3 13.67 1.00E-24 9 15 28.4 12.77 4.80E-23 32663.1 VF21_08575-T1 VF21_06727-T1 12 13 35.3 7.74 9.90E-14 18 22 39 6.38 3.20E-11 42227 VF21_06727-T1 VF21_05371-T1 14 16 35.4 9.32 1.20E-16 6 7 14.8 8.76 1.30E-15 8 8 14.8 6.23 6.20E-11 57971.1 VF21_05371-T1 VF21_02425-T1 11 13 32 10.62 4.60E-19 9 11 28.2 8.81 1.10E-15 10 11 23 7.59 1.90E-13 52012.4 VF21_02425-T1 VF21_02883-T1 9 9 13.2 9.53 4.90E-17 14 15 18.9 9.01 4.40E-16 98601.6 VF21_02883-T1 VF21_09729-T1 11 11 28 10.07 4.90E-18 3 4 7.6 4.95 1.40E-08 5 5 6.8 6.1 1.10E-10 67273.3 VF21_09729-T1 VF21_09781-T1 4 4 7.3 8.74 1.40E-15 12 13 15.9 9.09 3.10E-16 86405.5 VF21_09781-T1 VF21_00670-T1 14 15 33.4 8.24 1.20E-14 11 11 23.7 6.53 1.70E-11 10 10 23.1 5.8 3.90E-10 54805.7 VF21_00670-T1 VF21_07154-T1 8 8 45.5 8.67 1.90E-15 9 10 44.8 9.48 6.10E-17 32862.6 VF21_07154-T1 VF21_00789-T1 9 9 22.3 9.01 4.50E-16 11 12 21.5 9.21 1.90E-16 10 12 18.3 9.5 5.60E-17 53309.8 VF21_00789-T1 VF21_04017-T1 4 4 6.7 7.17 1.10E-12 11 11 20.1 7.57 2.10E-13 9 9 13.1 6.64 1.10E-11 81391.3 VF21_04017-T1 VF21_03726-T1 7 8 28.5 11.82 2.80E-21 2 2 8.4 6.73 7.30E-12 5 5 16.2 8.04 2.80E-14 47665.4 VF21_03726-T1 VF21_04199-T1 8 9 17.9 8.31 9.00E-15 9 10 20.9 8.47 4.40E-15 6 7 11 9.05 3.80E-16 67292.8 VF21_04199-T1 VF21_05104-T1 10 11 22.2 10.48 8.60E-19 11 17 22.8 9.39 8.70E-17 9 10 14.9 6.78 5.90E-12 47805.5 VF21_05104-T1 VF21_07835-T1 1 1 2.1 6.17 8.10E-11 11 11 20.7 7.66 1.40E-13 68459.8 VF21_07835-T1 VF21_07325-T1 8 12 56 9.7 2.30E-17 2 2 12.7 3.59 9.40E-09 2 2 18.7 7.78 8.40E-14 13992.1 VF21_07325-T1 VF21_00656-T1 8 8 53.7 10.3 1.80E-18 1 1 7.9 7.72 1.10E-13 6 6 33.1 7.93 4.40E-14 26480.4 VF21_00656-T1 VF21_07211-T1 6 6 14.7 7.89 5.20E-14 10 10 25.8 6.96 2.80E-12 53402.2 VF21_07211-T1 VF21_05370-T1 7 11 26.7 11.72 4.30E-21 6 12 23.8 13.13 1.00E-23 6 7 16.6 9.97 7.50E-18 28012.6 VF21_05370-T1 VF21_07707-T1 4 4 6.6 6.67 9.50E-12 10 10 13.9 6.83 4.90E-12 86298.5 VF21_07707-T1 VF21_08711-T1 5 6 11.6 10.93 1.20E-19 1 2 3.3 10.48 8.60E-19 67145.5 VF21_08711-T1 VF21_07458-T1 1 1 2.1 3.38 2.60E-07 8 9 17.3 8.81 1.00E-15 4 4 9.4 8.51 3.80E-15 52452.2 VF21_07458-T1 VF21_01222-T1 7 7 23.7 7.12 1.40E-12 2 2 7.9 8.46 4.60E-15 8 8 27.1 7.82 7.30E-14 44363.3 VF21_01222-T1 VF21_05927-T1 7 7 16.5 9.18 2.10E-16 1 1 1.2 2.27 3.70E-06 71461.3 VF21_05927-T1 VF21_04817-T1 6 7 37.7 9.37 9.40E-17 1 1 10.1 6.67 9.50E-12 6 7 35 9.32 1.20E-16 26917.7 VF21_04817-T1 VF21_00998-T1 5 8 21.5 11.61 6.80E-21 4 8 21.5 11.45 1.30E-20 2 4 9.1 9.44 7.00E-17 51073.4 VF21_00998-T1 VF21_07247-T1 6 9 57.4 8.31 8.70E-15 3 5 16.2 7.11 1.40E-12 4 6 37.5 5.83 3.50E-10 14578.8 VF21_07247-T1 VF21_01192-T1 6 6 17.4 9.52 5.00E-17 2 2 4.9 7.69 1.20E-13 6 7 21.5 8.46 4.60E-15 46806.7 VF21_01192-T1 VF21_00726-T1 7 10 26 7.95 4.10E-14 1 2 6.3 9.02 4.20E-16 4 4 11.8 6.19 7.40E-11 39024.8 VF21_00726-T1 VF21_01127-T1 6 7 17.3 10.53 6.90E-19 2 2 3.6 6.09 1.10E-10 62950.2 VF21_01127-T1 VF21_01961-T1 3 3 6 9.72 2.10E-17 7 7 13.4 9.03 4.20E-16 71179 VF21_01961-T1 VF21_06200-T1 6 8 19.2 8.63 2.30E-15 1 1 2.7 4.55 1.10E-08 3 3 8.1 6.41 2.90E-11 44721.1 VF21_06200-T1 VF21_07115-T1 6 6 42.5 7.89 5.20E-14 6 6 40.3 8.25 1.10E-14 6 7 24.9 7.48 3.10E-13 19468.1 VF21_07115-T1 VF21_00768-T1 6 8 9.5 8.12 2.00E-14 5 6 7.1 8.55 3.10E-15 7 8 10 6.73 7.30E-12 64482.7 VF21_00768-T1 VF21_07954-T1 7 8 10.5 10.07 4.90E-18 3 3 3.8 3.75 9.10E-07 79140.7 VF21_07954-T1 VF21_04683-T1 4 7 24.1 12.55 1.20E-22 4 7 18.5 11.22 3.60E-20 3 6 11.5 9.89 1.10E-17 41323.1 VF21_04683-T1 VF21_06980-T1 4 4 18.5 9.3 1.30E-16 7 8 25.5 7.87 5.70E-14 7 7 27.6 6.79 5.70E-12 34179.8 VF21_06980-T1 VF21_05813-T1 2 2 10.5 6.42 2.80E-11 6 7 27.2 9.05 3.80E-16 32748.5 VF21_05813-T1 VF21_01131-T1 6 6 22.8 6.58 1.40E-11 6 6 19.5 6.98 2.60E-12 6 6 18 6.59 1.40E-11 37632.9 VF21_01131-T1 VF21_00546-T1 5 6 18.1 7.84 6.40E-14 1 1 2.8 3 2.70E-06 8 9 22.6 7.97 3.80E-14 31852.1 VF21_00546-T1 VF21_02286-T1 3 4 9.9 6.41 1.70E-11 7 7 12.4 10.28 2.00E-18 72656.4 VF21_02286-T1 VF21_00769-T1 3 3 14.8 9.54 4.70E-17 6 6 15 7.12 1.40E-12 47190 VF21_00769-T1 VF21_04800-T1 2 2 3.6 7.42 4.00E-13 8 9 12.4 6.6 1.30E-11 79575.5 VF21_04800-T1 VF21_01342-T1 2 2 3.9 3.81 2.30E-07 7 9 20.5 8.37 6.80E-15 2 2 7.2 5.3 3.30E-09 49957.2 VF21_01342-T1 VF21_03592-T1 5 10 19.8 10.4 1.20E-18 3 6 11.8 8.37 6.80E-15 4 6 11.8 9.66 2.80E-17 40878.8 VF21_03592-T1 VF21_05354-T1 5 6 32.8 8.04 2.70E-14 2 3 20.7 7.75 9.70E-14 2 2 20.7 6.74 7.00E-12 11965.8 VF21_05354-T1 VF21_00029-T1 2 2 57.1 12.08 9.20E-22 2 5 57.1 20.18 9.00E-37 7471.8 VF21_00029-T1 VF21_08146-T1 4 5 10.4 8.09 2.30E-14 6 6 13 6.55 1.60E-11 56017 VF21_08146-T1 VF21_02670-T1 4 5 44.9 9.68 2.50E-17 5 5 45.7 8.74 1.40E-15 3 4 31.9 8.41 5.80E-15 14640.8 VF21_02670-T1 VF21_04846-T1 5 5 16.1 8.81 1.00E-15 3 3 8 10.76 2.60E-19 2 2 6.3 10.35 1.50E-18 55710.4 VF21_04846-T1 VF21_04344-T1 5 5 39.8 8.1 2.10E-14 1 1 10.8 6.44 2.60E-11 5 7 39.8 8.1 2.10E-14 23522 VF21_04344-T1 VF21_00879-T1 1 1 3.2 4.63 5.70E-08 8 8 24.5 5.35 2.70E-09 36524.9 VF21_00879-T1 VF21_02743-T1 5 5 27.4 6.98 2.50E-12 3 3 13.4 6.44 2.60E-11 32371.9 VF21_02743-T1 VF21_09775-T1 4 5 12.5 9.82 1.40E-17 2 2 8.7 8.47 4.40E-15 2 3 9.3 9.07 3.50E-16 34554 VF21_09775-T1 VF21_00770-T1 2 3 14.3 8.48 4.40E-15 5 6 32.7 8.45 4.80E-15 23484.6 VF21_00770-T1 VF21_00424-T1 4 6 39.3 10.52 7.10E-19 2 2 15.2 6.26 5.40E-11 4 5 35.1 7.71 1.10E-13 19722.3 VF21_00424-T1 VF21_03303-T1 5 5 15.5 7.67 1.30E-13 2 2 4.7 6.06 1.30E-10 5 5 11.4 6.79 5.70E-12 56081.4 VF21_03303-T1 VF21_06266-T1 4 4 32.5 10.53 6.70E-19 5 6 24.8 11.7 4.70E-21 4 5 24.8 12.4 2.30E-22 25420.3 VF21_06266-T1 VF21_07283-T1 3 4 45 14.22 1.00E-25 1 1 8.3 3.17 2.90E-06 11000.2 VF21_07283-T1 VF21_07349-T1 5 6 11.4 8.93 6.20E-16 4 4 4.9 3.74 1.50E-07 80624.6 VF21_07349-T1 VF21_07309-T1 4 4 8.2 10.3 1.80E-18 1 1 3.2 6.21 6.80E-11 3 4 9.3 9.57 4.00E-17 58233.6 VF21_07309-T1 VF21_01852-T1 4 6 19.9 8.61 2.40E-15 2 2 11.3 6.87 4.20E-12 3 3 15.1 7.08 1.70E-12 30222.4 VF21_01852-T1 VF21_05245-T1 2 2 13.9 6.96 2.80E-12 6 6 35.1 8.28 1.00E-14 25637.4 VF21_05245-T1 VF21_05493-T1 4 5 24.2 9.99 6.90E-18 1 1 1.9 1.91 7.30E-06 2 2 4.2 2.24 4.90E-06 36773.2 VF21_05493-T1 VF21_01513-T1 6 6 20.2 6.33 4.10E-11 4 4 13.4 6.06 1.30E-10 33177.3 VF21_01513-T1 VF21_00528-T1 5 6 16.7 7.43 3.80E-13 4 5 12.8 6.25 5.80E-11 4 5 12.8 7.09 1.60E-12 22526.2 VF21_00528-T1 VF21_08006-T1 4 5 25.8 8.01 3.20E-14 2 2 11.1 5.49 1.50E-09 1 1 4.5 4.76 3.20E-08 26089.4 VF21_08006-T1 VF21_00727-T1 1 1 3.8 3.61 1.80E-06 4 4 23.9 7.78 8.30E-14 26444.5 VF21_00727-T1 VF21_06573-T1 5 6 19 6.9 3.60E-12 1 1 6.3 5.2 5.00E-09 2 3 11.9 6.54 1.70E-11 27364.4 VF21_06573-T1 VF21_04743-T1 4 4 7 7.36 5.00E-13 5 5 6.3 6.16 8.60E-11 95112.8 VF21_04743-T1 VF21_07200-T1 1 1 2.9 5.91 1.50E-10 5 5 10.8 7.08 1.70E-12 2 2 4.7 4.07 2.60E-08 59780.2 VF21_07200-T1 VF21_06967-T1 5 6 15 5.87 2.90E-10 2 3 10.3 3.57 5.40E-06 3 4 15 4.52 9.20E-08 22048.1 VF21_06967-T1 VF21_02405-T1 2 2 6.5 5.63 8.20E-10 4 4 10 6.67 9.60E-12 43114.5 VF21_02405-T1 VF21_02704-T1 3 4 20.4 7.58 2.00E-13 4 4 17.9 7.51 2.70E-13 2 2 13.3 6.24 6.10E-11 21453.4 VF21_02704-T1 VF21_04909-T1 2 2 9.5 5.84 3.30E-10 3 3 16.5 7.49 2.90E-13 4 5 21 7.88 5.50E-14 23871.9 VF21_04909-T1

214

VF21_05024-T1 2 2 29 7.75 9.70E-14 4 4 59.8 6.06 1.30E-10 11937.7 VF21_05024-T1 VF21_00875-T1 4 5 10.7 7.04 2.00E-12 3 3 5.4 3 1.80E-06 55430.6 VF21_00875-T1 VF21_02356-T1 3 4 23.8 8.23 1.20E-14 4 4 23.8 7.32 6.10E-13 25421.4 VF21_02356-T1 VF21_02519-T1 4 5 30.2 8.98 5.00E-16 3 3 9.8 2.73 3.90E-07 25094.3 VF21_02519-T1 VF21_00410-T1 3 3 17.4 9.3 1.30E-16 3 3 7.8 5.64 3.70E-10 43064.8 VF21_00410-T1 VF21_04997-T1 2 3 4 7.08 1.70E-12 4 4 5.9 6.59 1.40E-11 4 4 5.6 3.66 7.50E-08 93382 VF21_04997-T1 VF21_06946-T1 1 1 4.3 8.06 2.50E-14 3 3 12 7.35 5.20E-13 4 5 14.4 6.87 4.10E-12 41566.8 VF21_06946-T1 VF21_05791-T1 3 5 6.8 8.16 1.70E-14 1 1 1.8 4.02 1.50E-07 60826.6 VF21_05791-T1 VF21_03989-T1 4 4 36 6.85 4.50E-12 3 3 24.3 4.28 2.10E-08 4 4 21.6 5.37 2.50E-09 11859.4 VF21_03989-T1 VF21_03027-T1 3 5 18.3 7.49 2.90E-13 3 4 18.3 7.29 6.70E-13 3 4 18.3 6.99 2.50E-12 20407.8 VF21_03027-T1 VF21_02960-T1 2 2 4.1 7.33 5.70E-13 3 3 8.5 7.16 1.20E-12 46868.3 VF21_02960-T1 VF21_01100-T1 4 4 12.7 5.12 7.00E-09 4 4 13 6.04 1.40E-10 2 2 6.5 4.18 3.90E-07 36853.7 VF21_01100-T1 VF21_03790-T1 1 1 2.3 3.82 4.20E-07 1 1 2.3 3.44 1.10E-06 5 5 12.7 4.67 4.80E-08 43948.2 VF21_03790-T1 VF21_00012-T1 3 3 36.9 7.97 3.70E-14 2 2 15 4.98 1.30E-08 17052 VF21_00012-T1 VF21_07972-T1 2 4 3 9.61 3.40E-17 1 1 2.7 7.14 1.30E-12 72757.1 VF21_07972-T1 VF21_05563-T1 2 5 14.9 7.07 1.70E-12 2 9 14.9 6.55 1.60E-11 3 7 21.3 7.29 6.90E-13 20986.6 VF21_05563-T1 VF21_05201-T1 3 3 8.8 7.26 7.80E-13 2 2 5.1 6.57 1.40E-11 1 1 3.2 6.16 8.50E-11 46308.5 VF21_05201-T1 VF21_07049-T1 2 2 11.5 8.65 2.00E-15 2 3 11.5 9.81 1.40E-17 2 2 11.5 9.09 3.20E-16 18091.4 VF21_07049-T1 VF21_09321-T1 2 4 6.8 8.62 2.40E-15 1 2 6.8 8.17 1.60E-14 26709.1 VF21_09321-T1 VF21_05482-T1 2 3 22.1 9.75 1.90E-17 1 1 12.8 12.65 8.20E-23 1 1 9.3 5.85 3.20E-10 16485.5 VF21_05482-T1 VF21_03746-T1 2 4 25.3 8.76 1.30E-15 2 4 25.3 9.73 2.10E-17 1 1 7.6 7.58 2.00E-13 29013 VF21_03746-T1 VF21_00775-T1 2 2 21.7 8.41 5.90E-15 1 1 11.8 2.71 5.80E-07 1 1 11.8 6.81 5.20E-12 16736.9 VF21_00775-T1 VF21_03897-T1 3 3 11.6 5.45 1.70E-09 3 3 11.6 5.62 8.40E-10 27187.5 VF21_03897-T1 VF21_02384-T1 2 3 34.3 8.48 4.20E-15 1 1 11.1 3.79 5.90E-08 1 1 11.1 5.86 1.50E-10 10602.4 VF21_02384-T1 VF21_06402-T1 2 2 4.1 3.9 1.30E-06 2 2 8.2 5.22 4.70E-09 4 4 7.7 4.43 1.40E-07 60960.3 VF21_06402-T1 VF21_04244-T1 2 2 7.1 8.12 2.00E-14 1 1 3.3 4.83 2.50E-08 40171.2 VF21_04244-T1 VF21_09173-T1 2 3 20.8 7.57 2.10E-13 1 1 10.1 6.65 3.90E-13 15978.9 VF21_09173-T1 VF21_00730-T1 2 3 17.9 7.22 9.10E-13 2 2 23.4 7 8.50E-13 1 1 9.2 4.6 1.60E-09 18721.6 VF21_00730-T1 VF21_00553-T1 2 2 20.4 6.73 7.40E-12 2 3 20.4 6.54 1.70E-11 28291.9 VF21_00553-T1 VF21_04163-T1 1 1 8.9 3.14 4.80E-06 1 1 8.9 3.45 2.70E-06 3 3 18.8 5.65 7.40E-10 11941 VF21_04163-T1 VF21_02018-T1 1 1 3.6 4.38 1.70E-07 2 2 10 8.67 1.90E-15 24722.7 VF21_02018-T1 VF21_05151-T1 1 1 13.2 5.2 5.10E-09 2 2 28.9 6.05 1.30E-10 3 3 38.6 5.4 2.20E-09 12498 VF21_05151-T1 VF21_08369-T1 1 1 2.1 4.78 1.10E-08 2 3 5.8 7.25 7.90E-13 2 2 5.2 7.17 1.10E-12 56027.7 VF21_08369-T1 VF21_03391-T1 3 3 19.5 5.72 5.40E-10 2 2 14.6 4.97 1.10E-09 2 2 14.6 6.55 1.60E-11 19656.2 VF21_03391-T1 VF21_00651-T1 2 2 11.1 2.78 1.40E-06 2 2 10.6 7.73 1.10E-13 22644.1 VF21_00651-T1 VF21_08224-T1 3 3 18.1 5.56 1.10E-09 1 1 9 6.32 4.20E-11 19953.2 VF21_08224-T1 VF21_05766-T1 2 2 18.3 6.54 1.70E-11 1 1 8.3 3.3 1.90E-06 1 1 8.3 5.01 1.10E-08 12911.9 VF21_05766-T1 VF21_03375-T1 1 1 3.7 4.09 2.00E-07 2 2 7.1 5.97 1.90E-10 34418.7 VF21_03375-T1 VF21_05119-T1 1 1 2.9 6.9 3.60E-12 2 2 4.8 6.14 9.20E-11 51713.5 VF21_05119-T1 VF21_03397-T1 2 2 10.3 6.52 1.80E-11 2 2 9.2 4.75 3.40E-08 2 2 9.5 5.55 1.10E-09 28505.8 VF21_03397-T1 VF21_05632-T1 1 1 5.1 1.38 2.60E-06 2 2 3.3 5.52 3.90E-10 61217.4 VF21_05632-T1 VF21_06108-T1 1 1 6.2 3.62 4.10E-08 3 3 12.7 3.88 8.70E-07 30766.6 VF21_06108-T1 VF21_09306-T1 3 3 6.2 3.55 5.70E-06 3 3 13.1 5.55 3.10E-11 1 1 5.2 6.22 3.70E-11 32783.6 VF21_09306-T1 VF21_01276-T1 1 1 4.7 5.33 3.00E-09 2 2 7.8 4.97 1.30E-08 28600.9 VF21_01276-T1 VF21_01825-T1 1 1 2.6 5.72 5.50E-10 2 2 4.2 5.41 2.10E-09 68228.1 VF21_01825-T1 VF21_04656-T1 1 1 3.6 9.55 4.40E-17 1 1 2.9 2.73 4.40E-06 40592.5 VF21_04656-T1 VF21_07604-T1 1 1 7.2 3.27 1.40E-06 2 2 24.7 7.35 5.20E-13 1 1 7.2 4.32 8.80E-08 17349.8 VF21_07604-T1 VF21_02295-T1 1 1 3.1 3.82 2.50E-07 1 1 3.9 1.94 9.80E-07 3 3 5.8 3.47 8.40E-07 39285.9 VF21_02295-T1 VF21_09842-T1 1 2 14.7 8.95 5.70E-16 1 1 14.7 6.47 2.10E-12 11331.2 VF21_09842-T1 VF21_07332-T1 1 1 3.4 5.05 7.80E-09 2 2 6.4 5.37 1.70E-09 33582.6 VF21_07332-T1 VF21_08425-T1 2 2 5.9 3.65 3.80E-06 2 2 4.4 5.22 4.70E-09 2 2 3.6 4.16 4.30E-07 53208.1 VF21_08425-T1 VF21_09441-T1 2 3 4.6 4.54 2.20E-08 1 1 2.2 3.6 1.20E-08 48534 VF21_09441-T1 VF21_03117-T1 1 1 5.3 8.37 6.70E-15 1 1 5.3 6.76 4.00E-14 26299.4 VF21_03117-T1 VF21_08106-T1 1 1 2.9 5.36 2.60E-09 2 2 4.4 6.1 1.10E-10 47751.9 VF21_08106-T1 VF21_07449-T1 2 2 6 4.51 3.80E-08 2 2 5.8 4.63 5.20E-09 2 2 6.5 3.23 7.20E-07 44905.1 VF21_07449-T1 VF21_07365-T1 1 1 26.9 3.93 3.30E-09 2 2 12.3 5.19 5.20E-09 13637.5 VF21_07365-T1 VF21_06508-T1 1 1 18.3 6.6 1.30E-11 1 1 18.3 5.21 9.70E-10 7506.1 VF21_06508-T1 VF21_03453-T1 1 2 3.7 6.57 1.40E-11 1 1 7.5 3.09 6.10E-08 1 1 1.8 2.93 4.40E-07 46079.5 VF21_03453-T1 VF21_08036-T1 1 1 1.4 2.15 8.40E-06 1 1 1.4 2.34 6.50E-06 2 2 3.3 3.39 2.30E-06 56459.7 VF21_08036-T1 VF21_07298-T1 1 2 8 6.32 4.20E-11 1 1 8 4.87 5.10E-09 1 1 8 5.73 5.30E-10 15545.5 VF21_07298-T1 VF21_05242-T1 1 1 6.4 5.69 6.30E-10 1 2 6.4 6.3 4.70E-11 26407.2 VF21_05242-T1 VF21_04229-T1 1 1 12.5 3.93 4.90E-08 2 2 36.1 3.38 2.20E-07 7634.5 VF21_04229-T1 VF21_07821-T1 1 1 6.1 5.85 3.20E-10 1 1 6.1 5.52 1.30E-09 21146.5 VF21_07821-T1 VF21_02256-T1 1 2 11.5 4.83 3.00E-09 1 1 11.5 5.79 5.10E-11 17713.7 VF21_02256-T1 VF21_03123-T1 1 1 4 4.94 1.50E-08 1 1 4 5.72 5.40E-10 29250.9 VF21_03123-T1 VF21_02354-T1 1 1 3.6 5.68 3.50E-10 1 1 4 5.55 1.10E-09 48764.6 VF21_02354-T1 VF21_08302-T1 1 1 4.8 5.49 1.10E-09 1 1 4.8 5.59 9.40E-10 20956.8 VF21_08302-T1 VF21_03241-T1 2 2 11.2 3.34 2.20E-06 1 1 5.3 5.33 2.50E-09 18361.7 VF21_03241-T1 VF21_05346-T1 1 1 1.9 4.86 1.90E-08 1 1 1.7 5.51 1.40E-09 67875.8 VF21_05346-T1 VF21_08913-T1 1 1 1.4 3.83 1.00E-07 1 1 1.4 5.01 3.40E-09 85723.7 VF21_08913-T1 VF21_02532-T1 1 1 6.5 4.93 1.60E-08 1 1 6.5 3.6 2.40E-07 23233.6 VF21_02532-T1 VF21_07405-T1 1 1 14.9 4.69 1.60E-08 1 1 14.9 4.86 1.70E-08 7895.2 VF21_07405-T1 VF21_08964-T1 1 1 12.7 3.83 7.00E-07 1 1 10.9 4.85 2.20E-08 10774.9 VF21_08964-T1 VF21_03128-T1 1 1 5.5 4.5 4.50E-08 1 1 5.5 4.54 6.30E-08 19565.2 VF21_03128-T1 VF21_06320-T1 2 2 21.5 2.19 5.50E-06 1 1 10.1 3.41 7.30E-08 9457.7 VF21_06320-T1 VF21_00060-T1 1 1 2.8 3.51 1.10E-06 1 1 2.8 4.36 1.80E-07 1 1 2.8 3.24 6.50E-07 44528.6 VF21_00060-T1 VF21_04474-T1 1 1 7.5 3.31 1.70E-06 1 1 7.5 3.63 4.70E-07 1 1 7.5 3.84 2.30E-07 13589.8 VF21_04474-T1 VF21_01126-T1 1 1 4.6 3.76 7.20E-08 1 1 4.6 2.91 1.10E-06 24209.7 VF21_01126-T1 VF21_01528-T1 1 1 4.5 3.75 2.10E-07 1 1 4.5 3.68 3.20E-07 23015.2 VF21_01528-T1 VF21_05889-T1 1 1 3.7 3.25 3.90E-06 1 1 3.7 3.02 9.30E-07 27458.3 VF21_05889-T1 VF21_03860-T1 1 1 2.6 2.31 1.90E-06 1 1 2.6 2.81 1.10E-06 35598.4 VF21_03860-T1 VF21_08365-T1 2 2 10.3 6.36 3.50E-11 3 3 11.1 3.31 2.90E-07 29374.1 VF21_08365-T1 VF21_09278-T1 3 3 9.4 4.66 5.00E-08 4 4 11.5 4.52 9.10E-08 3 3 8.3 4.3 2.30E-07 35269.6 VF21_09278-T1 VF21_04961-T1 3 3 4.5 5.26 1.20E-09 2 2 3.4 5.76 4.60E-10 2 2 2.8 3.03 3.10E-07 73239.5 VF21_04961-T1

Supplementary Table 4. Proteins identified in P. sp. 05NY08 secretome by LC-MS/MS

215

VE00_08172-T1 20 34 53.1 9.27 1.50E-16 24 32 41.9 9.98 7.10E-18 17 23 42.7 9.7 2.40E-17 50133 VE00_08172-T1 VE00_04293-T1 13 13 21 7.51 2.70E-13 24 32 34 10.16 3.30E-18 22 26 27.9 9.21 1.90E-16 86435.8 VE00_04293-T1 VE00_02782-T1 16 16 25.4 9.19 2.10E-16 16 18 20 7.63 1.60E-13 22 27 25.6 9.76 1.80E-17 101903.9 VE00_02782-T1 VE00_06034-T1 21 32 45.4 9.18 2.20E-16 8 8 14.4 7.62 1.70E-13 14 17 24.6 8.82 9.90E-16 72822.3 VE00_06034-T1 VE00_04993-T1 21 39 52.2 11.91 1.90E-21 19 33 41.8 11.46 1.30E-20 16 31 42.3 11.8 3.10E-21 39685.1 VE00_04993-T1 VE00_07229-T1 16 36 58.5 11.64 5.90E-21 11 27 38 11 9.30E-20 11 23 38 10.68 3.60E-19 42363.7 VE00_07229-T1 VE00_02683-T1 16 20 22.6 10.19 2.90E-18 19 22 24.9 10.95 1.10E-19 14 16 16.7 11.11 5.60E-20 118191.7 VE00_02683-T1 VE00_09062-T1 10 27 63.3 13.35 4.10E-24 13 31 67 14.54 2.60E-26 12 29 67 13.32 4.60E-24 26146.3 VE00_09062-T1 VE00_01827-T1 14 28 48.4 11.62 6.60E-21 12 21 35.7 11.87 2.20E-21 12 22 41.1 11.97 1.50E-21 44244.1 VE00_01827-T1 VE00_01457-T1 14 25 43.7 12.77 4.80E-23 14 25 43.7 14.72 1.20E-26 12 17 39.6 13.38 3.60E-24 56411.7 VE00_01457-T1 VE00_01254-T1 13 20 74.2 10.17 3.20E-18 10 13 51.3 9.8 1.50E-17 6 7 27.3 9.93 9.00E-18 29772.4 VE00_01254-T1 VE00_02947-T1 15 28 29.7 8.87 8.00E-16 5 5 8.4 6.13 9.70E-11 12 21 22.4 7.55 2.30E-13 72297.8 VE00_02947-T1 VE00_08911-T1 12 15 25.3 10.67 3.70E-19 12 12 24.1 9.12 2.70E-16 17 20 31.6 8.36 7.10E-15 68456.9 VE00_08911-T1 VE00_02282-T1 13 42 47.4 9.32 1.20E-16 13 60 37.8 12.93 2.40E-23 13 63 39 12.12 7.70E-22 34140.9 VE00_02282-T1 VE00_04173-T1 11 21 28.8 13.24 6.50E-24 12 27 29 15.15 1.90E-27 11 34 28.8 13.6 1.40E-24 45442.8 VE00_04173-T1 VE00_02104-T1 11 14 44.9 12.91 2.70E-23 11 18 40.6 12.03 1.10E-21 12 16 42.6 13.2 7.70E-24 32652 VE00_02104-T1 VE00_05454-T1 4 4 9.8 10.05 5.30E-18 13 16 26.3 11.39 1.80E-20 9 10 18.9 11.84 2.60E-21 69296.3 VE00_05454-T1 VE00_03536-T1 14 20 37 9.34 1.10E-16 10 13 24.7 9.66 2.70E-17 12 14 29.8 8.83 9.70E-16 54077.4 VE00_03536-T1 VE00_07406-T1 13 13 33.3 9.07 3.50E-16 11 14 30.5 10.47 8.70E-19 15 16 35.6 10.25 2.20E-18 51727 VE00_07406-T1 VE00_01585-T1 10 14 35.5 11.65 5.80E-21 9 13 23.1 9.5 5.60E-17 11 14 28.6 11.42 1.60E-20 47818.3 VE00_01585-T1 VE00_02047-T1 13 19 32.5 12.25 4.50E-22 11 16 28 8.89 7.30E-16 12 16 28 9.51 5.30E-17 58062.3 VE00_02047-T1 VE00_04663-T1 12 13 26 8.37 6.80E-15 5 6 8.6 7.16 1.20E-12 11 13 19.8 7.17 1.10E-12 72427.6 VE00_04663-T1 VE00_00868-T1 11 15 28.6 7.38 4.60E-13 13 19 28.6 6.29 4.90E-11 14 20 33.5 6.45 2.40E-11 42266.1 VE00_00868-T1 VE00_01460-T1 8 17 30.2 10.08 4.60E-18 6 16 21 9.31 1.20E-16 8 14 24.4 8.77 1.20E-15 39195.8 VE00_01460-T1 VE00_02820-T1 9 15 70.2 8.81 1.00E-15 8 15 70.2 10.9 1.40E-19 6 9 55.6 9.23 1.80E-16 18670.2 VE00_02820-T1 VE00_01879-T1 8 10 18.6 12.87 3.10E-23 7 10 21.6 10.98 9.80E-20 7 10 19 12.72 6.00E-23 58381.3 VE00_01879-T1 VE00_00372-T1 10 15 22.7 8.86 8.30E-16 11 20 22.9 7.71 1.10E-13 9 13 17.3 7.92 4.60E-14 48112.8 VE00_00372-T1 VE00_03106-T1 9 10 28.3 8.11 2.00E-14 10 10 36.4 7.63 1.60E-13 8 10 30.8 8.42 5.50E-15 46815.8 VE00_03106-T1 VE00_08340-T1 2 3 7.6 4.8 2.80E-08 3 5 22.1 7.22 9.10E-13 8 19 59.5 11.3 2.60E-20 24482.8 VE00_08340-T1 VE00_08904-T1 6 7 15.4 9.44 7.00E-17 7 11 19.9 11.52 1.00E-20 4 5 10.4 9.83 1.30E-17 55810.5 VE00_08904-T1 VE00_01572-T1 5 6 25.9 8.22 1.30E-14 3 3 10.2 5.04 1.00E-08 8 8 34 8.1 2.10E-14 35289.8 VE00_01572-T1 VE00_02926-T1 8 11 48.8 10.09 4.40E-18 6 7 39.4 10.97 1.00E-19 7 7 45.5 10.66 3.90E-19 25478.6 VE00_02926-T1 VE00_08234-T1 12 13 16 7.54 2.30E-13 3 3 5.1 6.49 2.10E-11 10 11 14.5 7.47 3.10E-13 86283.4 VE00_08234-T1 VE00_07666-T1 8 17 27.8 9.14 2.50E-16 8 11 21.8 8.66 2.00E-15 7 13 21.8 8.07 2.50E-14 42003.6 VE00_07666-T1 VE00_08367-T1 4 4 22.1 8.24 1.20E-14 4 4 20.2 11.21 3.80E-20 7 7 36 10.14 3.60E-18 27899.9 VE00_08367-T1 VE00_04096-T1 6 6 45.9 7.35 5.20E-13 6 6 31.5 5.89 2.70E-10 7 8 46.4 7.72 1.10E-13 19466.1 VE00_04096-T1 VE00_06468-T1 5 9 16.1 8.42 5.40E-15 6 12 23.6 11.59 7.40E-21 5 10 23.6 10.44 1.00E-18 28316 VE00_06468-T1 VE00_07478-T1 4 4 13.4 6.18 7.70E-11 7 10 21.7 7.4 4.30E-13 36558.5 VE00_07478-T1 VE00_07913-T1 7 8 21.2 7.18 1.10E-12 6 7 21.2 8.05 2.70E-14 9 9 27.2 7.23 8.90E-13 34955.7 VE00_07913-T1 VE00_03672-T1 3 3 10 6.33 4.10E-11 5 7 20.1 10.56 6.10E-19 1 1 5 11.25 3.20E-20 46487.9 VE00_03672-T1 VE00_05546-T1 6 6 27.8 7.96 3.90E-14 5 5 21.4 6.53 1.70E-11 32644.3 VE00_05546-T1 VE00_06608-T1 5 8 24.5 9.58 4.00E-17 4 6 19.9 8.53 3.40E-15 5 6 24.5 7.86 5.90E-14 50990.5 VE00_06608-T1 VE00_07980-T1 6 8 9.6 9 4.70E-16 6 7 8.2 8.25 1.10E-14 5 8 9.6 8.96 5.50E-16 63365.8 VE00_07980-T1 VE00_07423-T1 2 2 5.1 4.54 8.30E-08 3 3 8 6.62 1.20E-11 8 9 24.4 9.39 9.00E-17 36145.9 VE00_07423-T1 VE00_03479-T1 2 2 2.4 3.92 1.00E-07 3 3 3.7 4.35 1.70E-07 8 8 9.2 6.36 3.60E-11 93363 VE00_03479-T1 VE00_05626-T1 8 9 27.5 7.18 1.10E-12 1 1 3.4 4.73 3.70E-08 6 7 19.8 7.65 1.40E-13 35422.5 VE00_05626-T1 VE00_08277-T1 1 1 6.1 5.16 6.00E-09 5 7 28.6 11.95 1.60E-21 5 6 28.6 10.99 9.70E-20 28687.8 VE00_08277-T1 VE00_06392-T1 2 2 7.2 4.67 4.80E-08 5 6 23.4 8.34 7.60E-15 5 5 23.4 8.53 3.40E-15 30959.8 VE00_06392-T1 VE00_04325-T1 6 7 24.5 5.82 3.70E-10 8 8 30.7 6.56 1.50E-11 26821.9 VE00_04325-T1 VE00_03972-T1 3 3 5.8 6.14 9.10E-11 7 8 16.5 7.61 1.70E-13 4 5 9.5 9.33 1.20E-16 55432.6 VE00_03972-T1 VE00_05078-T1 3 3 4.8 7.14 1.30E-12 7 7 7.9 8.78 1.20E-15 89557.4 VE00_05078-T1 VE00_03079-T1 4 4 10.8 6.03 1.40E-10 6 6 18.9 10.21 2.60E-18 5 6 17.7 10.35 1.50E-18 44376.3 VE00_03079-T1 VE00_08102-T1 5 5 30.8 9.2 2.00E-16 2 2 13.6 7.98 2.00E-14 3 3 20.2 8.94 6.00E-16 20604.8 VE00_08102-T1 VE00_04783-T1 5 6 52.2 9.26 1.50E-16 2 2 21.2 7.17 1.20E-12 3 4 27.4 7.71 1.10E-13 11682.5 VE00_04783-T1 VE00_01534-T1 1 1 4.9 6.82 5.10E-12 3 3 18.5 8.37 6.70E-15 4 4 23.4 8.35 7.30E-15 32985.3 VE00_01534-T1 VE00_06750-T1 6 7 38.7 6.69 8.70E-12 3 3 11.3 3.94 1.40E-07 6 7 33.9 7.48 3.10E-13 19913.5 VE00_06750-T1 VE00_06685-T1 5 5 36.4 9 4.70E-16 2 2 12.1 5.68 6.50E-10 23592.3 VE00_06685-T1 VE00_00903-T1 6 6 18.2 7.19 1.00E-12 2 2 5.5 3.45 3.50E-08 47472.5 VE00_00903-T1 VE00_00170-T1 1 3 3 21.6 13.5 2.10E-24 3 3 21.6 7.52 2.60E-13 24701 VE00_00170-T1 VE00_01398-T1 3 4 8.8 6.13 9.60E-11 6 6 18.1 7.54 2.30E-13 5 6 15.2 7.61 1.70E-13 41566.8 VE00_01398-T1 VE00_05289-T1 3 5 9.4 9.96 7.80E-18 1 3 3.3 10.61 4.70E-19 1 2 3.3 10.12 4.00E-18 66593.1 VE00_05289-T1 VE00_04662-T1 5 5 13.5 6.36 3.60E-11 5 6 18 6.89 3.70E-12 4 5 11.4 6.74 7.20E-12 46713.2 VE00_04662-T1 VE00_04917-T1 2 2 2.4 3.79 5.40E-07 2 2 3.3 8.23 1.20E-14 5 5 7.7 8.99 4.80E-16 79579.4 VE00_04917-T1 VE00_04467-T1 3 5 11.9 7.37 4.90E-13 4 6 11.9 7.88 5.60E-14 4 9 11.9 9.06 3.50E-16 34598.1 VE00_04467-T1 VE00_02834-T1 3 7 14 10.01 6.10E-18 3 6 13.7 12.52 1.40E-22 3 4 7.8 7.78 8.30E-14 41022 VE00_02834-T1 VE00_03310-T1 2 2 9.7 6.27 5.30E-11 4 4 19 7.88 5.40E-14 2 2 9.3 7.71 1.20E-13 29175.2 VE00_03310-T1 VE00_01905-T1 6 6 11.3 5.53 1.20E-09 2 3 3.2 5.46 1.70E-09 2 2 2.7 4.4 1.20E-08 66598.6 VE00_01905-T1 VE00_02888-T1 5 5 41.9 6.6 1.30E-11 1 1 5.2 1.75 8.40E-06 3 3 21.3 6.73 7.40E-12 16835.2 VE00_02888-T1 VE00_04545-T1 4 4 10.5 6.74 7.00E-12 5 5 15.5 6.72 7.90E-12 50021.3 VE00_04545-T1 VE00_06946-T1 2 2 2.8 4.05 1.90E-07 3 3 5.1 5.75 4.80E-10 6 7 10.3 5.14 6.70E-09 73975.8 VE00_06946-T1 VE00_04695-T1 2 2 5 6.3 4.60E-11 5 5 11.2 7.48 3.10E-13 1 1 1.6 2.6 3.10E-06 58918.5 VE00_04695-T1 VE00_01934-T1 1 1 2.6 3.52 5.90E-08 2 6 7 11.8 6.21 6.80E-11 58896.7 VE00_01934-T1 VE00_00248-T1 4 4 16.1 5.88 2.80E-10 5 5 14.5 6.58 1.40E-11 2 2 7.9 5.72 5.50E-10 35384.6 VE00_00248-T1 VE00_03453-T1 4 4 47.7 7.35 5.30E-13 1 1 15.9 9.58 4.00E-17 2 2 29 6.76 6.60E-12 11897.7 VE00_03453-T1 VE00_06008-T1 2 2 2 6.9 3.43 1.20E-06 4 4 25.1 9.4 8.50E-17 25679.4 VE00_06008-T1 VE00_04842-T1 2 2 20.7 4.39 2.50E-08 2 2 18 4.3 2.90E-08 5 5 30.6 5.29 3.50E-09 11862.5 VE00_04842-T1 VE00_03845-T1 1 1 2.3 4.64 5.50E-08 4 4 8.8 6.99 2.50E-12 4 4 10.3 6.88 4.00E-12 59774 VE00_03845-T1 VE00_01861-T1 3 3 33.3 4.34 2.00E-07 5 5 65.8 5.73 5.30E-10 3 3 39.5 5.75 4.80E-10 12483.1 VE00_01861-T1 VE00_03756-T1 2 2 7.9 4.56 7.60E-08 1 6 6 24.1 5.49 1.50E-09 32802.7 VE00_03756-T1 VE00_00692-T1 4 7 20.7 7.2 9.80E-13 4 4 17.5 5.77 4.40E-10 4 4 17.5 5.21 4.90E-09 24290.4 VE00_00692-T1 VE00_07577-T1 2 2 5.7 5.14 6.50E-09 5 5 13.2 4.68 4.70E-08 45084.4 VE00_07577-T1 VE00_00961-T1 3 3 10.3 6.38 3.20E-11 4 4 16.4 6.65 1.00E-11 33163.3 VE00_00961-T1 VE00_00658-T1 1 1 5.4 3.07 1.60E-07 4 4 16.2 7.07 1.70E-12 34513.3 VE00_00658-T1 VE00_00659-T1 2 2 25 11.7 4.60E-21 2 2 25.8 12.44 2.00E-22 14026.1 VE00_00659-T1 VE00_02027-T1 3 4 9.1 8.04 2.80E-14 2 2 5.8 6.33 4.10E-11 49940.7 VE00_02027-T1 VE00_08790-T1 1 1 12.5 5.73 2.20E-10 2 3 29.5 9.26 1.50E-16 3 5 39.8 9.13 2.60E-16 9782.1 VE00_08790-T1 VE00_01618-T1 3 3 21.2 8.55 3.10E-15 1 2 7.4 7.02 2.10E-12 25361.4 VE00_01618-T1 VE00_03214-T1 5 5 26.4 5.19 2.00E-09 3 3 13.9 4.86 1.70E-08 22642.1 VE00_03214-T1 VE00_08622-T1 3 4 21.4 7.18 1.10E-12 2 3 7.4 9.29 1.40E-16 2 2 7.4 8.52 3.50E-15 43653.3 VE00_08622-T1 VE00_08741-T1 1 1 3.8 3.51 1.50E-07 2 2 19.5 11.15 4.90E-20 28558.1 VE00_08741-T1 VE00_03513-T1 2 2 2.4 4.7 1.40E-08 5 5 6.8 4.31 6.60E-08 2 2 2.3 4.05 1.10E-07 79142.7 VE00_03513-T1 VE00_02796-T1 1 1 2.4 4.6 6.40E-08 2 2 4 5.43 1.90E-09 3 3 6.7 9.31 1.30E-16 59342.9 VE00_02796-T1

216

VE00_04692-T1 3 5 8.1 6.82 5.10E-12 3 5 8.1 6.41 2.90E-11 3 4 8.1 5.85 3.10E-10 30496.1 VE00_04692-T1 VE00_01819-T1 1 1 3.8 3.65 1.50E-06 1 2 6.9 9.46 6.50E-17 2 4 6.9 8.82 1.00E-15 26361.8 VE00_01819-T1 VE00_02632-T1 3 4 29.7 7.33 5.80E-13 3 3 32.4 7.23 8.70E-13 3 3 27.9 7.75 9.40E-14 11803.8 VE00_02632-T1 VE00_02272-T1 4 4 17.3 7.36 5.00E-13 1 4 6 16.9 3.23 3.00E-06 29212.4 VE00_02272-T1 VE00_06384-T1 3 3 20.4 6.48 2.20E-11 2 2 13.3 6.45 2.50E-11 1 1 4.6 3.07 1.00E-06 21410.3 VE00_06384-T1 VE00_02210-T1 1 1 7.5 2.68 8.60E-07 3 3 18.7 7.08 1.70E-12 1 1 7.5 3.57 6.50E-07 13663.9 VE00_02210-T1 VE00_04615-T1 3 3 9 7.08 1.70E-12 2 2 5.7 6.85 4.40E-12 2 2 5.7 5.75 4.80E-10 47208.1 VE00_04615-T1 VE00_03976-T1 1 1 2.6 2.46 1.70E-07 1 1 2.6 2.4 7.70E-07 4 5 11.8 5.59 9.70E-10 36609.9 VE00_03976-T1 VE00_06372-T1 2 2 9.5 5.77 4.50E-10 3 3 11 3.73 2.30E-07 4 4 15.6 5.96 2.00E-10 29445.2 VE00_06372-T1 VE00_07632-T1 1 1 2.4 5.11 7.40E-09 3 3 9.2 6.4 3.00E-11 48391.8 VE00_07632-T1 VE00_01368-T1 1 1 3 3.25 4.70E-07 3 4 12.4 6.21 6.80E-11 2 2 12.4 4.87 2.10E-08 34175.9 VE00_01368-T1 VE00_04672-T1 2 3 36.7 8.41 5.60E-15 1 1 12.8 6.22 6.50E-11 1 1 12.8 5.78 4.30E-10 10956.2 VE00_04672-T1 VE00_06632-T1 3 3 30.1 6.03 1.50E-10 2 2 24.7 4.71 4.10E-08 17397.9 VE00_06632-T1 VE00_03646-T1 2 2 11.8 6.44 2.60E-11 1 1 3.6 4.99 2.80E-09 2 2 10.1 8.38 6.50E-15 39212.7 VE00_03646-T1 VE00_03886-T1 1 1 2.8 6.5 2.00E-11 3 3 6.9 5.97 1.90E-10 2 2 4.5 5.85 3.20E-10 56057.5 VE00_03886-T1 VE00_03778-T1 2 2 17.7 8.25 1.10E-14 2 2 17.7 8.33 8.10E-15 1 1 6.6 3.85 5.80E-08 17063.1 VE00_03778-T1 VE00_05736-T1 3 2 2 7 5.35 2.70E-09 3 3 9.4 6.68 9.20E-12 49951.2 VE00_05736-T1 VE00_02315-T1 2 8 14.9 6.61 1.30E-11 2 7 14.9 6.73 7.50E-12 2 6 14.9 7.23 8.60E-13 20967.6 VE00_02315-T1 VE00_06755-T1 2 2 10.3 7.51 2.60E-13 2 2 9.2 5.47 1.60E-09 28505.8 VE00_06755-T1 VE00_08104-T1 2 2 14.3 4.8 2.80E-08 3 4 15.5 4.46 1.20E-07 16917.3 VE00_08104-T1 VE00_01381-T1 3 3 12.7 3.85 1.60E-06 2 2 8.9 3.65 3.80E-06 2 2 10.3 3.33 8.40E-06 21868 VE00_01381-T1 VE00_04608-T1 1 1 4.4 5.91 2.40E-10 2 2 7.9 5.94 2.20E-10 2 2 7.9 5.64 7.70E-10 24861.8 VE00_04608-T1 VE00_00277-T1 1 1 6.4 4.94 2.60E-09 1 1 6 10.06 5.10E-18 27696.6 VE00_00277-T1 VE00_04968-T1 2 2 3 4.96 3.10E-09 1 1 1.7 4.55 6.80E-09 80652.8 VE00_04968-T1 VE00_02910-T1 2 2 6.4 5.03 1.00E-08 1 2 3.6 2.16 2.60E-07 41576.3 VE00_02910-T1 VE00_08560-T1 1 1 2.1 2.6 4.70E-06 3 3 6.6 3.92 1.60E-07 3 3 5.1 3.7 1.30E-06 54079 VE00_08560-T1 VE00_01643-T1 1 1 4.3 4.06 4.80E-09 1 1 6.4 5.93 2.20E-10 2 2 9 6.43 2.70E-11 25117 VE00_01643-T1 VE00_08523-T1 2 2 5.5 3.98 5.40E-09 2 2 5.8 5.7 5.90E-10 2 44905.1 VE00_08523-T1 VE00_07218-T1 1 1 6.3 5.27 3.50E-10 1 1 6.3 8.93 6.30E-16 1 1 6.3 7.44 3.70E-13 39968.8 VE00_07218-T1 VE00_00564-T1 1 1 3.2 3.53 3.90E-06 2 2 7 6.14 9.30E-11 31019.4 VE00_00564-T1 VE00_04008-T1 2 2 12 4.85 7.60E-09 2 3 12 4.1 2.40E-08 14198.2 VE00_04008-T1 VE00_06479-T1 1 1 10.2 3.94 1.10E-06 2 2 17.6 4.18 3.90E-07 12084.8 VE00_06479-T1 VE00_02726-T1 2 2 9.6 4.93 9.00E-09 2 2 9.6 3.36 9.80E-07 1 17914 VE00_02726-T1 VE00_08258-T1 1 1 9.3 7.78 8.30E-14 1 1 9.3 6.58 2.70E-13 20458.1 VE00_08258-T1 VE00_02354-T1 2 2 21.5 4.51 1.70E-08 1 1 8.9 2.59 1.10E-06 9457.7 VE00_02354-T1 VE00_06226-T1 1 1 9.9 6.79 5.60E-12 1 1 11.8 6.75 6.80E-12 16736.9 VE00_06226-T1 VE00_04686-T1 1 1 8 6.57 1.50E-11 1 1 8 5.84 3.20E-10 1 1 8 5.99 1.80E-10 15538.6 VE00_04686-T1 VE00_04218-T1 1 1 10.1 6.49 4.50E-13 1 1 10.1 5.37 7.70E-11 1 1 10.1 2.33 1.30E-07 15884.7 VE00_04218-T1 VE00_03360-T1 1 1 1 10.7 5.72 5.50E-10 1 1 10.7 5.29 2.90E-09 16966.8 VE00_03360-T1 VE00_07106-T1 1 1 5.9 5.62 8.50E-10 1 1 5.9 5.44 1.40E-09 23150.1 VE00_07106-T1 VE00_07646-T1 1 1 4.1 5.14 6.70E-09 1 1 4.1 4.86 2.20E-08 28880.6 VE00_07646-T1 VE00_04028-T1 1 1 3.5 4.49 1.00E-07 1 1 3.5 4.7 4.20E-08 35850 VE00_04028-T1 VE00_04043-T1 1 1 7.1 4.41 1.50E-07 1 1 7.1 4.56 7.80E-08 18714.3 VE00_04043-T1 VE00_04274-T1 1 1 1.8 4.47 4.50E-09 1 1 1.9 3.63 2.20E-07 60572.2 VE00_04274-T1 VE00_01234-T1 1 1 3.6 4.45 1.20E-07 2 3 6.6 2.4 1.30E-06 36224.3 VE00_01234-T1 VE00_03370-T1 1 1 5.5 4.42 6.00E-10 1 1 8.5 2.3 1.50E-06 17953.5 VE00_03370-T1 VE00_04983-T1 1 1 2.7 1.75 8.80E-06 2 2 5.7 2.15 4.90E-06 33536.5 VE00_04983-T1 VE00_07252-T1 1 1 3.2 3.93 1.10E-06 1 1 3.2 4.13 1.90E-07 37139.6 VE00_07252-T1 VE00_03517-T1 1 1 6 3.73 2.70E-06 1 1 6 3.42 9.90E-06 1 18225.8 VE00_03517-T1 VE00_04638-T1 1 1 5.3 3.66 1.10E-06 1 1 5.3 3.42 4.00E-07 18411.9 VE00_04638-T1 VE00_02780-T1 1 1 2.7 3.6 6.10E-08 1 1 2.7 3.56 4.50E-08 31486.8 VE00_02780-T1 VE00_05866-T1 1 1 7.3 3.35 1.60E-07 1 1 6.3 1.31 8.40E-06 20305.9 VE00_05866-T1 VE00_06620-T1 1 1 1.8 2.92 3.30E-06 1 1 1.8 2.45 1.50E-06 61046.5 VE00_06620-T1 VE00_02941-T1 1 1 3.9 2.35 5.60E-06 1 1 3.9 2.64 7.50E-07 25086.4 VE00_02941-T1 VE00_04507-T1 2 2 7.8 3.92 1.20E-06 1 1 3.7 2.84 1.80E-06 4 4 10.7 3.89 1.10E-07 30224 VE00_04507-T1 VE00_05141-T1 2 2 5.9 4.13 4.70E-07 4 4 11.2 3.82 1.80E-06 3 3 9.1 4.04 7.10E-07 35269.6 VE00_05141-T1

Supplementary Table 5. Proteins identified in P. sp. WSF3629 secretome by LC-MS/MS

217

Gene ID cluster_GSZ cluster_RM1sum cluster_size cluster_desccount RM2 val_avg jac_avg description GMDG_01891T0 1390.739997 64.942831 99 2890 1.148906315 0.004925 2.25E-06 1,3-beta-glucanosyltransferase GMDG_03130T0 1399.06024 66.97561108 99 2874 1.149624096 0 0 1,3-beta-glucanosyltransferase GMDG_08350T0 594.4636007 74.97930526 97 15657 1.072372513 0.461433 0.001372 6-phosphogluconate dehydrogenase, decarboxylating GMDG_01434T0 1154.148545 21.43359374 34 1453 1.076392031 0.046665 6.37E-06 Acyl-CoA-binding protein, ACBP GMDG_05500T0 873.7705951 70.2435746 97 7240 1.195373141 0.482578 0.004927 Arginase GMDG_03638T0 787.5580713 23.66871536 43 3959 0.926469428 0.040463 3.90E-05 Aspartic-type endopeptidase ctsD GMDG_05333T0 477.8959427 49.89131137 78 19478 0.787827945 0.012584 2.96E-07 Beta-glucosidase GMDG_08104T0 3.06E-14 0.549355761 1 5 0.592937831 0.005543 1.98E-06 Carbohydrate-binding WSC domain protein GMDG_00646T0 456.9413892 62.13670916 91 24753 0.846955722 0.139951 0.000257 Carboxylic ester hydrolase GMDG_00651T0 479.3071992 63.25178538 97 23875 0.863973257 0.155178 0.000308 Carboxylic ester hydrolase GMDG_01515T0 884.812262 69.08824393 99 7176 0.946559175 0.00497 2.33E-09 Catalase-peroxidase GMDG_05222T0 646.2944245 2.287793764 4 547 0.861663502 0.0248 9.06E-05 CFEM domain-containing protein GMDG_03116T0 1023.843504 23.8683599 40 2178 1.026462661 0.050801 4.32E-05 Copper radical oxidase GMDG_01066T0 196.4129064 10.26641263 17 25154 0.672843513 0.008072 5.84E-06 Copper-dependent polysaccharide monooxygenase GMDG_05832T0 219.669774 13.99780545 23 27170 0.717584106 0.074935 1.92E-05 Cupin GMDG_06417T0 207.6713041 16.54769695 26 34132 0.683249837 0.01921 3.94E-06 Cuticle-degrading serine protease GMDG_08491T0 708.2441746 19.87605419 32 3616 0.884128864 0.020436 9.37E-06 Cuticle-degrading serine protease GMDG_07037T0 320.9912226 26.33737298 46 25475 0.762935355 0.084032 9.02E-05 Cu-Zn superoxide dismutase GMDG_07199T0 2124.715785 25.23816827 40 506 1.449510162 0.018151 0.000199 Disintegrin and metalloproteinase domain-containing protein B GMDG_06914T0 6168.099133 5.0206981 8 12 3.062550218 0.01002 3.63E-06 DNA damage-responsive protein 48 GMDG_06420T0 815.3978404 17.96307013 30 2566 0.92270347 0.012344 8.93E-05 Eukaryotic aspartyl protease GMDG_08378T0 903.3424569 1.767159049 3 210 0.952318458 0.001851 2.86E-05 Expression library immunization antigen 1 GMDG_03914T0 590.1032952 23.71572238 40 6560 0.831069537 0.009486 2.67E-05 Extracellular cell wall glucanase GMDG_07064T0 992.4768861 1.157273561 2 116 0.989837282 0.005371 5.80E-06 Extracellular membrane protein, 8-cysteine region, CFEM GMDG_08552T0 689.4526088 22.48532884 37 4438 0.875379293 0.01811 1.28E-06 FAD-binding domain and SignalP-predicted secretion signal GMDG_07491T0 483.9601236 25.47318794 39 9499 0.799376605 0.029797 2.25E-06 FAD-dependent oxygenase GMDG_03077T0 182.5855993 19.18237894 31 53065 0.676342389 0.025092 0.000482 FAD-dependent pyridine nucleotide-disulphide oxidoreductase GMDG_04925T0 368.8115165 64.7698064 95 39715 0.857802241 0.226928 0.000277 Fkbp-type peptidyl-prolyl cis-trans isomerase GMDG_07208T0 475.2596935 40.20797631 60 15070 0.785316789 0.009836 1.07E-07 Gamma-glutamyltranspeptidase GMDG_04446T0 913.7930264 26.28814938 39 2667 0.973698624 0.0343 0.000126 Glucan-beta-glucosidase GMDG_07699T0 215.6529287 42.80001057 64 78244 0.698362681 0.0417 1.60E-05 Glucan-beta-glucosidase GMDG_05916T0 1232.613017 58.12749738 87 3261 1.283738341 0.378421 0.006497 Glucoamylase GMDG_03414T0 3652.358211 22.90443342 32 137 2.070239613 0.036246 0.004303 Glucose-repressible protein Grg1 GMDG_08145T0 219.4580798 19.48564876 31 36743 0.692044538 0.026908 7.23E-06 Glutamyl-trna amidotransferase subunit a GMDG_01630T0 265.2280703 26.64031415 45 36449 0.710462843 0.026976 0.00371 Glycoside hydrolase GMDG_07937T0 314.218873 32.71682622 51 29491 0.729164625 0.025424 0.000112 Glycoside hydrolase GMDG_07779T0 845.1042859 36.04952648 58 4637 0.947374012 0.036403 0.001923 Glycoside hydrolase subgroup catalytic core GMDG_02704T0 3609.205782 7.679543586 13 57 2.045080819 0.021501 0.000149 Gpi-anchored cell wall organization protein ecm33 GMDG_02357T0 423.2239586 35.40367746 64 20299 0.822756927 0.11974 0.000253 Heme-dependent peroxidase GMDG_02429T0 1609.912103 1.155451415 2 44 1.233964841 0 0 IgE-binding protein GMDG_04735T0 521.36815 44.41405512 62 13005 0.874066471 0.142481 0.00021 Inorganic pyrophosphatase GMDG_02454T0 2609.902506 29.40919672 50 419 1.648084968 0.026625 0.000629 Major allergen Asp f 2 GMDG_00372T0 270.5131282 21.05287909 34 26516 0.730486371 0.060907 1.71E-05 Malate dehydrogenase GMDG_05533T0 455.4517065 73.22715252 96 26387 0.896288232 0.234158 0.000191 Malate dehydrogenase, NAD-dependent GMDG_07480T0 782.2954832 67.00305947 99 9176 0.913306401 0.0196 3.01E-06 Mannosyl-oligosaccharide alpha-1,2-mannosidase GMDG_00092T0 158.7078295 24.83110696 42 95073 0.71696438 0.119773 6.90E-05 Mono-and diacylglycerol lipase GMDG_04818T0 338.576902 26.32990253 44 21914 0.744391901 0.03577 0.000145 Murein transglycosylase GMDG_05440T0 646.7650304 13.32482179 22 3004 0.870929834 0.041927 0.000135 Murein transglycosylase GMDG_08409T0 284.8870959 2.763031864 5 3518 0.705850322 0.003576 1.38E-06 Murein transglycosylase GMDG_02498T0 1394.150183 34.50881729 53 1557 1.195244463 0.089713 0.001831 Neutral alkaline non-lysosomal ceramidase GMDG_06268T0 575.2184405 71.34405707 98 16818 0.90332521 0.15705 7.68E-05 Nucleoside diphosphate kinase GMDG_04457T0 677.122454 65.38788162 100 12356 0.863298923 0.004623 4.22E-07 Pectinesterase GMDG_00903T0 653.4281885 6.87950105 12 1605 0.856142545 0.008999 8.83E-05 Peptidase inhibitor 16 GMDG_05358T0 288.7036674 72.64280519 99 67549 0.95776135 0.475996 0.000105 Peptidyl-prolyl cis-trans isomerase GMDG_06522T0 281.0325903 67.74371039 94 67759 0.941550337 0.451199 9.55E-05 Peptidyl-prolyl cis-trans isomerase B GMDG_01295T0 362.2487305 60.82857271 92 39581 0.848424099 0.214188 0.000256 Peptidylprolyl isomerase GMDG_06638T0 535.3791238 71.72063689 96 19092 1.053524344 0.470463 0.001365 Peroxisomal catalase GMDG_00384T0 523.3439067 74.39896985 99 20613 0.925468967 0.237979 0.000123 Phosphoglycerate kinase GMDG_01408T0 616.2012414 54.87436318 71 10676 0.888283745 0.097742 1.22E-05 p-loop containing nucleoside triphosphate hydrolase GMDG_00087T0 2736.094589 5.088099874 8 61 1.692661689 0.015517 2.71E-06 Protease propeptide/inhibitor GMDG_08270T0 1163.73334 68.20663404 100 4190 1.134032181 0.148148 0.001033 Purple acid phosphatase GMDG_04626T0 107.9503335 36.23099296 51 248756 0.644460751 0.021264 0.000541 Reductase with broad range of substrate specificity GMDG_07510T0 1824.641214 11.65517462 21 360 1.32566526 0.010959 1.35E-05 Riboflavin aldehyde-forming enzyme GMDG_00797T0 875.8497712 6.146301931 11 816 0.946476779 0.011579 7.57E-06 Ribonuclease M GMDG_01608T0 146.2198323 4.944289706 8 21363 0.658634384 0.019144 9.38E-06 Ribosome maturation protein SBDS GMDG_05452T0 711.1879929 68.57970344 100 11204 1.145138884 0.510664 0.000576 Serine carboxypeptidase GMDG_00633T0 385.4465877 27.45144305 47 17988 0.763518399 0.036489 2.62E-05 Six-hairpin glycosidase GMDG_03526T0 1857.896724 63.15577959 97 1586 1.459857161 0.238915 0.003683 Sphingomyelin phosphodiesterase GMDG_06714T0 1874.892489 34.65395314 48 780 1.366474798 0.050031 8.07E-05 Structural constituent of ribosome GMDG_05256T0 1621.97049 10.5207606 16 347 1.266254398 0.051804 0.000496 Subtilisin cleaved region like protein GMDG_03955T0 346.988975 2.413504012 4 1898 0.736845297 0.015188 1.84E-06 Translation machinery-associated protein 20 (Fragment) GMDG_08205T0 496.9524761 34.05812077 52 12019 0.803414134 0.027598 0.000309 Transmembrane transport GMDG_04989T0 782.1741066 29.71896409 48 4479 0.914612271 0.022131 0.000657 Tripeptidyl aminopeptidase GMDG_07602T0 1428.160353 14.4374968 24 672 1.207983753 0.088096 0.001445 WSC domain-containing protein (Fragment) Supplementary Table 6. Proteins in P. destructans secretome functionally annotated using 218

PANNZER

Gene ID cluster_GSZ cluster_RM1sum cluster_size cluster_desccount RM2 val_avg jac_avg description VE01_01168-T1 1404.481955 69.17292301 100 2878 1.159670592 0.014697407 0.004409222 1,3-beta-glucanosyltransferase VE01_06364-T1 1399.013729 66.84733816 99 2874 1.149605492 0 0 1,3-beta-glucanosyltransferase VE01_06727-T1 1390.761077 65.31255357 99 2890 1.14892767 0.004949423 2.26E-06 1,3-beta-glucanosyltransferase VE01_08855-T1 594.4621613 74.65779823 97 15657 1.078470406 0.472938539 0.001405796 6-phosphogluconate dehydrogenase, decarboxylating VE01_07248-T1 1196.846952 23.74801707 36 1435 1.088073513 0.036480594 8.45E-07 Acyl-CoA-binding protein, ACBP VE01_07730-T1 2.76E-13 0.507885261 1 30 0.591584009 0.002988602 2.52E-06 Adhesin protein Mad1 VE01_00003-T1 899.806114 32.44397056 52 3662 0.985944296 0.067964101 4.38E-05 Alginate lyase VE01_07492-T1 815.1109576 23.77157765 39 3343 0.940266302 0.045701172 1.49E-05 Alginate lyase VE01_00429-T1 1166.264831 2.203139351 4 168 1.057519561 0.001910653 4.92E-05 Alkali-sensitive linkage protein 1 VE01_05095-T1 597.7197255 42.95982749 65 10383 0.85503419 0.048934793 0.000542955 Alpha-glucosidase VE01_08809-T1 3119.533342 17.02245657 30 176 1.8650705 0.051428248 9.55E-06 Asparagine amidase a VE01_02707-T1 3.06E-14 0.704474119 1 8 0.591259921 0.002377206 7.50E-08 Bacterial-type extracellular deoxyribonuclease VE01_02280-T1 466.9386793 49.80713537 78 20400 0.785056354 0.0156243 1.82E-07 Beta-glucosidase A VE01_07713-T1 358.1656686 31.77335636 49 21809 0.744472762 0.021144274 1.45E-06 Beta-glucosidase G VE01_03033-T1 770.5520411 70.40716192 100 9579 0.900854834 0.004969844 1.51E-09 Beta-hexosaminidase VE01_05202-T1 486.7437426 24.29542648 40 9634 0.798361988 0.025777543 0.000119671 Bifunctional solanapyrone synthase VE01_06516-T1 411.7971707 34.07527272 55 18513 0.783502633 0.054306818 5.76E-05 Bifunctional solanapyrone synthase VE01_03427-T1 442.1929295 33.19087961 50 14603 0.829723288 0.118570545 0.000186371 Carbohydrate binding VE01_03503-T1 669.3987996 1.038744763 2 255 0.866737605 0.01693964 3.79E-06 Carbohydrate-binding module family 50 protein VE01_02532-T1 3.06E-14 0.563360071 1 5 0.592783734 0.005252257 1.88E-06 Carbohydrate-binding WSC domain protein VE01_00132-T1 227.8168839 63.03382303 92 100228 0.758053789 0.145134934 0.000276047 Carboxylic ester hydrolase VE01_00133-T1 389.6680745 49.67873614 85 31522 0.797226637 0.096899136 0.000143287 Carboxylic ester hydrolase VE01_05143-T1 462.2766364 49.08573073 82 21905 0.834855312 0.113096258 0.000182053 Carboxylic ester hydrolase VE01_00679-T1 698.1551211 72.26885113 99 11557 1.076772273 0.391512292 0.000435503 Carboxypeptidase VE01_05992-T1 884.7269891 69.20109853 99 7176 0.946499125 0.004921376 2.30E-09 Catalase-peroxidase VE01_03554-T1 311.5978692 20.76522842 38 22333 0.744073296 0.055534246 4.99E-05 Cell surface Cu-only superoxide dismutase 5 VE01_06755-T1 2930.624872 13.50784329 20 133 1.765930877 0.006924848 0.000537953 Cerato-platanin VE01_04945-T1 9.17E-11 0.587832697 1 547 0.59439099 0.0082846 7.57E-06 CFEM domain-containing protein VE01_05856-T1 2.40E-13 0.509968175 1 28 0.591041361 0.00196483 7.59E-08 Chitin recognition protein VE01_03504-T1 305.4270044 9.480642758 16 9735 0.717158906 0.009411461 1.49E-06 Chitin-binding type 1 VE01_08982-T1 495.4844934 30.6694567 48 11165 0.807777563 0.036948678 4.83E-05 Choline dehydrogenase mitochondrial VE01_00710-T1 1015.441638 25.26987669 41 2271 1.019065855 0.043181057 0.000161989 Common central domain of tyrosinase VE01_05132-T1 196.4256435 10.28060629 17 25155 0.672964725 0.008291016 1.15E-05 Copper-dependent polysaccharide monooxygenase VE01_03196-T1 196.5410518 15.39079037 24 35218 0.682480178 0.026157979 1.43E-06 Cuticle-degrading serine protease VE01_06111-T1 1110.551558 13.91499514 23 1065 1.037923434 0.006985482 2.53E-05 Cysteine, histidine-dependent amidohydrolase/peptidase VE01_07751-T1 1183.442118 17.86653345 27 1095 1.068422949 0.009519816 2.99E-05 Cysteine, histidine-dependent amidohydrolase/peptidase VE01_02923-T1 307.2952859 21.10924415 37 22350 0.753076558 0.075768184 6.53E-05 Cytosolic cu zn superoxide dismutase VE01_08339-T1 928.5206615 19.9707916 32 2119 0.979396085 0.033936113 8.40E-05 Di-copper centre-containing VE01_05013-T1 354.1590044 44.7353855 60 27245 0.804043916 0.13656646 4.55E-06 Electron carrier VE01_07152-T1 1962.728062 38.02158712 61 904 1.391369309 0.030704611 0.000232003 Endo-beta-glucanase VE01_07383-T1 1036.753336 34.10646722 52 2762 1.020323198 0.029474901 8.28E-06 Endo-beta--glucanase VE01_04627-T1 289.576214 7.937815413 14 9532 0.727440477 0.040773271 7.89E-06 Endoglucanase VE01_02953-T1 463.9228852 58.01735049 75 19888 0.862285599 0.163602932 0.000344544 Eukaryotic translation initiation factor 3 subunit J VE01_06806-T1 222.1846 38.02336359 55 63557 0.713291773 0.064938472 2.71E-05 Eukaryotic translation initiation factor subunit VE01_07926-T1 360.1973191 24.02213716 39 17160 0.75294228 0.035590951 7.41E-06 Eukaryotic translation initiation factor subunit VE01_04855-T1 426.9793855 33.64234584 53 16598 0.779015555 0.034384385 3.82E-06 Exo-beta-D-glucosaminidase VE01_02715-T1 1560.974359 5.22627786 9 211 1.217644829 0.006123681 0.000476751 Expression library immunization antigen 1 VE01_05800-T1 1057.28578 37.06787072 60 3063 1.027303088 0.027144232 0.00011665 Extracellular cell wall glucanase VE01_08061-T1 569.6416452 22.58703535 38 6686 0.823487011 0.010622459 2.25E-05 Extracellular cell wall glucanase VE01_02884-T1 269.8644657 3.92413854 7 5481 0.700676853 0.005152954 5.21E-08 FAD linked oxidase domain-containing protein VE01_06488-T1 110.9410087 16.65942832 29 134261 0.644206947 0.018548171 6.69E-07 FAD-dependent oxygenase VE01_05385-T1 182.5854779 19.4106589 31 53065 0.677320045 0.026934718 0.000522656 FAD-dependent pyridine nucleotide-disulphide oxidoreductase VE01_07389-T1 226.448338 26.85297878 44 48799 0.715941984 0.06671724 0.000125603 FAD-dependent pyridine nucleotide-disulphide oxidoreductase VE01_01353-T1 369.1446149 67.1199531 95 39715 0.821226451 0.157669351 0.000192486 Fkbp-type peptidyl-prolyl cis-trans isomerase VE01_07945-T1 729.4187061 74.34879653 98 10510 1.01097351 0.24377937 0.000148064 Formate dehydrogenase VE01_07469-T1 669.2963113 4.088443835 7 892 0.859534392 0.003425114 2.79E-05 Fruiting body protein SC7 VE01_05051-T1 579.8992271 42.72009031 62 10529 0.874927319 0.09993264 0.000166417 Fungal type ribonuclease VE01_00252-T1 708.325775 63.88591826 98 11070 1.071423291 0.373698035 0.001651135 Glucanase VE01_01811-T1 911.7043994 25.72901069 39 2679 0.974033712 0.036508242 0.00012918 Glucan-beta-glucosidase VE01_02196-T1 217.3227364 43.39011235 65 78253 0.69718301 0.038213497 3.81E-05 Glucan-beta-glucosidase VE01_04418-T1 166.1413708 14.83349695 22 45491 0.667413484 0.02067335 3.02E-06 Glutamyl-tRNA amidotransferase subunit A VE01_02284-T1 350.81817 21.1758327 36 16660 0.779940132 0.093056012 0.014658865 Glycoside hydrolase VE01_05479-T1 266.5515015 26.53983548 45 36089 0.713442619 0.031519688 0.005829187 Glycoside hydrolase VE01_06361-T1 307.9656286 22.78657532 40 24062 0.725680381 0.023572049 4.72E-05 Glycoside hydrolase VE01_07963-T1 313.238673 30.19796543 47 27348 0.726458509 0.02105902 8.80E-05 Glycoside hydrolase VE01_00639-T1 1425.073343 36.61286349 59 1659 1.182365169 0.042041993 0.002678767 Glycoside hydrolase subgroup catalytic core VE01_05195-T1 1360.462878 16.16611696 27 833 1.16036124 0.049362797 0.000690324 Glyoxal oxidase VE01_08297-T1 3510.147119 6.497364017 11 51 2.004672812 0.02001952 0.00018096 Gpi-anchored cell wall organization protein ecm33 VE01_06376-T1 1028.286008 26.78843131 44 2375 1.029271053 0.052747185 3.20E-05 Gram-negative bacteria-binding protein 1 VE01_01925-T1 1293.024782 48.86906295 61 2084 1.146879139 0.074845723 4.96E-05 Guanine nucleotide-binding protein beta subunit VE01_00313-T1 225.2007588 21.95734703 28 31520 0.695894193 0.0298372 8.68E-06 Heat shock protein 70 VE01_03394-T1 2525.204418 14.51089072 22 197 1.610105052 0.018909081 7.36E-05 Heat shock protein 90-associated VE01_09076-T1 886.5160295 3.433812958 6 435 0.991691546 0.088825259 0.000387361 Hydrophobin VE01_09010-T1 1972.381254 1.711736427 3 44 1.378952502 0 0 IgE-binding protein

219

VE01_05361-T1 442.3098811 29.43242795 46 13421 0.789091837 0.041824972 3.25E-05 Intradiol ring-cleavage dioxygenase VE01_01732-T1 1042.43043 11.16867573 18 946 1.01618042 0.017373991 1.68E-06 Isonitrile hydratase VE01_02502-T1 3779.227917 4.458970469 7 28 2.148920293 0.089099569 0.000317723 Kazal domain-containing protein VE01_06555-T1 800.6375229 21.99848758 38 3379 0.944045762 0.06375152 0.000122395 Kynurenine formamidase VE01_02778-T1 284.1515437 2.313625996 4 2827 0.720970545 0.03265998 6.89E-06 LysM domain-containing protein-like protein 6 VE01_06762-T1 286.3591073 25.46174032 43 29929 0.713478182 0.016856847 2.05E-05 Lytic polysaccharide monooxygenase VE01_02701-T1 453.1037201 72.64300521 95 26387 0.894056345 0.231719082 0.000187185 Malate dehydrogenase, NAD-dependent VE01_03332-T1 782.4590116 68.37995029 99 9173 0.918568251 0.029404737 6.77E-06 Mannosyl-oligosaccharide alpha-1,2-mannosidase VE01_08417-T1 947.3944697 26.26592584 44 2798 0.983152159 0.026748293 0.000888788 Metallopeptidase VE01_00419-T1 156.8234645 24.18251847 41 95073 0.716232995 0.11981561 6.68E-05 Mono-and diacylglycerol lipase VE01_08262-T1 2073.375064 16.26673205 21 279 1.425583551 0.011750153 0.000297196 Multifunctional chaperone VE01_01013-T1 309.5857004 3.393538238 6 3575 0.719876261 0.011398846 2.96E-05 Murein transglycosylase VE01_01505-T1 365.0494505 33.35355961 52 22278 0.751851981 0.029870817 3.34E-05 Murein transglycosylase VE01_05425-T1 348.6996413 28.79673743 48 22536 0.745516568 0.030253746 0.000111312 Murein transglycosylase VE01_06213-T1 674.7269089 14.5685751 24 3011 0.88578582 0.048852024 0.000174151 Murein transglycosylase VE01_03624-T1 2836.541002 46.46282442 76 527 1.804877876 0.151423843 0.000341915 Neutral protease 2 VE01_00516-T1 588.4980397 10.48086861 16 2636 0.834216686 0.016621129 0.000413584 Nucleic acid-binding protein VE01_09038-T1 578.0253161 72.03439265 99 16818 0.906527897 0.160973927 7.95E-05 Nucleoside diphosphate kinase VE01_05790-T1 870.9544616 29.45953842 43 3226 0.95097758 0.023765531 3.18E-06 Nucleotide-binding, alpha-beta plait VE01_01585-T1 76.81221665 5.340787761 8 77297 0.654927409 0.064502536 0.000808887 PAF acetylhydrolase VE01_02780-T1 643.0912038 33.21329346 48 6619 0.879988617 0.061791195 0.000140104 Pectate lyase catalytic VE01_05199-T1 645.9569996 33.30533813 49 6695 0.878651747 0.057107386 0.000101645 Pectate lyase catalytic VE01_05850-T1 1006.17752 2.641197754 4 225 0.994312874 0.003475211 2.00E-07 Pectin lyase fold/virulence factor VE01_06449-T1 1104.394032 21.83756367 41 1920 1.049540891 0.033546769 0.000174534 Pectin lyase fold/virulence factor VE01_01861-T1 677.1100622 65.52021333 100 12356 0.863463841 0.004943033 4.51E-07 Pectinesterase VE01_03617-T1 4252.278426 12.92082559 20 63 2.343601751 0.099415627 4.90E-06 Peptidoglycan endopeptidase RipB VE01_06509-T1 1607.021864 14.83381628 23 506 1.274181894 0.078062197 9.23E-06 Peptidoglycan endopeptidase RipB VE01_08881-T1 288.8470203 73.13313166 99 67549 0.954089474 0.468959613 0.000103548 Peptidyl-prolyl cis-trans isomerase VE01_06973-T1 284.728605 68.98530893 97 68081 0.928748343 0.424254808 9.26E-05 Peptidyl-prolyl cis-trans isomerase B VE01_06014-T1 372.4556881 66.99650573 97 39581 0.860698273 0.229641766 0.000293087 Peptidylprolyl isomerase VE01_08424-T1 231.8983073 15.48944877 26 27563 0.701991522 0.036286418 1.99E-05 Periplasmic alpha-carbonic anhydrase VE01_06819-T1 750.1301118 18.25328975 29 2943 0.897859543 0.014730808 8.51E-06 Periplasmic component of the Tol biopolymer transport system VE01_07752-T1 477.9520626 32.92963567 51 12692 0.813831836 0.061603871 4.79E-05 Phage lysozyme (Glycoside hydrolase family 24) VE01_03592-T1 418.2278381 21.46439667 35 11384 0.775363052 0.034096386 4.16E-05 Phospholipase VE01_06978-T1 345.7548617 39.02540495 60 28656 0.753559676 0.047655737 9.55E-06 Phosphorylated carbohydrates phosphatase VE01_03133-T1 140.7624786 40.44620227 62 177973 0.681118003 0.065684707 5.82E-06 Pimeloyl-ACP methyl ester carboxylesterase VE01_00423-T1 1542.514944 5.069297713 8 192 1.216832533 0.018540544 3.36E-06 Protease propeptide/inhibitor VE01_08259-T1 898.9539888 68.07624581 97 6841 0.95096091 0.002602479 1.38E-08 Protein disulfide-isomerase VE01_09050-T1 1453.138107 24.31214189 39 1055 1.203096129 0.060076244 2.38E-05 Purine nucleoside permease NUP VE01_04077-T1 1236.318398 5.639926734 9 336 1.087736003 0.006053546 1.32E-05 Putative Chloride intracellular channel protein 6 VE01_07557-T1 4140.011848 1.801152401 3 7 2.24729788 0.002438688 3.18E-05 Related to TOS1 Target of SBF VE01_04925-T1 3450.314516 68.85791692 98 468 2.092317742 0.230014421 0.014214602 Rhamnogalacturonate lyase A VE01_04998-T1 1837.564033 11.70637452 21 355 1.332091444 0.013330799 2.54E-05 Riboflavin aldehyde-forming enzyme VE01_05459-T1 171.4021942 6.716028924 11 21368 0.673717583 0.028596937 1.64E-05 Ribosome maturation protein SBDS VE01_01092-T1 477.2582515 56.7057992 74 18530 0.867614294 0.163602707 7.79E-05 S-adenosyl-L-methionine-dependent methyltransferase VE01_04878-T1 116.9368999 18.50469623 27 112515 0.65292845 0.030478637 6.32E-07 S-adenosyl-L-methionine-dependent methyltransferase VE01_02047-T1 1053.201114 32.88713409 55 2831 1.028322529 0.032033921 0.003205252 Secreted aspartic proteinase VE01_06761-T1 1.73E-09 0.61440798 1 2376 0.592251565 0.004248229 1.92E-07 SH3 domain-containing protein VE01_05710-T1 459.8877301 32.65704838 55 14803 0.793116245 0.036150617 6.63E-05 Six-hairpin glycosidase VE01_00201-T1 662.6792944 22.44054392 37 4811 0.874428335 0.036521344 1.52E-05 Soluble quino protein glucose dehydrogenase VE01_06422-T1 1461.75579 55.99600333 70 1871 1.218062374 0.081755544 0.00148101 Structural constituent of ribosome VE01_08822-T1 363.55736 35.36822029 51 22031 0.758262118 0.043073541 0.000509858 Subtilisin serine protease VE01_01196-T1 318.4237576 60.13810744 91 50673 0.914650898 0.372218076 0.000290726 Thioredoxin TrxA VE01_00397-T1 1560.13237 40.82373921 56 1314 1.259629259 0.085990044 7.94E-05 Translation elongation factor 1 beta VE01_08610-T1 823.8007326 33.06886855 53 4459 0.932641997 0.024686757 0.001886154 Tripeptidyl aminopeptidase VE01_07429-T1 2291.263695 24.24249109 31 337 1.527582559 0.039766938 3.02E-05 Tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein VE01_08127-T1 149.8430099 4.367126787 8 20341 0.662536989 0.023773133 1.25E-06 Ureidoglycolate dehydrogenase VE01_09066-T1 323.3499947 3.866791474 6 3271 0.72143999 0.003962226 6.39E-07 Zn-dependent exopeptidase Supplementary Table 7. Proteins in P. verrucosus secretome functionally annotated using PANNZER

220

Gene ID cluster_GSZ cluster_RM1sum cluster_size cluster_desccount RM2 val_avg jac_avg description VE02_00111-T1 1405.363903 65.87143257 100 2994 1.152145561 0 0 1,3-beta-glucanosyltransferase VE02_00413-T1 1395.956287 65.83055936 98 2994 1.148382515 0 0 1,3-beta-glucanosyltransferase VE02_05113-T1 737.7666416 76.86315388 100 10972 1.150133643 0.499994832 0.001486266 6-phosphogluconate dehydrogenase, decarboxylating VE02_07376-T1 2455.394369 42.12985952 60 594 1.650253656 0.14713015 0.005846475 Actin-binding, cofilin/tropomyosin type VE02_02686-T1 1176.617905 23.91983791 37 1595 1.080668283 0.037775587 2.99E-06 Acyl-CoA-binding protein ACBP VE02_00285-T1 163.5626729 4.342598462 8 17869 0.657686771 0.00426634 2.71E-05 Alkali-sensitive linkage protein 1 VE02_02081-T1 234.8193475 18.70751024 28 30351 0.691320833 0.013947549 4.46E-05 Alkyl hydroperoxide reductase/peroxiredoxin VE02_08638-T1 358.5097592 39.14993364 59 27405 0.753044922 0.037056673 4.91E-05 Aromatic compound dioxygenase VE02_03940-T1 3344.626745 19.63740712 35 187 1.954448668 0.050184455 1.04E-05 Asparagine amidase a VE02_05149-T1 1035.769323 30.15412808 55 3064 1.023457698 0.036120445 0.000306627 Aspartic-type endopeptidase ctsD VE02_08089-T1 233.8450916 24.94859777 32 34973 0.701055184 0.033050849 9.87E-06 ATP binding VE02_00826-T1 537.8880084 79.12330135 100 20650 0.871693721 0.125544109 7.00E-06 ATP synthase subunit alpha VE02_06828-T1 457.9204283 77.20357116 100 28468 0.8390945 0.12438905 6.60E-06 ATP synthase subunit beta VE02_07142-T1 807.7031807 66.96156539 95 8672 0.915715294 0.004969851 1.47E-09 Beta-hexosaminidase VE02_00509-T1 411.2069984 33.44232893 54 19083 0.782022698 0.051959914 5.72E-05 Bifunctional solanapyrone synthase VE02_00871-T1 207.187955 25.23501212 40 55609 0.695609973 0.042895567 7.00E-06 Bifunctional solanapyrone synthase VE02_07542-T1 654.7590269 1.048032277 2 279 0.852925883 0.001928814 6.87E-08 Carbohydrate-binding module family 18 protein VE02_04801-T1 2.80E-14 0.563204032 1 5 0.59186233 0.003513785 1.18E-06 Carbohydrate-binding WSC domain protein VE02_02323-T1 1036.928575 34.12293841 55 3056 1.016045576 0.021266799 0.000137126 Cell wall glucanase VE02_03721-T1 1388.635533 20.5350512 36 1116 1.153965988 0.01605761 6.21E-05 Cell wall integrity and stress response component VE02_04174-T1 202.0529243 26.79309517 44 64285 0.686911298 0.030358123 1.61E-05 Cellulose-binding gdsl lipase VE02_00004-T1 3797.11774 3.86164491 7 29 2.12439587 0.029337248 1.64E-06 Conidiation-specific protein 13 VE02_02851-T1 217.5125449 12.75890923 21 26528 0.681877781 0.009192532 3.60E-05 Copper-dependent polysaccharide monooxygenase VE02_04099-T1 337.7105344 18.56696019 29 15120 0.742683986 0.033161251 0.001215448 Cuticle-degrading serine protease VE02_00001-T1 994.0569341 12.02998797 20 1210 0.991590216 0.007484534 3.20E-05 Cysteine, histidine-dependent amidohydrolase/peptidase VE02_08573-T1 742.6424486 5.773321462 10 1084 0.889920353 0.00540173 2.28E-05 Cysteine-rich secretory protein family VE02_01018-T1 467.7019949 58.76997147 76 20757 0.864948408 0.165774967 0.000343858 Eukaryotic translation initiation factor 3 subunit J VE02_07304-T1 420.8005518 32.27043046 51 17214 0.775914076 0.033195822 3.47E-06 Exo-beta-D-glucosaminidase VE02_01345-T1 922.0389402 1.753685451 3 211 0.959771287 0.001802146 2.87E-05 Expression library immunization antigen 1 VE02_05772-T1 180.5967959 18.62615663 30 54951 0.676466355 0.02682345 0.000560401 FAD-dependent pyridine nucleotide-disulphide oxidoreductase VE02_05742-T1 1214.507433 6.834158276 12 486 1.081315943 0.010396406 0.000143701 F-box/WD repeat-containing protein pof10 VE02_06038-T1 213.8228152 42.71981175 64 83335 0.694856037 0.036465354 1.37E-05 Glucan-beta-glucosidase VE02_04837-T1 396.0030332 31.81942077 48 18296 0.781287262 0.062047951 3.17E-05 Glucose-methanol-choline oxidoreductase VE02_07527-T1 3851.603895 24.47003292 34 137 2.148050874 0.032739276 0.00287497 Glucose-repressible protein Grg1 VE02_01241-T1 263.6557909 26.46171866 45 38599 0.711663638 0.030367119 0.005337404 Glycoside hydrolase VE02_02941-T1 310.5932444 29.07024281 46 28499 0.727552962 0.025121403 6.60E-05 Glycoside hydrolase VE02_05425-T1 841.8699744 39.27604255 64 5397 0.950465485 0.044630926 0.003155239 Glycoside hydrolase subgroup catalytic core VE02_04151-T1 550.2814391 23.35259198 39 7698 0.816436366 0.011930619 2.81E-05 Glycosylphosphatidylinositol-glucanosyltransferase VE02_07822-T1 1352.393305 17.43568658 29 948 1.180547965 0.093503802 0.001681378 Glyoxal oxidase VE02_02185-T1 4051.465913 8.244644348 14 51 2.224043702 0.02537728 0.000368944 GPI-anchored cell wall organization protein ecm33 VE02_00402-T1 982.9903121 25.44799567 42 2595 1.009587957 0.049794756 3.06E-05 Gram-negative bacteria-binding protein 1 VE02_04269-T1 415.8008664 35.5108429 64 22028 0.921904578 0.312414879 0.000217281 Heme-dependent peroxidase VE02_08921-T1 918.897254 3.557606107 6 424 1.022357312 0.122236616 0.000650195 Hydrophobin VE02_04065-T1 739.2652904 64.88122757 95 10336 1.13398592 0.468378467 0.001960816 Lysophospholipase VE02_03887-T1 2533.133414 26.78293263 45 419 1.618746542 0.029199669 0.000867599 Major allergen Asp f 2 VE02_05228-T1 260.7522885 19.15965127 31 27243 0.723223837 0.054571045 1.34E-05 Malate dehydrogenase VE02_03433-T1 108.7805294 2.177978783 4 20207 0.643227713 0.018331112 5.89E-07 Malate dehydrogenase VE02_01358-T1 468.6883978 76.10628272 100 27174 0.90932616 0.248767452 0.000202558 Malate dehydrogenase, NAD-dependent VE02_06386-T1 2723.960613 9.432565771 17 137 1.690749118 0.021065702 2.55E-06 Microtubule-actin cross-linking factor 1 VE02_00100-T1 626.908531 13.30764607 22 3347 0.860095254 0.036471158 0.000106389 Murein transglycosylase VE02_01208-T1 849.8605635 26.40581819 44 3642 0.945879283 0.030061689 0.000118118 Murein transglycosylase VE02_01490-T1 545.7494036 11.28304036 20 4015 0.832230884 0.045136342 0.000443079 Murein transglycosylase VE02_02804-T1 205.2346795 32.3813916 46 65226 0.69559129 0.044334436 8.36E-06 NADP-dependent oxidoreductase domain VE02_08168-T1 576.5284791 71.2073986 98 17513 0.905899351 0.160917844 7.51E-05 Nucleoside diphosphate kinase VE02_07523-T1 641.5606271 31.94465104 47 6818 0.876576868 0.056510463 0.00010361 Pectate lyase catalytic VE02_06960-T1 293.2659994 73.75152392 100 69287 0.959653929 0.476123608 0.000100816 Peptidyl-prolyl cis-trans isomerase VE02_00693-T1 375.6797146 69.94935722 100 42237 0.853039908 0.212759758 0.000267526 Peptidylprolyl isomerase VE02_01744-T1 528.5510798 75.47981306 100 21371 0.929441327 0.24154429 0.000121076 Phosphoglycerate kinase VE02_06498-T1 343.3861079 38.36578819 59 29907 0.75160223 0.045750218 8.60E-06 Phosphorylated carbohydrates phosphatase VE02_04325-T1 1025.280494 6.311720724 11 625 1.009950831 0.018562514 2.50E-05 Pleckstrin homology VE02_00279-T1 1578.369729 5.034383785 8 192 1.230520232 0.017306185 3.10E-06 Protease propeptide/inhibitor VE02_02270-T1 2.80E-14 0.5360569 1 4 0.591170337 0.00220818 8.08E-08 Putative chitin recognition protein VE02_07717-T1 1582.264781 8.904820726 16 382 1.227169799 0.008044871 5.23E-06 Riboflavin aldehyde-forming enzyme VE02_01260-T1 144.1369731 4.907020672 8 23010 0.654628543 0.013157876 3.99E-06 Ribosome maturation protein SBDS VE02_05105-T1 481.2972205 56.06520748 73 18818 0.867124349 0.159630275 7.07E-05 S-adenosyl-L-methionine-dependent methyltransferase VE02_08838-T1 2.80E-14 0.499015536 1 2 0.591846669 0.003482348 5.12E-05 Similar to beta-1,6-glucan boisynthesis protein (Knh1) VE02_05466-T1 437.370277 30.99149045 52 16193 0.784593516 0.037064483 6.15E-05 Six-hairpin glycosidase VE02_00476-T1 316.6074791 59.23283567 89 52545 0.81558348 0.186675023 0.000136308 Thioredoxin TrxA VE02_00758-T1 135.5629038 2.526901506 5 16267 0.647230832 0.005671051 6.40E-07 Unsaturated rhamnogalacturonyl hydrolase YteR Supplementary Table 8. Proteins in P. sp. 03VT05 secretome functionally annotated using PANNZER

221

Gene ID cluster_GSZ cluster_RM1sum cluster_size cluster_desccount RM2 val_avg jac_avg description VF21_02425-T1 1405.164889 65.91731614 100 2994 1.152065955 0 0 1,3-beta-glucanosyltransferase VF21_04846-T1 1400.853089 65.1877252 99 2995 1.152858765 0.004732198 0.00047322 1,3-beta-glucanosyltransferase VF21_08415-T1 1395.663947 66.14197909 98 2994 1.148265579 0 0 1,3-beta-glucanosyltransferase VF21_08106-T1 1387.444398 67.28301105 97 2997 1.150035064 0.009470607 0.001894121 1,3-beta-glucanosyltransferase VF21_05889-T1 1853.6737 77.21316812 99 1721 1.415151152 0.157878813 0.000295031 40S ribosomal protein S6-B VF21_07707-T1 2143.917638 52.03384541 68 884 1.484630676 0.06993123 3.45E-06 5-methyltetrahydropteroyltriglutamate--homocyste ine S-methyltransferase VF21_03303-T1 737.7482586 76.87352551 100 10972 1.147502262 0.495044391 0.001471551 6-phosphogluconate dehydrogenase, decarboxylating VF21_00775-T1 2455.394369 42.12985952 60 594 1.650253656 0.14713015 0.005846475 Actin-binding, cofilin/tropomyosin type VF21_05024-T1 1176.617905 23.91983791 37 1595 1.080668283 0.037775587 2.99E-06 Acyl-CoA-binding protein ACBP VF21_02354-T1 683.24925 78.59659853 100 12800 1.128372923 0.499994425 0.003808901 Adenosylhomocysteinase VF21_07211-T1 234.7202793 38.64714705 52 56322 0.734027914 0.094601854 4.10E-05 Aldehyde dehydrogenase VF21_05245-T1 193.5316813 29.49382098 39 62178 0.683881051 0.031042705 0.000787222 Alkyl hydroperoxide reductase subunit C/ Thiol specific antioxidant VF21_07604-T1 230.5815253 18.00440136 27 30351 0.688797749 0.012386237 2.17E-05 Alkyl hydroperoxide reductase/peroxiredoxin VF21_00789-T1 489.8138609 49.29405798 77 18868 0.789150272 0.006084369 6.07E-07 Alpha-amylase B VF21_09729-T1 345.0196108 38.85894247 65 32405 0.771537464 0.082125552 0.000153876 Alpha-galactosidase VF21_01961-T1 3217.229258 17.54599118 31 179 1.906102321 0.055113997 9.97E-06 Asparagine amidase a VF21_07309-T1 988.1997456 26.32926995 48 2938 1.001515295 0.030629873 7.82E-05 Aspartic-type endopeptidase ctsD VF21_07200-T1 537.8880084 79.12330135 100 20650 0.871693721 0.125544109 7.00E-06 ATP synthase subunit alpha VF21_00875-T1 457.9140986 77.2159145 100 28468 0.839423392 0.125014376 6.64E-06 ATP synthase subunit beta VF21_07835-T1 807.6362846 67.02066861 95 8672 0.915688529 0.00496984 1.47E-09 Beta-hexosaminidase VF21_04199-T1 417.2184432 29.09847526 47 16138 0.78163915 0.046698793 7.06E-05 Bifunctional solanapyrone synthase VF21_08224-T1 661.3452275 53.11310206 68 9289 0.998356259 0.271324808 0.000800991 Calmodulin A VF21_02391-T1 282.6985689 15.43035796 28 20931 0.711570707 0.01602006 3.24E-05 Carbohydrate metabolic process VF21_09012-T1 654.7477566 1.045872108 2 279 0.852941483 0.001966753 7.01E-08 Carbohydrate-binding module family 18 protein VF21_05201-T1 2.80E-14 0.543238926 1 5 0.592592192 0.004890865 1.64E-06 Carbohydrate-binding WSC domain protein VF21_01127-T1 486.8030706 66.76865234 98 24510 0.867964238 0.157050635 0.000308664 Carboxylic ester hydrolase VF21_01276-T1 153.2337297 25.12323344 41 104220 0.687042527 0.067447957 8.09E-05 Carboxymethylenebutenolidase VF21_05632-T1 706.5943199 73.13686397 100 11931 1.080767869 0.392682653 0.00041672 Carboxypeptidase VF21_09781-T1 896.2274144 70.16217539 100 7399 0.951048891 0.004826275 2.17E-09 Catalase-peroxidase VF21_06200-T1 1837.010201 1.836342009 3 52 1.326763473 0.003696956 2.98E-07 Cell wall glycosyl hydrolase VF21_04683-T1 206.8948127 11.56417166 19 26528 0.67665624 0.007353883 3.78E-05 Copper-dependent polysaccharide monooxygenase VF21_02018-T1 1610.58557 14.51131363 25 576 1.262526437 0.053380659 2.30E-05 Cupredoxin VF21_01100-T1 916.4495236 10.22294636 17 1210 0.95956017 0.005623099 5.90E-06 Cysteine, histidine-dependent amidohydrolase/peptidase VF21_07405-T1 3015.753207 26.89178842 42 276 1.807727234 0.021558329 1.84E-06 Cytochrome c oxidase copper chaperone VF21_06320-T1 2379.458334 73.57673163 99 1044 1.603751112 0.11692012 5.74E-06 Cytochrome c oxidase subunit VF21_09306-T1 507.7690524 43.29132674 58 13421 0.860452652 0.127059621 0.000171615 Cytoplasmic inorganic pyrophosphatase VF21_03397-T1 560.195094 38.69577595 49 9330 0.840924507 0.050651336 6.31E-05 Cytoplasmic ribosomal protein 10 VF21_04909-T1 335.4619992 25.16407451 44 23353 0.766545695 0.079923268 7.82E-05 Cytosolic cu zn superoxide dismutase VF21_00410-T1 957.3025533 21.12818467 34 2217 0.997205474 0.045815028 0.000124411 Di-copper centre-containing VF21_08964-T1 291.635464 37.49050546 51 35832 0.730826276 0.045606377 3.55E-05 DNA-(Apurinic or apyrimidinic site) lyase VF21_02532-T1 899.0523763 16.15600609 26 1923 0.964175845 0.027457183 0.000129395 EF-hand domain pair VF21_01342-T1 319.8190849 79.07130903 100 58363 0.815432123 0.183969127 4.26E-05 Elongation factor 1-alpha VF21_02960-T1 1060.068731 31.30629926 45 2393 1.034746378 0.039092128 2.92E-06 Elongation factor EF-1 gamma subunit VF21_03726-T1 467.7019949 58.76997147 76 20757 0.864948408 0.165774967 0.000343858 Eukaryotic translation initiation factor 3 subunit J VF21_02883-T1 299.0770128 32.92180462 52 34735 0.727526838 0.03376596 3.70E-06 Exo-beta-D-glucosaminidase VF21_06967-T1 922.1178391 1.740737767 3 211 0.959794867 0.001787102 2.84E-05 Expression library immunization antigen 1 VF21_05493-T1 1030.778952 39.35592188 64 3599 1.016203473 0.026207349 9.99E-05 Extracellular cell wall glucanase VF21_06874-T1 600.7967893 24.17666658 42 6956 0.836998338 0.01260171 3.58E-05 Extracellular cell wall glucanase VF21_07458-T1 672.986984 18.16165104 29 3826 0.866395938 0.013557807 0.000775311 Extracellular exo-inulinase inuE VF21_00670-T1 492.4118632 25.97732319 39 9606 0.803901096 0.031955288 2.42E-06 FAD-dependent oxygenase VF21_05813-T1 292.387584 24.57874173 40 27858 0.741059412 0.064344913 7.87E-05 FAD-dependent pyridine nucleotide-disulphide oxidoreductase VF21_06727-T1 183.577983 19.28852771 31 54951 0.678345171 0.028119143 0.000541596 FAD-dependent pyridine nucleotide-disulphide oxidoreductase VF21_05371-T1 791.9724106 34.73948146 56 5329 0.954153975 0.08936302 0.000130531 Flavin adenine dinucleotide binding VF21_04244-T1 727.8870321 75.9152836 100 11275 1.008560556 0.24038295 0.000139004 Formate dehydrogenase VF21_02295-T1 458.7477813 60.17606791 79 22423 0.834472171 0.115042429 2.86E-05 Fructose-bisphosphate aldolase VF21_09775-T1 462.0866787 5.209514597 9 2520 0.777080626 0.004236741 2.41E-05 Fruiting body protein SC7 VF21_06946-T1 1054.944742 28.35202625 35 1880 1.06064112 0.091815964 3.81E-05 Gamma-actin act-Penicillium chrysogenum VF21_04743-T1 891.067103 25.78446751 39 2936 0.964124856 0.033390022 6.52E-05 Glucan-beta-glucosidase VF21_08575-T1 212.1492812 42.2035341 63 83335 0.695515818 0.038973241 1.44E-05 Glucan-beta-glucosidase VF21_05346-T1 1227.537472 58.19074257 86 3402 1.292512145 0.398797067 0.006735519 Glucoamylase VF21_06508-T1 4279.175351 29.83629415 42 137 2.318570657 0.031842421 0.001201678 Glucose-repressible protein Grg1 VF21_00879-T1 392.0155685 76.73381782 100 38843 1.001632534 0.480803457 2.37E-05 Glyceraldehyde-3-phosphate dehydrogenase VF21_05173-T1 261.2405263 26.32546804 44 38504 0.711389818 0.031700288 0.004622732 Glycoside hydrolase VF21_08412-T1 328.3761755 19.51859376 34 18834 0.732958919 0.021901652 2.86E-05 Glycoside hydrolase VF21_05104-T1 1297.029934 38.08104955 62 2203 1.133453316 0.046374045 0.00315493 Glycoside hydrolase subgroup catalytic core VF21_04730-T1 1230.292057 14.46952005 24 948 1.122772733 0.076666874 0.001123322 Glyoxal oxidase VF21_03592-T1 3548.174718 7.126832215 12 57 2.020018461 0.020274388 0.000157398 Gpi-anchored cell wall organization protein ecm33 VF21_06666-T1 959.3735254 24.3395858 40 2595 0.997719086 0.045224764 2.76E-05 Gram-negative bacteria-binding protein 1 VF21_00546-T1 1285.694436 46.18636836 59 2134 1.143498591 0.073999666 4.97E-05 Guanine nucleotide-binding protein beta subunit VF21_02286-T1 215.879654 21.19167671 27 34624 0.691528648 0.028635144 8.00E-06 Heat shock protein 70 kDa VF21_02519-T1 2359.595501 9.805787289 15 161 1.542071098 0.015533273 1.32E-05 Heat shock protein 90-associated VF21_07954-T1 1396.30946 20.7446537 29 889 1.168506213 0.037651999 0.00134347 Heat shock protein Hsp88 VF21_04800-T1 426.5803559 45.28390357 58 19047 0.7805566 0.037590821 6.61E-05 Heat shock protein sspB-Aspergillus niger VF21_02670-T1 2827.231688 14.92730105 23 172 1.727389926 0.012211566 0.001256079 Heat-stable antigen

222

VF21_04017-T1 410.2581699 33.21985872 59 20855 0.791919002 0.071333184 0.000457343 Heme-dependent peroxidase VF21_09842-T1 839.3383366 3.002764505 5 424 0.993566118 0.127961207 0.000567204 Hydrophobin VF21_05927-T1 382.6684784 27.31862524 42 17124 0.760637487 0.033009666 0.003748603 Isoamyl alcohol oxidase VF21_05119-T1 501.6926701 30.5255297 48 11402 0.806858483 0.030526972 0.000105963 Isoamyl alcohol oxidase (FAD binding domain-containing protein) VF21_06573-T1 1068.472761 11.77996682 19 995 1.02438289 0.013195556 7.02E-06 Isonitrile hydratase VF21_00060-T1 3248.971688 75.6941263 99 560 2.148062379 0.48767823 0.000212079 Ketol-acid reductoisomerase, mitochondrial VF21_03860-T1 405.3292012 28.74526876 40 14447 0.948431556 0.370361807 0.000405908 Lactoylglutathione lyase VF21_09646-T1 2533.138033 26.58391457 45 419 1.618737781 0.029179778 0.000864259 Major allergen Asp f 2 VF21_04817-T1 264.9116385 19.85816845 32 27243 0.722134617 0.049376814 1.25E-05 Malate dehydrogenase VF21_04344-T1 94.2069153 1.639817864 3 20207 0.633435896 0.010854952 2.61E-07 Malate dehydrogenase VF21_09278-T1 540.9094495 75.51768462 98 19997 0.934930886 0.242572243 0.000190868 Malate dehydrogenase VF21_06980-T1 541.3194156 75.41826878 99 20169 0.933891945 0.240302461 0.000193743 Malate dehydrogenase, NAD-dependent VF21_05242-T1 71.77259915 4.149571075 7 80615 0.621922194 0.006062535 5.17E-07 Metal dependent phosphohydrolase VF21_04961-T1 904.5632472 26.89196353 35 2558 0.966570599 0.027821097 5.95E-06 Mitochondrial matrix ATPase subunit VF21_01131-T1 252.4119891 19.04890322 30 27730 0.707976424 0.032096559 2.26E-05 Monophenol monooxygenase (Tyrosinase) VF21_08365-T1 1964.275103 13.98778994 18 279 1.381896432 0.01166161 0.00028686 Multifunctional chaperone VF21_01430-T1 585.0670126 12.90003868 23 4015 0.855476187 0.05931037 0.000744292 Murein transglycosylase VF21_00998-T1 612.5097998 12.67000498 21 3347 0.856945043 0.041393893 0.000117997 Murein transglycosylase VF21_03453-T1 358.899187 32.64274635 51 23662 0.749760656 0.0305666 3.42E-05 Murein transglycosylase VF21_07390-T1 820.1245048 24.68119129 41 3644 0.931785235 0.025912394 9.32E-05 Murein transglycosylase VF21_07332-T1 124.3419025 29.50606962 40 154293 0.657335992 0.03320601 2.28E-06 Nad dependent epimerase dehydratase VF21_02704-T1 576.5284791 71.2073986 98 17513 0.905899351 0.160917844 7.51E-05 Nucleoside diphosphate kinase VF21_05482-T1 931.9227769 30.17434234 44 3020 0.977148733 0.027131219 3.80E-06 Nucleotide-binding, alpha-beta plait VF21_09441-T1 328.9734765 49.30370666 66 36401 0.73274332 0.021020009 0.000666233 O-acetylhomoserine aminocarboxypropyltransferase VF21_08302-T1 1.44E-11 0.601657449 1 227 0.599781167 0.018454441 1.57E-05 PAN domain-containing protein VF21_06266-T1 648.2707207 32.60829695 48 6818 0.878862816 0.055759491 9.99E-05 Pectate lyase catalytic VF21_00726-T1 598.4058082 38.26821148 57 9511 0.867777688 0.072474012 0.000206922 Pectin lyase fold/virulence factor VF21_08006-T1 4350.7115 12.92174075 20 63 2.376513984 0.087224896 9.45E-06 Peptidoglycan endopeptidase RipB VF21_07115-T1 293.2692908 73.67362697 100 69287 0.959652166 0.476117799 0.000100815 Peptidyl-prolyl cis-trans isomerase VF21_00651-T1 290.4157677 70.40257295 99 69917 0.935034536 0.431823401 9.13E-05 Peptidyl-prolyl cis-trans isomerase B VF21_03128-T1 374.3920663 69.21465161 100 42293 0.870357363 0.246404532 0.000306705 Peptidylprolyl isomerase VF21_03989-T1 375.6804826 70.23435452 100 42237 0.852562415 0.211858291 0.000266392 Peptidylprolyl isomerase VF21_01222-T1 528.5545897 75.47463475 100 21371 0.929442731 0.241544291 0.000121076 Phosphoglycerate kinase VF21_00656-T1 346.2841451 39.01094746 60 29907 0.753027458 0.046252125 8.67E-06 Phosphorylated carbohydrates phosphatase VF21_08425-T1 1008.104668 7.359977962 13 764 1.004507557 0.021254718 3.45E-05 Pleckstrin homology VF21_04997-T1 627.1515885 54.07158214 70 10637 0.891270176 0.095111916 1.13E-05 p-loop containing nucleoside triphosphate hydrolase VF21_07449-T1 405.572628 59.02181489 73 26527 0.795748814 0.081994423 0.003135941 p-loop containing nucleoside triphosphate hydrolase VF21_08913-T1 1562.919455 70.90533782 99 2410 1.219416167 0.008015807 3.49E-07 Polyadenylate-binding protein, cytoplasmic and nuclear VF21_07365-T1 1541.323829 68.65046468 100 2504 1.449947992 0.458691569 0.015596449 Profilin VF21_04229-T1 1660.143831 11.03456967 17 368 1.284046066 0.056564564 0.000465725 Protease B inhibitor 2 VF21_02384-T1 1578.369729 5.034383785 8 192 1.230520232 0.017306185 3.10E-06 Protease propeptide/inhibitor VF21_08369-T1 911.2394032 71.31793175 100 7171 0.954495761 0 0 Protein disulfide-isomerase VF21_06402-T1 490.673456 74.91243874 100 24782 0.919436125 0.251254981 8.01E-05 Pyruvate kinase VF21_01825-T1 526.8103186 1.072771345 2 431 0.801386292 0.001248998 9.77E-06 Related to mixed-linked glucanase MLG1 VF21_05370-T1 1911.147838 12.23880615 22 360 1.361908307 0.014054051 2.62E-05 Riboflavin aldehyde-forming enzyme VF21_02256-T1 1516.080616 32.95133377 42 1092 1.227144095 0.057945403 3.93E-05 Ribosomal protein L11, C-terminal VF21_05151-T1 161.0654532 6.108156612 10 23034 0.661358369 0.013079437 4.29E-06 Ribosome maturation protein SBDS VF21_03375-T1 1475.942761 17.03953548 28 768 1.19568193 0.028876641 1.03E-05 Rna recognition domain-containing protein (Fragment) VF21_01513-T1 481.2972205 56.06520748 73 18818 0.867124349 0.159630275 7.07E-05 S-adenosyl-L-methionine-dependent methyltransferase VF21_07325-T1 1.87E-09 0.577382694 1 2583 0.592232403 0.004212074 1.86E-07 SH3 domain-containing protein VF21_01852-T1 2.80E-14 0.499015536 1 2 0.592316233 0.004367826 6.42E-05 Similar to beta-1,6-glucan boisynthesis protein (Knh1) VF21_01931-T1 440.5556393 31.6110806 53 16267 0.786553266 0.038358019 6.30E-05 Six-hairpin glycosidase VF21_03123-T1 462.7945047 50.08865582 64 17856 0.810004961 0.065821813 7.99E-05 Small subunit ribosomal protein S2e VF21_01192-T1 647.8350921 21.16031766 35 4985 0.868000173 0.03559586 1.65E-05 Soluble quino protein glucose dehydrogenase VF21_07283-T1 1903.144857 37.19724589 49 809 1.377150731 0.048851275 8.06E-05 Structural constituent of ribosome VF21_08146-T1 374.7288249 38.14297137 55 23411 0.764450563 0.046310408 0.000725844 Subtilisin serine protease VF21_01126-T1 476.7400979 65.49858447 99 25894 0.895884105 0.217315722 0.00053666 Superoxide dismutase VF21_04163-T1 316.5457652 59.19930741 89 52545 0.815573245 0.186702287 0.000136322 Thioredoxin TrxA VF21_02356-T1 1560.093652 39.84534586 55 1351 1.263568714 0.093453581 4.27E-05 Translation elongation factor 1 beta VF21_06108-T1 2257.861966 25.09739592 32 375 1.511867289 0.035324358 2.96E-05 Tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein VF21_02743-T1 226.9640705 13.18625182 19 22047 0.686568027 0.010910163 6.18E-07 Uracil-DNA glycosylase VF21_03790-T1 3457.102024 13.97771458 24 120 2.000247538 0.05161065 0.00265421 Woronin body major protein

Supplementary Table 9. Proteins in P. sp. 05NY08 secretome functionally annotated using PANNZER

223

Gene ID cluster_GSZ cluster_RM1sum cluster_size cluster_desccount RM2 val_avg jac_avg description VE00_01585-T1 1058.40838 1.093481316 2 102 1.013823417 0.000867551 1.32E-05 (Trans)glycosidase VE00_01457-T1 1399.454122 66.86381879 99 2874 1.149781649 0 0 1,3-beta-glucanosyltransferase VE00_00903-T1 1404.88768 69.22424985 100 2878 1.159950469 0.014916786 0.004475036 1,3-beta-glucanosyltransferase VE00_07406-T1 1391.02875 65.22250082 99 2890 1.149009022 0.0049009 2.24E-06 1,3-beta-glucanosyltransferase VE00_06620-T1 862.1231021 56.17077371 75 5755 1.10456099 0.320208565 6.05E-05 2-methylcitrate dehydratase PrpD VE00_08234-T1 2209.264588 52.86164267 69 807 1.513346082 0.07479278 3.68E-06 5-methyltetrahydropteroyltriglutamate--homocyste ine S-methyltransferase VE00_03886-T1 594.4582937 74.53626933 97 15657 1.072389384 0.461469118 0.001371704 6-phosphogluconate dehydrogenase, decarboxylating VE00_06226-T1 2480.488114 42.12985952 60 556 1.660296389 0.14713015 0.006108217 Actin-binding, cofilin/tropomyosin type VE00_03453-T1 1177.182914 23.98285259 37 1522 1.080879248 0.03774721 3.06E-06 Acyl-CoA-binding protein ACBP VE00_01234-T1 1256.908087 42.61825188 62 2240 1.147576733 0.103420409 3.41E-05 Adenosine kinase VE00_07980-T1 2.76E-13 0.579113774 1 30 0.591538824 0.002903348 2.45E-06 Adhesin protein Mad1 VE00_06008-T1 194.614363 29.44408933 39 58738 0.684515337 0.031418385 0.000892374 Alkyl hydroperoxide reductase subunit C/ Thiol specific antioxidant VE00_06632-T1 255.2676113 21.43965245 32 28039 0.700287522 0.015433323 4.08E-05 Alkyl hydroperoxide reductase/peroxiredoxin VE00_02796-T1 630.6985865 62.85757099 99 14117 0.845448573 0.005979472 9.48E-07 Alpha-galactosidase VE00_02782-T1 538.7485322 33.98748434 51 10031 0.826633759 0.03986779 0.000220887 Alpha-glucosidase VE00_00248-T1 869.3302511 69.51135171 96 7240 1.188463337 0.472897494 0.004778251 Arginase VE00_06392-T1 353.3809115 37.12346937 57 26030 0.749217805 0.033706516 4.94E-05 Aromatic compound dioxygenase VE00_04662-T1 757.7524892 22.10839171 34 3380 0.920757779 0.052181837 2.05E-05 Aromatic peroxygenase VE00_04695-T1 861.6526803 27.46322583 50 3845 0.952467748 0.033594548 7.83E-05 Aspartic-type endopeptidase ctsD VE00_06946-T1 231.7303995 23.36219074 30 31895 0.698802273 0.030396097 9.09E-06 ATP binding VE00_03845-T1 527.0675893 77.28443146 98 20132 0.866421392 0.123762671 7.04E-06 ATP synthase subunit alpha VE00_03972-T1 453.248443 77.27331494 100 27761 0.836897305 0.123769415 6.90E-06 ATP synthase subunit beta VE00_05078-T1 477.8669384 49.94248151 78 19479 0.785948616 0.009060071 1.77E-07 Beta-glucosidase VE00_08911-T1 770.5551877 70.40976545 100 9579 0.900817356 0.004896756 1.49E-09 Beta-hexosaminidase VE00_03360-T1 621.6987232 55.34906209 69 10191 0.964722657 0.237789703 0.000731273 Calmodulin A VE00_01934-T1 63.72070947 4.024316571 7 98086 0.626303347 0.020405683 2.56E-06 Carboxylic ester hydrolase VE00_04293-T1 884.9333587 69.41606886 99 7176 0.94654369 0.004849711 2.27E-09 Catalase-peroxidase VE00_00277-T1 1432.800217 18.85562365 33 918 1.171052438 0.014965438 3.35E-05 Cell wall integrity and stress response component VE00_08102-T1 1238.547831 41.380078 67 2472 1.111142708 0.048524052 0.000291388 Cellular nucleic acid-binding protein like VE00_08277-T1 272.8533378 17.48483366 27 20695 0.707265684 0.015321291 0.000203251 Chloride peroxidase VE00_07423-T1 891.3979451 5.244959678 9 645 0.955801723 0.017437291 3.90E-05 Circumsporozoite protein VE00_02683-T1 952.3162403 21.63147169 37 2329 0.99234694 0.040415322 1.62E-05 Copper radical oxidase VE00_02834-T1 207.6279334 11.46659827 19 25155 0.678070893 0.009470767 1.07E-05 Copper-dependent polysaccharide monooxygenase VE00_04608-T1 1668.666583 16.30917546 28 574 1.310784322 0.100597598 4.81E-05 Cupredoxin VE00_00692-T1 293.6995613 19.300694 34 22492 0.751703952 0.083439259 6.60E-05 Cu-Zn superoxide dismutase VE00_07478-T1 1106.057034 13.25024166 22 1027 1.03625402 0.007227709 2.60E-05 Cysteine, histidine-dependent amidohydrolase/peptidase VE00_04467-T1 668.8491698 4.6131371 8 1021 0.859140986 0.003020471 2.34E-05 Cysteine-rich secretory protein family VE00_02354-T1 2320.477481 72.74680513 98 1038 1.577746897 0.112369413 5.77E-06 Cytochrome c oxidase subunit VE00_03756-T1 526.9571641 46.77484649 63 12926 0.876771007 0.143365297 0.000226711 Cytoplasmic inorganic pyrophosphatase VE00_06755-T1 561.5835084 37.93824723 48 8689 0.840940674 0.049633812 6.75E-05 Cytoplasmic ribosomal protein 10 VE00_02820-T1 1.08E-10 0.585651785 1 594 0.594500965 0.008492325 1.63E-06 DNAse VE00_06479-T1 347.9669232 44.11860465 59 27756 0.797708811 0.129286712 4.20E-06 Electron carrier VE00_03517-T1 1756.627919 68.13250264 98 1805 1.343337142 0.095625604 0.000220203 Eukaryotic translation initiation factor eIF-5A VE00_04663-T1 215.7933787 37.34548582 54 66146 0.711919751 0.067173248 2.89E-05 Eukaryotic translation initiation factor subunit VE00_06034-T1 360.1631475 24.00344255 39 17163 0.749487795 0.02909893 5.14E-06 Eukaryotic translation initiation factor subunit VE00_01381-T1 1471.615506 4.687848938 8 211 1.181442356 0.005262304 0.000356648 Expression library immunization antigen 1 VE00_04993-T1 634.7537379 26.40625965 44 6235 0.849213197 0.010021131 2.51E-05 Extracellular cell wall glucanase VE00_04545-T1 295.9469564 45.64788179 68 44263 0.730611793 0.041919001 0.000796971 Extracellular exo-polygalacturonase VE00_03536-T1 507.3833701 28.39989395 43 9530 0.810617328 0.033328155 2.89E-06 FAD-dependent oxygenase VE00_00868-T1 182.5741616 19.3183646 31 53065 0.68456639 0.04063265 7.10E-05 FAD-dependent pyridine nucleotide-disulphide oxidoreductase VE00_05546-T1 229.0049821 27.58256971 45 48799 0.716576143 0.065984478 0.000118828 FAD-dependent pyridine nucleotide-disulphide oxidoreductase VE00_04842-T1 369.0951111 67.02081077 95 39715 0.857296527 0.225760321 0.000275605 Fkbp-type peptidyl-prolyl cis-trans isomerase VE00_03646-T1 122.2446368 15.36887031 23 87758 0.652222576 0.024996698 0.00382356 Fusarubin cluster-esterase VE00_01398-T1 1075.106298 29.97301148 37 1828 1.078248646 0.109820904 5.24E-05 Gamma-actin act-Penicillium chrysogenum VE00_02104-T1 215.6597236 42.74981092 64 78244 0.698149391 0.0412928 1.58E-05 Glucan-beta-glucosidase VE00_01905-T1 1179.97352 51.63052658 79 3215 1.228145988 0.313324697 0.004724505 Glucoamylase P VE00_03976-T1 388.10828 76.66304435 100 37855 0.982696758 0.448024494 2.32E-05 Glyceraldehyde-3-phosphate dehydrogenase VE00_01460-T1 288.7550017 20.04491141 35 23946 0.71665405 0.021040504 2.91E-05 Glycoside hydrolase VE00_01879-T1 274.1383112 28.49112134 48 36402 0.715014603 0.028842761 0.003630747 Glycoside hydrolase VE00_00372-T1 1334.537912 38.74177778 63 2020 1.146472184 0.042631926 0.003104906 Glycoside hydrolase subgroup catalytic core VE00_08172-T1 1273.266602 13.2585558 22 775 1.118639233 0.036461165 0.000408716 Glyoxal oxidase VE00_07913-T1 1293.013214 48.88865578 61 2084 1.147120778 0.075310394 4.92E-05 Guanine nucleotide-binding protein beta subunit VE00_01643-T1 2891.497806 18.89100839 29 198 1.760337955 0.025916685 0.000149479 Heat shock protein 90-associated VE00_03513-T1 1546.152226 24.23017188 34 812 1.230405591 0.041360643 0.001177977 Heat shock protein Hsp88 VE00_04917-T1 445.0773115 47.69595881 61 17579 0.789913681 0.041284672 9.40E-05 Heat shock protein sspB-Aspergillus niger VE00_02315-T1 1609.995366 1.154022468 2 44 1.233998146 0 0 IgE-binding protein VE00_07218-T1 438.3622671 28.61597089 45 13367 0.788293107 0.043297202 3.42E-05 Intradiol ring-cleavage dioxygenase VE00_05454-T1 753.5199768 67.31146626 99 9908 1.144244305 0.4769681 0.002161079 Lysophospholipase VE00_08622-T1 433.6599551 25.81343346 44 13359 0.771210699 0.014615803 1.71E-05 Lytic polysaccharide monooxygenase VE00_02282-T1 2478.722567 26.8094506 45 418 1.597960829 0.031045157 0.000893435 Major allergen Asp f 2 VE00_05141-T1 538.169109 75.48096849 98 19297 0.931979258 0.239070904 0.000201772 Malate dehydrogenase VE00_01368-T1 453.0825602 72.41213167 95 26387 0.895257901 0.234002069 0.000189033 Malate dehydrogenase, NAD-dependent VE00_06372-T1 2027.415857 14.71203123 19 264 1.406863462 0.011116349 0.000272676 Multifunctional chaperone VE00_01827-T1 870.8190131 26.45888449 44 3314 0.953743964 0.02908297 0.000119237 Murein transglycosylase

224

VE00_02047-T1 550.4143147 10.66096104 19 3582 0.824767985 0.027542383 0.000239818 Murein transglycosylase VE00_03672-T1 361.5945424 32.57518245 51 22268 0.750607539 0.030130245 3.46E-05 Murein transglycosylase VE00_06608-T1 645.9893326 13.30226926 22 3011 0.871026268 0.042694111 0.000132815 Murein transglycosylase VE00_04983-T1 125.5712663 29.43834772 40 144523 0.657483512 0.032556526 2.36E-06 Nad dependent epimerase dehydratase VE00_06384-T1 575.1144731 71.3308382 98 16818 0.902100151 0.154816695 7.57E-05 Nucleoside diphosphate kinase VE00_07632-T1 332.8918699 50.80365235 68 34989 0.734377097 0.021154412 0.00042555 O-acetylhomoserine aminocarboxypropyltransferase VE00_02027-T1 1060.925353 47.59897791 66 3342 1.076389231 0.11701466 6.60E-05 Ornithine-oxo-acid transaminase VE00_02926-T1 632.5689198 31.97219567 47 6700 0.874536787 0.059446921 0.000117558 Pectate lyase catalytic VE00_00170-T1 733.5350167 46.8629395 66 7003 0.925188108 0.078813583 0.000145115 Pectate lyase F VE00_08904-T1 677.193994 65.75742252 100 12356 0.863272181 0.004518066 4.12E-07 Pectinesterase VE00_04096-T1 288.8454785 73.13069165 99 67549 0.954088857 0.468959613 0.000103548 Peptidyl-prolyl cis-trans isomerase VE00_03214-T1 284.7633667 69.40803782 97 68081 0.932035101 0.430429953 9.40E-05 Peptidyl-prolyl cis-trans isomerase B VE00_02272-T1 193.1523199 10.52788722 18 27540 0.681176333 0.02625507 1.09E-05 Periplasmic alpha-carbonic anhydrase VE00_04325-T1 491.6869207 34.82365177 54 12712 0.816358494 0.056005425 4.25E-05 Phage lysozyme (Glycoside hydrolase family 24) VE00_03079-T1 520.7175441 73.59135654 98 20613 0.92435654 0.23786245 0.000121209 Phosphoglycerate kinase VE00_00658-T1 449.4111988 47.7662042 67 18938 0.854484951 0.159838536 0.000302382 Phosphopyruvate hydratase VE00_05626-T1 143.3588197 40.23866654 63 174462 0.684924208 0.070906703 6.36E-06 Pimeloyl-ACP methyl ester carboxylesterase VE00_08560-T1 1182.185767 8.549645134 15 612 1.073747635 0.02051446 3.32E-05 Pleckstrin homology VE00_03479-T1 607.4611467 53.28141343 69 10676 0.883453775 0.095224692 1.15E-05 p-loop containing nucleoside triphosphate hydrolase VE00_08523-T1 393.5440466 59.00030197 73 26912 0.787742186 0.07596864 0.003059399 p-loop containing nucleoside triphosphate hydrolase VE00_08741-T1 201.3460163 27.75662568 49 68941 0.697159059 0.050226648 2.64E-05 Pregnancy-associated plasma protein-A VE00_07666-T1 1453.110283 24.30897486 39 1055 1.203122541 0.060147064 2.42E-05 Purine nucleoside permease NUP VE00_01534-T1 1481.356984 8.113085373 13 338 1.189734363 0.013567833 3.09E-05 Putative Chloride intracellular channel protein 6 VE00_00564-T1 2330.885361 32.40311597 47 494 1.558482127 0.068136971 0.000769373 Ran-specific GTPase-activating protein 1 VE00_02780-T1 105.898595 34.8380956 49 248355 0.646160959 0.025997237 0.001149253 Reductase with broad range of substrate specificity VE00_05866-T1 3.71E-14 0.556720903 1 11 0.591632631 0.003080303 3.49E-06 Related to cylicin I VE00_03310-T1 4139.98604 1.833367803 3 4 2.248699072 0.005100828 6.08E-05 Related to KRE9 Cell wall synthesis protein VE00_06468-T1 1894.494326 12.18232049 22 350 1.356420165 0.016267585 3.07E-05 Riboflavin aldehyde-forming enzyme VE00_08790-T1 2066.651235 75.58325555 98 1310 1.501667006 0.160371607 0.000477987 Ribosomal protein S21e VE00_01861-T1 171.4522539 6.769967204 11 21363 0.671198605 0.023806574 1.10E-05 Ribosome maturation protein SBDS VE00_07252-T1 874.3278145 25.79001595 36 2689 0.949429179 0.018297879 8.85E-06 Rna recognition domain-containing protein (Fragment) VE00_00961-T1 477.269663 56.68555408 74 18530 0.86978793 0.167695221 7.99E-05 S-adenosyl-L-methionine-dependent methyltransferase VE00_08367-T1 116.9304621 18.47884169 27 112515 0.66161621 0.046875482 9.72E-07 S-adenosyl-L-methionine-dependent methyltransferase VE00_04615-T1 1074.460754 34.24878914 57 2819 1.037292806 0.032939922 0.002517283 Secreted aspartic proteinase VE00_04692-T1 1452.035124 9.823523145 17 460 1.190818194 0.037742388 3.39E-05 Serine-threonine rich VE00_08104-T1 1158.470096 5.573182179 9 383 1.063709043 0.019473537 1.52E-06 Small secreted protein VE00_07646-T1 470.9362806 52.63261799 67 17247 0.815265204 0.069601732 8.87E-05 Small subunit ribosomal protein S2e VE00_03106-T1 628.073409 19.99693587 33 4777 0.856767673 0.029317074 1.30E-05 Soluble quino protein glucose dehydrogenase VE00_02726-T1 1461.75579 55.99600333 70 1871 1.218062374 0.081755544 0.00148101 Structural constituent of ribosome VE00_04672-T1 1913.572194 37.91808601 50 780 1.380167919 0.04667441 8.02E-05 Structural constituent of ribosome VE00_02941-T1 503.8280419 72.64370511 98 21978 0.91462209 0.232224632 0.000590926 Superoxide dismutase VE00_04028-T1 2262.273814 11.04888983 18 200 1.498848521 0.007431762 8.11E-06 Telomere and ribosome associated protein/elicitor protein VE00_02632-T1 308.274975 57.01506407 86 51187 0.815381444 0.192582209 0.000144191 Thioredoxin TrxA VE00_05736-T1 311.5305118 78.32721307 99 58178 0.808899917 0.177899738 4.25E-05 Translation elongation factor VE00_01618-T1 1547.308261 40.03991074 55 1312 1.258071416 0.092731471 2.16E-05 Translation elongation factor 1 beta VE00_04043-T1 1781.294077 30.41527192 43 773 1.35783017 0.10435378 0.000251816 Translationally controlled tumor-associated VE00_04507-T1 2057.627983 22.85982767 29 391 1.430911361 0.03369732 2.94E-05 Tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein VE00_06685-T1 140.1728442 3.821653887 7 20341 0.656393269 0.019479461 8.71E-07 Ureidoglycolate dehydrogenase VE00_04274-T1 374.9488834 34.03874373 54 21931 0.762834457 0.043112581 0.000261756 Vacuolar serine protease PrtB VE00_07577-T1 4186.563817 23.05169755 39 127 2.303047907 0.072263428 0.006138135 Woronin body major protein (Fragment)

Supplementary Table 10. Proteins in P. sp. WSF3629 secretome functionally annotated using PANNZER

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Table Legends

Supplementary Tables I-V. Tables of proteins identified by LC-MS/MS in P. destructans (Table S1), P. verrucosus (S2), 03VT05 (S3), 05NY08 (S4), WSF3629 (S5) N=3 replicates per species. MS/MS-related metrics from Protein Prospector software are included in table columns (for each replicate in order from left to right: the number of uniquely mapped peptides, the number of observed spectra for a given protein identified—peptide count, the percent sequence coverage, the best individual peptide score, the best discriminant score, and the best expectation value for an individual peptide in each secretome analysis). The accession number of each sequence within the corresponding proteome database is also provided (first column) as well as the protein predicted molecular weight and name (last two columns).

Supplementary Tables VI-X. Table of proteins identified by LC-MS/MS with functional annotations provided by PANNZER software1 (webtool) for P. destructans (Table S6), P. verrucosus (S7), 03VT05 (S8), 05NY08 (S9), WSF3629 (S10). Annotation statistics and descriptions are included in table columns (from left to right: Gene ID, cluster_GSZ, cluster_RM1sum, cluster_size, cluster_desccount, RM2, val_avg, jac_avg, description, genename). See PANNZER publication1 for more information on associated statistical analyses.

References:

1 Koskinen, P., Toronen, P., Nokso-Koivisto, J. & Holm, L. PANNZER: high- throughput functional annotation of uncharacterized proteins in an error-prone environment. Bioinformatics 31, 1544-1552, doi:10.1093/bioinformatics/btu851 (2015).

226

APPENDIX C: Supplementary Data Table for Chapter 3

227 plate_id well_id chemical name moa co_id avg activity PM21 A01 Guanidine hydrochloride membrane, chaotropic agent 5.25 PM21 A02 Guanidine hydrochloride membrane, chaotropic agent 5 PM21 A03 Guanidine hydrochloride membrane, chaotropic agent 0.5 PM21 A04 Guanidine hydrochloride membrane, chaotropic agent 0.25 PM21 A05 2,2`-Dipyridyl chelator, lipophilic 5 PM21 A06 2,2`-Dipyridyl chelator, lipophilic 5 PM21 A07 2,2`-Dipyridyl chelator, lipophilic 5.25 PM21 A08 2,2`-Dipyridyl chelator, lipophilic 5.25 PM21 A09 Promethazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic D00480 4.75 PM21 A10 Promethazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic D00480 0 PM21 A11 Promethazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic D00480 1 PM21 A12 Promethazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic D00480 6 PM21 B01 Nystatin membrane, increase permeability, Na+ K+ H+ C06572 1.75 PM21 B02 Nystatin membrane, increase permeability, Na+ K+ H+ C06572 0 PM21 B03 Nystatin membrane, increase permeability, Na+ K+ H+ C06572 0 PM21 B04 Nystatin membrane, increase permeability, Na+ K+ H+ C06572 0 PM21 B05 Dodecyltrimethyl ammonimembrane, detergent, cationic 4.25 PM21 B06 Dodecyltrimethyl ammonimembrane, detergent, cationic 0 PM21 B07 Dodecyltrimethyl ammonimembrane, detergent, cationic 0 PM21 B08 Dodecyltrimethyl ammonimembrane, detergent, cationic 1 PM21 B09 Protamine sulfate membrane, nonspecific binding C01609 6 PM21 B10 Protamine sulfate membrane, nonspecific binding C01609 4 PM21 B11 Protamine sulfate membrane, nonspecific binding C01609 2 PM21 B12 Protamine sulfate membrane, nonspecific binding C01609 2 PM21 C01 Cetylpyridinium chloride membrane, detergent, cationic C11307 5.25 PM21 C02 Cetylpyridinium chloride membrane, detergent, cationic C11307 0 PM21 C03 Cetylpyridinium chloride membrane, detergent, cationic C11307 0 PM21 C04 Cetylpyridinium chloride membrane, detergent, cationic C11307 0 PM21 C05 Domiphen bromide membrane, detergent, cationic, fungicide D01588 4 PM21 C06 Domiphen bromide membrane, detergent, cationic, fungicide D01588 2 PM21 C07 Domiphen bromide membrane, detergent, cationic, fungicide D01588 0 PM21 C08 Domiphen bromide membrane, detergent, cationic, fungicide D01588 0 PM21 C09 L-Aspartic acid b-hydroxamtRNA synthetase C03124 4 PM21 C10 L-Aspartic acid b-hydroxamtRNA synthetase C03124 5 PM21 C11 L-Aspartic acid b-hydroxamtRNA synthetase C03124 5.25 PM21 C12 L-Aspartic acid b-hydroxamtRNA synthetase C03124 5 PM21 D01 1-Hydroxypyridine-2-thionbiofilm inhibitor, chelator, anti-fungal 5.25 PM21 D02 1-Hydroxypyridine-2-thionbiofilm inhibitor, chelator, anti-fungal 4 PM21 D03 1-Hydroxypyridine-2-thionbiofilm inhibitor, chelator, anti-fungal 4.25 PM21 D04 1-Hydroxypyridine-2-thionbiofilm inhibitor, chelator, anti-fungal 2.25 PM21 D05 EDTA chelator, hydrophilic C00284 5 PM21 D06 EDTA chelator, hydrophilic C00284 5 PM21 D07 EDTA chelator, hydrophilic C00284 5 PM21 D08 EDTA chelator, hydrophilic C00284 5 PM21 D09 Sodium Dichromate toxic anion, SO4 analog C15227 4.25 PM21 D10 Sodium Dichromate toxic anion, SO4 analog C15227 5 PM21 D11 Sodium Dichromate toxic anion, SO4 analog C15227 5 PM21 D12 Sodium Dichromate toxic anion, SO4 analog C15227 4.25 PM21 E01 Compound 48/80 cyclic AMP phosphodiesterase inhibitor 5 PM21 E02 Compound 48/80 cyclic AMP phosphodiesterase inhibitor 4 PM21 E03 Compound 48/80 cyclic AMP phosphodiesterase inhibitor 5.5 PM21 E04 Compound 48/80 cyclic AMP phosphodiesterase inhibitor 5.25 PM21 E05 Manganese chloride toxic cation C00034 4 PM21 E06 Manganese chloride toxic cation C00034 5.25 PM21 E07 Manganese chloride toxic cation C00034 4.5 PM21 E08 Manganese chloride toxic cation C00034 4.5 PM21 E09 Magnesium chloride toxic cation C07755 4 PM21 E10 Magnesium chloride toxic cation C07755 4 PM21 E11 Magnesium chloride toxic cation C07755 4 PM21 E12 Magnesium chloride toxic cation C07755 2.25 PM21 F01 Copper (II) sulfate toxic cation C00070 6 PM21 F02 Copper (II) sulfate toxic cation C00070 6 PM21 F03 Copper (II) sulfate toxic cation C00070 6 PM21 F04 Copper (II) sulfate toxic cation C00070 6 PM21 F05 Neomycin protein synthesis, 30S ribosomal subunit, aminoglycoside C00384 5.25 PM21 F06 Neomycin protein synthesis, 30S ribosomal subunit, aminoglycoside C00384 5 PM21 F07 Neomycin protein synthesis, 30S ribosomal subunit, aminoglycoside C00384 2.5 PM21 F08 Neomycin protein synthesis, 30S ribosomal subunit, aminoglycoside C00384 1.25 PM21 F09 D-Cycloserine wall C08057 4 PM21 F10 D-Cycloserine wall C08057 5 PM21 F11 D-Cycloserine wall C08057 4.25 PM21 F12 D-Cycloserine wall C08057 0 PM21 G01 Sodium Selenite toxic anion C05684 6 PM21 G02 Sodium Selenite toxic anion C05684 5.25 PM21 G03 Sodium Selenite toxic anion C05684 6 PM21 G04 Sodium Selenite toxic anion C05684 4.75

228

PM21 G05 Nickel chloride toxic cation C00291 5 PM21 G06 Nickel chloride toxic cation C00291 4.25 PM21 G07 Nickel chloride toxic cation C00291 4.5 PM21 G08 Nickel chloride toxic cation C00291 5 PM21 G09 Trifluoperazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic C07168 0 PM21 G10 Trifluoperazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic C07168 0.25 PM21 G11 Trifluoperazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic C07168 3.5 PM21 G12 Trifluoperazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic C07168 6.25 PM21 H01 Diamide oxidizes sulfhydryls, depletes glutathione 5.75 PM21 H02 Diamide oxidizes sulfhydryls, depletes glutathione 5 PM21 H03 Diamide oxidizes sulfhydryls, depletes glutathione 0 PM21 H04 Diamide oxidizes sulfhydryls, depletes glutathione 0 PM21 H05 Thiourea membrane, chaotropic agent C14415 2 PM21 H06 Thiourea membrane, chaotropic agent C14415 0 PM21 H07 Thiourea membrane, chaotropic agent C14415 0 PM21 H08 Thiourea membrane, chaotropic agent C14415 0 PM21 H09 Zinc chloride toxic cation C00038 5.75 PM21 H10 Zinc chloride toxic cation C00038 6.75 PM21 H11 Zinc chloride toxic cation C00038 7 PM21 H12 Zinc chloride toxic cation C00038 6.25 PM22 A01 L-Glutamic acid g-hydroxatRNA synthetase 3.666666667 PM22 A02 L-Glutamic acid g-hydroxatRNA synthetase 0.666666667 PM22 A03 L-Glutamic acid g-hydroxatRNA synthetase 0 PM22 A04 L-Glutamic acid g-hydroxatRNA synthetase 0 PM22 A05 Sodium metavanadate toxic anion, PO4 analog 5 PM22 A06 Sodium metavanadate toxic anion, PO4 analog 4.333333333 PM22 A07 Sodium metavanadate toxic anion, PO4 analog 5 PM22 A08 Sodium metavanadate toxic anion, PO4 analog 3.666666667 PM22 A09 Caffeine cyclic AMP phosphodiesterase inhibitor C07481 5 PM22 A10 Caffeine cyclic AMP phosphodiesterase inhibitor C07481 4 PM22 A11 Caffeine cyclic AMP phosphodiesterase inhibitor C07481 0 PM22 A12 Caffeine cyclic AMP phosphodiesterase inhibitor C07481 0 PM22 B01 L-Arginine hydroxamate tRNA synthetase 5.666666667 PM22 B02 L-Arginine hydroxamate tRNA synthetase 5 PM22 B03 L-Arginine hydroxamate tRNA synthetase 3.333333333 PM22 B04 L-Arginine hydroxamate tRNA synthetase 0.666666667 PM22 B05 Glycine hydroxamate tRNA synthetase 5 PM22 B06 Glycine hydroxamate tRNA synthetase 3.666666667 PM22 B07 Glycine hydroxamate tRNA synthetase 2 PM22 B08 Glycine hydroxamate tRNA synthetase 0 PM22 B09 Potassium iodide toxic anion C08219 0 PM22 B10 Potassium iodide toxic anion C08219 0 PM22 B11 Potassium iodide toxic anion C08219 0 PM22 B12 Potassium iodide toxic anion C08219 0 PM22 C01 3-Amino-1,2,4-triazole inhibits catalase, inhibits histidine synthesis C11261 0 PM22 C02 3-Amino-1,2,4-triazole inhibits catalase, inhibits histidine synthesis C11261 0 PM22 C03 3-Amino-1,2,4-triazole inhibits catalase, inhibits histidine synthesis C11261 0 PM22 C04 3-Amino-1,2,4-triazole inhibits catalase, inhibits histidine synthesis C11261 0 PM22 C05 Miltefosine phosphatidyl choline synthesis, protein kinase C inhibitor, fungicide 5 PM22 C06 Miltefosine phosphatidyl choline synthesis, protein kinase C inhibitor, fungicide 3 PM22 C07 Miltefosine phosphatidyl choline synthesis, protein kinase C inhibitor, fungicide 0 PM22 C08 Miltefosine phosphatidyl choline synthesis, protein kinase C inhibitor, fungicide 0 PM22 C09 DL-Serine hydroxamate tRNA synthetase C00716 4.333333333 PM22 C10 DL-Serine hydroxamate tRNA synthetase C00716 4 PM22 C11 DL-Serine hydroxamate tRNA synthetase C00716 0 PM22 C12 DL-Serine hydroxamate tRNA synthetase C00716 0 PM22 D01 Polymyxin B membrane, cyclic peptide C11613 0.666666667 PM22 D02 Polymyxin B membrane, cyclic peptide C11613 0 PM22 D03 Polymyxin B membrane, cyclic peptide C11613 0 PM22 D04 Polymyxin B membrane, cyclic peptide C11613 1.333333333 PM22 D05 Urea hydrogen peroxide oxidizing agent 4.666666667 PM22 D06 Urea hydrogen peroxide oxidizing agent 4 PM22 D07 Urea hydrogen peroxide oxidizing agent 4.666666667 PM22 D08 Urea hydrogen peroxide oxidizing agent 4.333333333 PM22 D09 Sodium Arsenate toxic anion, PO4 analog C01478 4.666666667 PM22 D10 Sodium Arsenate toxic anion, PO4 analog C01478 4.333333333 PM22 D11 Sodium Arsenate toxic anion, PO4 analog C01478 4 PM22 D12 Sodium Arsenate toxic anion, PO4 analog C01478 2 PM22 E01 Ceftriaxone wall, cephalosporin C06683 2 PM22 E02 Ceftriaxone wall, cephalosporin C06683 0 PM22 E03 Ceftriaxone wall, cephalosporin C06683 0 PM22 E04 Ceftriaxone wall, cephalosporin C06683 0 PM22 E05 BAPTA chelator, Ca++ 4.666666667 PM22 E06 BAPTA chelator, Ca++ 1.666666667 PM22 E07 BAPTA chelator, Ca++ 1.333333333 PM22 E08 BAPTA chelator, Ca++ 0.666666667

229

PM22 E09 D-Serine inhibits 3PGA dehydrogenase (L-serine and pantothenate synthesis) C00740 5 PM22 E10 D-Serine inhibits 3PGA dehydrogenase (L-serine and pantothenate synthesis) C00740 4.666666667 PM22 E11 D-Serine inhibits 3PGA dehydrogenase (L-serine and pantothenate synthesis) C00740 4 PM22 E12 D-Serine inhibits 3PGA dehydrogenase (L-serine and pantothenate synthesis) C00740 4 PM22 F01 Azaserine nucleic acid inhibitor, purine, glutamine analog 5 PM22 F02 Azaserine nucleic acid inhibitor, purine, glutamine analog 4.333333333 PM22 F03 Azaserine nucleic acid inhibitor, purine, glutamine analog 4.666666667 PM22 F04 Azaserine nucleic acid inhibitor, purine, glutamine analog 4.666666667 PM22 F05 Lithium chloride toxic cation 0 PM22 F06 Lithium chloride toxic cation 0 PM22 F07 Lithium chloride toxic cation 0 PM22 F08 Lithium chloride toxic cation 0 PM22 F09 Boric acid toxic anion C12486 5.333333333 PM22 F10 Boric acid toxic anion C12486 0.333333333 PM22 F11 Boric acid toxic anion C12486 0 PM22 F12 Boric acid toxic anion C12486 0 PM22 G01 Benzamidine peptidase inhibitor, fungicide C01784 4 PM22 G02 Benzamidine peptidase inhibitor, fungicide C01784 0 PM22 G03 Benzamidine peptidase inhibitor, fungicide C01784 0 PM22 G04 Benzamidine peptidase inhibitor, fungicide C01784 0 PM22 G05 Cycloheximide protein synthesis, 30S ribosomal subunit C06685 5 PM22 G06 Cycloheximide protein synthesis, 30S ribosomal subunit C06685 4 PM22 G07 Cycloheximide protein synthesis, 30S ribosomal subunit C06685 4.333333333 PM22 G08 Cycloheximide protein synthesis, 30S ribosomal subunit C06685 4.333333333 PM22 G09 Thallium (I) acetate toxic cation C15226 0 PM22 G10 Thallium (I) acetate toxic cation C15226 0.333333333 PM22 G11 Thallium (I) acetate toxic cation C15226 0 PM22 G12 Thallium (I) acetate toxic cation C15226 0 PM22 H01 Cephalotin wall, cephalosporin C07761 5.666666667 PM22 H02 Cephalotin wall, cephalosporin C07761 5 PM22 H03 Cephalotin wall, cephalosporin C07761 5 PM22 H04 Cephalotin wall, cephalosporin C07761 1 PM22 H05 Paromomycin protein synthesis, 30S ribosomal subunit, aminoglycoside C08048 1 PM22 H06 Paromomycin protein synthesis, 30S ribosomal subunit, aminoglycoside C08048 1 PM22 H07 Paromomycin protein synthesis, 30S ribosomal subunit, aminoglycoside C08048 0.666666667 PM22 H08 Paromomycin protein synthesis, 30S ribosomal subunit, aminoglycoside C08048 0 PM22 H09 Myclobutanil membrane function, inhibits sterol biosynthesis 5.333333333 PM22 H10 Myclobutanil membrane function, inhibits sterol biosynthesis 0.666666667 PM22 H11 Myclobutanil membrane function, inhibits sterol biosynthesis 0 PM22 H12 Myclobutanil membrane function, inhibits sterol biosynthesis 0 PM23 A01 Benzethonium Chloride membrane, detergent, cationic D01140 5 PM23 A02 Benzethonium Chloride membrane, detergent, cationic D01140 0 PM23 A03 Benzethonium Chloride membrane, detergent, cationic D01140 0 PM23 A04 Benzethonium Chloride membrane, detergent, cationic D01140 0 PM23 A05 Chlorpromazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic C06906 5 PM23 A06 Chlorpromazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic C06906 0 PM23 A07 Chlorpromazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic C06906 0 PM23 A08 Chlorpromazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic C06906 2 PM23 A09 Ammonium Sulfate toxic cation 4.333333333 PM23 A10 Ammonium Sulfate toxic cation 5.333333333 PM23 A11 Ammonium Sulfate toxic cation 4 PM23 A12 Ammonium Sulfate toxic cation 2.666666667 PM23 B01 Cadmium Chloride toxic cation C01413 4.333333333 PM23 B02 Cadmium Chloride toxic cation C01413 2 PM23 B03 Cadmium Chloride toxic cation C01413 0.666666667 PM23 B04 Cadmium Chloride toxic cation C01413 0 PM23 B05 Dequalinium chloride ion channel inhibitor, K+ D01575 4 PM23 B06 Dequalinium chloride ion channel inhibitor, K+ D01575 4.666666667 PM23 B07 Dequalinium chloride ion channel inhibitor, K+ D01575 2 PM23 B08 Dequalinium chloride ion channel inhibitor, K+ D01575 1 PM23 B09 Doxycycline protein synthesis, 30S ribosomal subunit, tetracycline C06973 4.666666667 PM23 B10 Doxycycline protein synthesis, 30S ribosomal subunit, tetracycline C06973 5 PM23 B11 Doxycycline protein synthesis, 30S ribosomal subunit, tetracycline C06973 5.333333333 PM23 B12 Doxycycline protein synthesis, 30S ribosomal subunit, tetracycline C06973 6 PM23 C01 Glycine hydrochloride wall C00037 5 PM23 C02 Glycine hydrochloride wall C00037 4 PM23 C03 Glycine hydrochloride wall C00037 2 PM23 C04 Glycine hydrochloride wall C00037 2 PM23 C05 Hydroxylamine DNA damage, mutagen, antifolate (inhibits thymine and methionine synthesis) C00192 4 PM23 C06 Hydroxylamine DNA damage, mutagen, antifolate (inhibits thymine and methionine synthesis) C00192 4 PM23 C07 Hydroxylamine DNA damage, mutagen, antifolate (inhibits thymine and methionine synthesis) C00192 2.666666667 PM23 C08 Hydroxylamine DNA damage, mutagen, antifolate (inhibits thymine and methionine synthesis) C00192 0 PM23 C09 Poly-L-lysine membrane, detergent, cationic 4.333333333 PM23 C10 Poly-L-lysine membrane, detergent, cationic 6 PM23 C11 Poly-L-lysine membrane, detergent, cationic 4.666666667 PM23 C12 Poly-L-lysine membrane, detergent, cationic 4.666666667

230

PM23 D01 Chromium (III) chloride toxic cation C06268 5.333333333 PM23 D02 Chromium (III) chloride toxic cation C06268 4 PM23 D03 Chromium (III) chloride toxic cation C06268 2 PM23 D04 Chromium (III) chloride toxic cation C06268 3.333333333 PM23 D05 Cobalt (II) chloride toxic cation C00175 3.333333333 PM23 D06 Cobalt (II) chloride toxic cation C00175 4 PM23 D07 Cobalt (II) chloride toxic cation C00175 4.333333333 PM23 D08 Cobalt (II) chloride toxic cation C00175 1 PM23 D09 Cupric chloride toxic cation C00070 4.333333333 PM23 D10 Cupric chloride toxic cation C00070 5.333333333 PM23 D11 Cupric chloride toxic cation C00070 6 PM23 D12 Cupric chloride toxic cation C00070 6 PM23 E01 Sodium metaborate toxic anion 4.333333333 PM23 E02 Sodium metaborate toxic anion 2.666666667 PM23 E03 Sodium metaborate toxic anion 0.666666667 PM23 E04 Sodium metaborate toxic anion 0 PM23 E05 Sodium metaperiodate toxic anion, oxidizing agent 2 PM23 E06 Sodium metaperiodate toxic anion, oxidizing agent 2 PM23 E07 Sodium metaperiodate toxic anion, oxidizing agent 4.333333333 PM23 E08 Sodium metaperiodate toxic anion, oxidizing agent 5 PM23 E09 Sodium Arsenite toxic anion 4 PM23 E10 Sodium Arsenite toxic anion 4.666666667 PM23 E11 Sodium Arsenite toxic anion 4.333333333 PM23 E12 Sodium Arsenite toxic anion 5 PM23 F01 Sodium Azide respiration, uncoupler 2.666666667 PM23 F02 Sodium Azide respiration, uncoupler 1 PM23 F03 Sodium Azide respiration, uncoupler 0 PM23 F04 Sodium Azide respiration, uncoupler 0.333333333 PM23 F05 Sodium Caprylate respiration, ionophore, H+ C06423 1 PM23 F06 Sodium Caprylate respiration, ionophore, H+ C06423 3.333333333 PM23 F07 Sodium Caprylate respiration, ionophore, H+ C06423 4 PM23 F08 Sodium Caprylate respiration, ionophore, H+ C06423 4 PM23 F09 Sodium Cyanate toxic anion C01417 5 PM23 F10 Sodium Cyanate toxic anion C01417 4.666666667 PM23 F11 Sodium Cyanate toxic anion C01417 4.333333333 PM23 F12 Sodium Cyanate toxic anion C01417 4.666666667 PM23 G01 Sodium Nitrite toxicity, nitrite C00088 4.333333333 PM23 G02 Sodium Nitrite toxicity, nitrite C00088 2.666666667 PM23 G03 Sodium Nitrite toxicity, nitrite C00088 1 PM23 G04 Sodium Nitrite toxicity, nitrite C00088 1.333333333 PM23 G05 Sodium orthovanadate toxic anion, PO4 analog 2 PM23 G06 Sodium orthovanadate toxic anion, PO4 analog 2.666666667 PM23 G07 Sodium orthovanadate toxic anion, PO4 analog 4 PM23 G08 Sodium orthovanadate toxic anion, PO4 analog 2.666666667 PM23 G09 2-Deoxy-D-glucose transport, sugar C00586 4.666666667 PM23 G10 2-Deoxy-D-glucose transport, sugar C00586 5 PM23 G11 2-Deoxy-D-glucose transport, sugar C00586 5 PM23 G12 2-Deoxy-D-glucose transport, sugar C00586 4.333333333 PM23 H01 Sodium Selenate toxic anion, SO4 analog C05697 5 PM23 H02 Sodium Selenate toxic anion, SO4 analog C05697 4 PM23 H03 Sodium Selenate toxic anion, SO4 analog C05697 2.666666667 PM23 H04 Sodium Selenate toxic anion, SO4 analog C05697 3 PM23 H05 Sodium Cyanide toxic anion C00177 3.666666667 PM23 H06 Sodium Cyanide toxic anion C00177 4 PM23 H07 Sodium Cyanide toxic anion C00177 4 PM23 H08 Sodium Cyanide toxic anion C00177 2 PM23 H09 Sodium Thiosulfate toxic anion, reducing agent C00320 5 PM23 H10 Sodium Thiosulfate toxic anion, reducing agent C00320 4.333333333 PM23 H11 Sodium Thiosulfate toxic anion, reducing agent C00320 1.666666667 PM23 H12 Sodium Thiosulfate toxic anion, reducing agent C00320 0 PM24 A01 Apramycin protein synthesis, 30S ribosomal subunit, aminoglycoside C01555 5 PM24 A02 Apramycin protein synthesis, 30S ribosomal subunit, aminoglycoside C01555 4.333333333 PM24 A03 Apramycin protein synthesis, 30S ribosomal subunit, aminoglycoside C01555 7.666666667 PM24 A04 Apramycin protein synthesis, 30S ribosomal subunit, aminoglycoside C01555 4.333333333 PM24 A05 9-Aminoacridine DNA intercalator, inhibits RNA synthesis D02905 5 PM24 A06 9-Aminoacridine DNA intercalator, inhibits RNA synthesis D02905 5.333333333 PM24 A07 9-Aminoacridine DNA intercalator, inhibits RNA synthesis D02905 7 PM24 A08 9-Aminoacridine DNA intercalator, inhibits RNA synthesis D02905 9 PM24 A09 Zaragozic acid A sterol synthesis inhibitor, inhibits squalene synthase 5.666666667 PM24 A10 Zaragozic acid A sterol synthesis inhibitor, inhibits squalene synthase 5.333333333 PM24 A11 Zaragozic acid A sterol synthesis inhibitor, inhibits squalene synthase 0.666666667 PM24 A12 Zaragozic acid A sterol synthesis inhibitor, inhibits squalene synthase 0 PM24 B01 Blasticidin S protein synthesis C02010 4.666666667 PM24 B02 Blasticidin S protein synthesis C02010 4.333333333 PM24 B03 Blasticidin S protein synthesis C02010 4 PM24 B04 Blasticidin S protein synthesis C02010 4

231

PM24 B05 Thioridazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic D00798 3.333333333 PM24 B06 Thioridazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic D00798 1.333333333 PM24 B07 Thioridazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic D00798 0 PM24 B08 Thioridazine membrane, phenothiazine, efflux pump inhibitor, anti-psychotic D00798 1.333333333 PM24 B09 Sodium Benzoate respiration, ionophore, H+ C00180 4 PM24 B10 Sodium Benzoate respiration, ionophore, H+ C00180 5 PM24 B11 Sodium Benzoate respiration, ionophore, H+ C00180 4.666666667 PM24 B12 Sodium Benzoate respiration, ionophore, H+ C00180 4.666666667 PM24 C01 Chlortetracycline protein synthesis, 30S ribosomal subunit, tetracycline C06571 5.666666667 PM24 C02 Chlortetracycline protein synthesis, 30S ribosomal subunit, tetracycline C06571 4.333333333 PM24 C03 Chlortetracycline protein synthesis, 30S ribosomal subunit, tetracycline C06571 6 PM24 C04 Chlortetracycline protein synthesis, 30S ribosomal subunit, tetracycline C06571 7 PM24 C05 Sodium metasilicate toxic anion 4.333333333 PM24 C06 Sodium metasilicate toxic anion 2.666666667 PM24 C07 Sodium metasilicate toxic anion 2.666666667 PM24 C08 Sodium metasilicate toxic anion 3.333333333 PM24 C09 Pentamidine Isethionate antifungal C07420 0.333333333 PM24 C10 Pentamidine Isethionate antifungal C07420 5.333333333 PM24 C11 Pentamidine Isethionate antifungal C07420 8 PM24 C12 Pentamidine Isethionate antifungal C07420 8 PM24 D01 6-Azauracil nucleic acid analog, pyrimidine 4.666666667 PM24 D02 6-Azauracil nucleic acid analog, pyrimidine 4 PM24 D03 6-Azauracil nucleic acid analog, pyrimidine 4.666666667 PM24 D04 6-Azauracil nucleic acid analog, pyrimidine 2.666666667 PM24 D05 Potassium Chromate toxic anion 3.333333333 PM24 D06 Potassium Chromate toxic anion 4 PM24 D07 Potassium Chromate toxic anion 4 PM24 D08 Potassium Chromate toxic anion 4.333333333 PM24 D09 Thialysine inhibitor of lysine-2,3-aminomutase 4 PM24 D10 Thialysine inhibitor of lysine-2,3-aminomutase 4 PM24 D11 Thialysine inhibitor of lysine-2,3-aminomutase 4.666666667 PM24 D12 Thialysine inhibitor of lysine-2,3-aminomutase 5 PM24 E01 a-Monothioglycerol reducing agent, thiol, adenosyl methionine antagonist 4.333333333 PM24 E02 a-Monothioglycerol reducing agent, thiol, adenosyl methionine antagonist 4 PM24 E03 a-Monothioglycerol reducing agent, thiol, adenosyl methionine antagonist 2 PM24 E04 a-Monothioglycerol reducing agent, thiol, adenosyl methionine antagonist 1.333333333 PM24 E05 EGTA chelator, Ca++ 4.333333333 PM24 E06 EGTA chelator, Ca++ 4.333333333 PM24 E07 EGTA chelator, Ca++ 4 PM24 E08 EGTA chelator, Ca++ 4 PM24 E09 Sodium pyrophosphate chelator, hydrophilic C00013 5.666666667 PM24 E10 Sodium pyrophosphate chelator, hydrophilic C00013 4.666666667 PM24 E11 Sodium pyrophosphate chelator, hydrophilic C00013 4.666666667 PM24 E12 Sodium pyrophosphate chelator, hydrophilic C00013 4 PM24 F01 Propiconazole membrane function and sterol synthesis, azole, antifungal C11121 4.666666667 PM24 F02 Propiconazole membrane function and sterol synthesis, azole, antifungal C11121 2 PM24 F03 Propiconazole membrane function and sterol synthesis, azole, antifungal C11121 1 PM24 F04 Propiconazole membrane function and sterol synthesis, azole, antifungal C11121 0.333333333 PM24 F05 Methyl viologen oxidizing agent C00225 1 PM24 F06 Methyl viologen oxidizing agent C00225 0 PM24 F07 Methyl viologen oxidizing agent C00225 0.333333333 PM24 F08 Methyl viologen oxidizing agent C00225 0.666666667 PM24 F09 Sodium Fluoride toxic anion, respiration, uncoupler C08142 4.333333333 PM24 F10 Sodium Fluoride toxic anion, respiration, uncoupler C08142 4.666666667 PM24 F11 Sodium Fluoride toxic anion, respiration, uncoupler C08142 5.333333333 PM24 F12 Sodium Fluoride toxic anion, respiration, uncoupler C08142 4.666666667 PM24 G01 Cisplatin DNA damage, crosslinker C06911 5 PM24 G02 Cisplatin DNA damage, crosslinker C06911 4.666666667 PM24 G03 Cisplatin DNA damage, crosslinker C06911 4 PM24 G04 Cisplatin DNA damage, crosslinker C06911 4 PM24 G05 Aluminum sulfate toxic cation 2 PM24 G06 Aluminum sulfate toxic cation 4 PM24 G07 Aluminum sulfate toxic cation 6 PM24 G08 Aluminum sulfate toxic cation 7 PM24 G09 Berberine Chloride NULL C00757 4 PM24 G10 Berberine Chloride NULL C00757 4.666666667 PM24 G11 Berberine Chloride NULL C00757 0.333333333 PM24 G12 Berberine Chloride NULL C00757 0.666666667 PM24 H01 Isoniazid inhibitor of fatty acid synthesis C07054 5 PM24 H02 Isoniazid inhibitor of fatty acid synthesis C07054 5 PM24 H03 Isoniazid inhibitor of fatty acid synthesis C07054 4 PM24 H04 Isoniazid inhibitor of fatty acid synthesis C07054 0.333333333 PM24 H05 Amphotericin B membrane, increase permeability, Na+ K+ H+ C06573 0 PM24 H06 Amphotericin B membrane, increase permeability, Na+ K+ H+ C06573 1.333333333 PM24 H07 Amphotericin B membrane, increase permeability, Na+ K+ H+ C06573 0 PM24 H08 Amphotericin B membrane, increase permeability, Na+ K+ H+ C06573 0

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PM24 H09 Miconazole Nitrate membrane function and sterol synthesis, azole, antifungal C08070 4.666666667 PM24 H10 Miconazole Nitrate membrane function and sterol synthesis, azole, antifungal C08070 0.333333333 PM24 H11 Miconazole Nitrate membrane function and sterol synthesis, azole, antifungal C08070 0.333333333 PM24 H12 Miconazole Nitrate membrane function and sterol synthesis, azole, antifungal C08070 0 PM25 A01 Hydroxyurea ribonucleotide DP reductase inhibitor, antifolate (inhibits thymine and methionine synthesis) C07044 1 PM25 A02 Hydroxyurea ribonucleotide DP reductase inhibitor, antifolate (inhibits thymine and methionine synthesis) C07044 0.333333333 PM25 A03 Hydroxyurea ribonucleotide DP reductase inhibitor, antifolate (inhibits thymine and methionine synthesis) C07044 0 PM25 A04 Hydroxyurea ribonucleotide DP reductase inhibitor, antifolate (inhibits thymine and methionine synthesis) C07044 0 PM25 A05 Tobramycin protein synthesis, 30S ribosomal subunit, aminoglycoside C00397 5 PM25 A06 Tobramycin protein synthesis, 30S ribosomal subunit, aminoglycoside C00397 5 PM25 A07 Tobramycin protein synthesis, 30S ribosomal subunit, aminoglycoside C00397 5 PM25 A08 Tobramycin protein synthesis, 30S ribosomal subunit, aminoglycoside C00397 1 PM25 A09 Niaproof membrane, detergent, anionic 5 PM25 A10 Niaproof membrane, detergent, anionic 5 PM25 A11 Niaproof membrane, detergent, anionic 5 PM25 A12 Niaproof membrane, detergent, anionic 1.333333333 PM25 B01 Chloroalanine hydrochlorialanine analog 4.666666667 PM25 B02 Chloroalanine hydrochlorialanine analog 3.333333333 PM25 B03 Chloroalanine hydrochlorialanine analog 3.333333333 PM25 B04 Chloroalanine hydrochlorialanine analog 4 PM25 B05 Tetrazolium violet respiration, uncoupler 4.333333333 PM25 B06 Tetrazolium violet respiration, uncoupler 3 PM25 B07 Tetrazolium violet respiration, uncoupler 0 PM25 B08 Tetrazolium violet respiration, uncoupler 2 PM25 B09 Kanamycin protein synthesis, 30S ribosomal subunit, aminoglycoside C08046 5 PM25 B10 Kanamycin protein synthesis, 30S ribosomal subunit, aminoglycoside C08046 5 PM25 B11 Kanamycin protein synthesis, 30S ribosomal subunit, aminoglycoside C08046 5 PM25 B12 Kanamycin protein synthesis, 30S ribosomal subunit, aminoglycoside C08046 4.333333333 PM25 C01 4-Aminopyridine ion channel inhibitor, K+ C13728 4.666666667 PM25 C02 4-Aminopyridine ion channel inhibitor, K+ C13728 4 PM25 C03 4-Aminopyridine ion channel inhibitor, K+ C13728 4.333333333 PM25 C04 4-Aminopyridine ion channel inhibitor, K+ C13728 1.333333333 PM25 C05 Amitriptyline membrane, transport D00809 0 PM25 C06 Amitriptyline membrane, transport D00809 0 PM25 C07 Amitriptyline membrane, transport D00809 2.666666667 PM25 C08 Amitriptyline membrane, transport D00809 6.333333333 PM25 C09 Citric acid Trisodium Salt NULL C00158 4 PM25 C10 Citric acid Trisodium Salt NULL C00158 4.666666667 PM25 C11 Citric acid Trisodium Salt NULL C00158 4 PM25 C12 Citric acid Trisodium Salt NULL C00158 5.666666667 PM25 D01 Mechlorethamine hydrochalkylating agent C07115 0 PM25 D02 Mechlorethamine hydrochalkylating agent C07115 0 PM25 D03 Mechlorethamine hydrochalkylating agent C07115 0 PM25 D04 Mechlorethamine hydrochalkylating agent C07115 0 PM25 D05 Hygromycin B protein synthesis, 30S ribosomal subunit, aminoglycoside C01925 2.666666667 PM25 D06 Hygromycin B protein synthesis, 30S ribosomal subunit, aminoglycoside C01925 2 PM25 D07 Hygromycin B protein synthesis, 30S ribosomal subunit, aminoglycoside C01925 1 PM25 D08 Hygromycin B protein synthesis, 30S ribosomal subunit, aminoglycoside C01925 0 PM25 D09 Fluorodeoxyuridine DNA synthesis inhibitor C11736 4.666666667 PM25 D10 Fluorodeoxyuridine DNA synthesis inhibitor C11736 3.666666667 PM25 D11 Fluorodeoxyuridine DNA synthesis inhibitor C11736 4.333333333 PM25 D12 Fluorodeoxyuridine DNA synthesis inhibitor C11736 3 PM25 E01 Sodium Salicylate biofilm inhibitor, anti-capsule agent, chelator, prostaglandin syntetase inhibitor, mar inducer C00805 4.666666667 PM25 E02 Sodium Salicylate biofilm inhibitor, anti-capsule agent, chelator, prostaglandin syntetase inhibitor, mar inducer C00805 3.333333333 PM25 E03 Sodium Salicylate biofilm inhibitor, anti-capsule agent, chelator, prostaglandin syntetase inhibitor, mar inducer C00805 4 PM25 E04 Sodium Salicylate biofilm inhibitor, anti-capsule agent, chelator, prostaglandin syntetase inhibitor, mar inducer C00805 4 PM25 E05 Succinic acid organic acid C00042 3.333333333 PM25 E06 Succinic acid organic acid C00042 3.333333333 PM25 E07 Succinic acid organic acid C00042 3.666666667 PM25 E08 Succinic acid organic acid C00042 3 PM25 E09 Ferulic acid antioxidant C06917 1 PM25 E10 Ferulic acid antioxidant C06917 0 PM25 E11 Ferulic acid antioxidant C06917 0 PM25 E12 Ferulic acid antioxidant C06917 1 PM25 F01 Malic acid organic acid C00497 4 PM25 F02 Malic acid organic acid C00497 5 PM25 F03 Malic acid organic acid C00497 2.666666667 PM25 F04 Malic acid organic acid C00497 1.333333333 PM25 F05 Tartaric acid organic acid C02107 3.333333333 PM25 F06 Tartaric acid organic acid C02107 2 PM25 F07 Tartaric acid organic acid C02107 4.333333333 PM25 F08 Tartaric acid organic acid C02107 1.333333333 PM25 F09 Fumaric acid organic acid C00122 4.333333333 PM25 F10 Fumaric acid organic acid C00122 4 PM25 F11 Fumaric acid organic acid C00122 4 PM25 F12 Fumaric acid organic acid C00122 4

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PM25 G01 5-Fluorocytosine DNA synthesis inhibitor D00323 5 PM25 G02 5-Fluorocytosine DNA synthesis inhibitor D00323 4 PM25 G03 5-Fluorocytosine DNA synthesis inhibitor D00323 4.333333333 PM25 G04 5-Fluorocytosine DNA synthesis inhibitor D00323 4 PM25 G05 Palladium(II) Chloride toxic cation 4.333333333 PM25 G06 Palladium(II) Chloride toxic cation 4 PM25 G07 Palladium(II) Chloride toxic cation 4.666666667 PM25 G08 Palladium(II) Chloride toxic cation 5 PM25 G09 Ibuprofen biofilm inhibitor, anti-capsule agent, prostaglandin syntetase inhibitor C01588 4.333333333 PM25 G10 Ibuprofen biofilm inhibitor, anti-capsule agent, prostaglandin syntetase inhibitor C01588 4.666666667 PM25 G11 Ibuprofen biofilm inhibitor, anti-capsule agent, prostaglandin syntetase inhibitor C01588 4 PM25 G12 Ibuprofen biofilm inhibitor, anti-capsule agent, prostaglandin syntetase inhibitor C01588 5 PM25 H01 Chloroquine iron depleter C07625 7 PM25 H02 Chloroquine iron depleter C07625 4.333333333 PM25 H03 Chloroquine iron depleter C07625 2.666666667 PM25 H04 Chloroquine iron depleter C07625 2.666666667 PM25 H05 Cinnamic acid respiration, ionophore, H+ C10438 5 PM25 H06 Cinnamic acid respiration, ionophore, H+ C10438 4.666666667 PM25 H07 Cinnamic acid respiration, ionophore, H+ C10438 5 PM25 H08 Cinnamic acid respiration, ionophore, H+ C10438 5 PM25 H09 5-Fluorouracil nucleic acid analog, pyrimidine C07649 4.666666667 PM25 H10 5-Fluorouracil nucleic acid analog, pyrimidine C07649 5 PM25 H11 5-Fluorouracil nucleic acid analog, pyrimidine C07649 5.333333333 PM25 H12 5-Fluorouracil nucleic acid analog, pyrimidine C07649 6

Supplementary Table 1. Biolog chemical inhibitor screen. A full list of each chemical compounds tested in Biolog drug screen for ability to inhibit growth of P. destructans with average activity index of P. destructans (rightmost column) in each well.

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APPENDIX D: Supplementary Data Tables for Chapter 4

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#plate_id well_id chemical moa co_id 24MN13 3VT5 5NY8 Pverr WSF3629 Pdest PM01-A01 PM01 A01 Negative Control C-Source, negative control 3 1 2 2 1 2 PM01-A02 PM01 A02 L-Arabinose C-Source, carbohydrate C00259 9 6 6 8 5 1 PM01-A03 PM01 A03 N-Acetyl-D-Glucosamine C-Source, carbohydrate C00140 9 6 6 8 6 4 PM01-A04 PM01 A04 D-Saccharic acid C-Source, carboxylic acid C00818 6 1 2 5 3 2 PM01-A05 PM01 A05 Succinic acid C-Source, carboxylic acid C00042 6 3 3 5 2 2 PM01-A06 PM01 A06 D-Galactose C-Source, carbohydrate C00124 9 7 4 8 6 2 PM01-A07 PM01 A07 L-Aspartic acid C-Source, amino acid C00049 6 3 6 3 6 3 PM01-A08 PM01 A08 L-Proline C-Source, amino acid C00148 5 3 3 6 3 4 PM01-A09 PM01 A09 D-Alanine C-Source, amino acid C00133 1 1 1 1 1 1 PM01-A10 PM01 A10 D-Trehalose C-Source, carbohydrate C01083 8 6 6 9 6 7 PM01-A11 PM01 A11 D-Mannose C-Source, carbohydrate C00159 9 9 4 8 6 7 PM01-A12 PM01 A12 Dulcitol C-Source, carbohydrate C01697 8 5 5 8 2 2 PM01-B01 PM01 B01 D-Serine C-Source, amino acid C00740 1 2 2 1 0 0 PM01-B02 PM01 B02 D-Sorbitol C-Source, carbohydrate C00794 8 3 4 8 5 2 PM01-B03 PM01 B03 Glycerol C-Source, carbohydrate C00116 6 3 2 5 1 4 PM01-B04 PM01 B04 L-Fucose C-Source, carbohydrate C01019 5 2 3 3 1 2 PM01-B05 PM01 B05 D-Glucuronic acid C-Source, carboxylic acid C00191 6 3 6 5 4 2 PM01-B06 PM01 B06 D-Gluconic acid C-Source, carboxylic acid C00257 9 3 4 9 6 2 PM01-B07 PM01 B07 DL-a-Glycerol Phosphate C-Source, carbohydrate C00093 3 2 2 2 1 2 PM01-B08 PM01 B08 D-Xylose C-Source, carbohydrate C00181 8 7 4 9 4 1 PM01-B09 PM01 B09 L-Lactic acid C-Source, carboxylic acid C01432 3 3 3 3 2 3 PM01-B10 PM01 B10 Formic acid C-Source, carboxylic acid C00058 3 2 2 2 1 2 PM01-B11 PM01 B11 D-Mannitol C-Source, carbohydrate C00392 8 8 5 8 3 4 PM01-B12 PM01 B12 L-Glutamic acid C-Source, amino acid C00025 6 6 6 6 3 4 PM01-C01 PM01 C01 D-Glucose-6-Phosphate C-Source, carbohydrate C00092 3 1 2 2 1 2 PM01-C02 PM01 C02 D-Galactonic acid-g-Lactone C-Source, carboxylic acid C03383 2 2 1 3 1 0 PM01-C03 PM01 C03 DL-Malic acid C-Source, carboxylic acid C00497 6 3 3 6 4 3 PM01-C04 PM01 C04 D-Ribose C-Source, carbohydrate C00121 9 3 4 9 6 2 PM01-C05 PM01 C05 Tween 20 C-Source, fatty acid C11624 2 3 3 2 3 3 PM01-C06 PM01 C06 L-Rhamnose C-Source, carbohydrate C00507 5 3 3 5 3 4 PM01-C07 PM01 C07 D-Fructose C-Source, carbohydrate C00095 9 5 4 8 6 7 PM01-C08 PM01 C08 Acetic acid C-Source, carboxylic acid C00033 3 2 2 3 2 3 PM01-C09 PM01 C09 a-D-Glucose C-Source, carbohydrate C00031 9 6 6 8 5 7 PM01-C10 PM01 C10 Maltose C-Source, carbohydrate C00208 9 8 5 8 6 2 PM01-C11 PM01 C11 D-Melibiose C-Source, carbohydrate C05402 8 5 5 8 6 2 PM01-C12 PM01 C12 Thymidine C-Source, carbohydrate C00214 3 2 2 2 1 2 PM01-D01 PM01 D01 L-Asparagine C-Source, amino acid C00152 6 5 3 6 3 4 PM01-D02 PM01 D02 D-Aspartic acid C-Source, amino acid C00402 2 3 2 2 1 2 PM01-D03 PM01 D03 D-Glucosaminic acid C-Source, carboxylic acid C03752 2 1 2 2 1 2 PM01-D04 PM01 D04 1,2-Propanediol C-Source, alcohol C00583 2 1 2 2 1 2 PM01-D05 PM01 D05 Tween 40 C-Source, fatty acid 6 5 6 6 5 5 PM01-D06 PM01 D06 a-Ketoglutaric acid C-Source, carboxylic acid C00026 6 2 4 2 2 2 PM01-D07 PM01 D07 a-Ketobutyric acid C-Source, carboxylic acid C00109 6 3 2 6 1 2 PM01-D08 PM01 D08 a-Methyl-D-Galactoside C-Source, carbohydrate C03619 3 2 2 2 1 2 PM01-D09 PM01 D09 a-D-Lactose C-Source, carbohydrate C00243 8 9 5 8 1 2 PM01-D10 PM01 D10 Lactulose C-Source, carbohydrate C07064 6 9 2 3 1 2 PM01-D11 PM01 D11 Sucrose C-Source, carbohydrate C00089 9 7 6 9 6 2 PM01-D12 PM01 D12 Uridine C-Source, carbohydrate C00299 3 3 2 2 1 2 PM01-E01 PM01 E01 L-Glutamine C-Source, amino acid C00064 5 3 6 5 3 4 PM01-E02 PM01 E02 m-Tartaric acid C-Source, carboxylic acid C00552 6 2 1 3 0 2 PM01-E03 PM01 E03 D-Glucose-1-Phosphate C-Source, carbohydrate C00103 2 2 2 2 1 2 PM01-E04 PM01 E04 D-Fructose-6-Phosphate C-Source, carbohydrate C00085 3 1 2 2 1 2 PM01-E05 PM01 E05 Tween 80 C-Source, fatty acid C11625 6 7 6 6 3 6 PM01-E06 PM01 E06 a-Hydroxyglutaric acid-g-Lactone C-Source, carboxylic acid 3 2 2 3 1 2 PM01-E07 PM01 E07 a-Hydroxybutyric acid C-Source, carboxylic acid C05984 6 2 2 3 1 3 PM01-E08 PM01 E08 b-Methyl-D-Glucoside C-Source, carbohydrate 9 8 4 8 6 3 PM01-E09 PM01 E09 Adonitol C-Source, carbohydrate C00474 9 9 6 8 6 2 PM01-E10 PM01 E10 Maltotriose C-Source, carbohydrate C01835 9 5 5 9 9 2 PM01-E11 PM01 E11 2`-Deoxyadenosine C-Source, carbohydrate C00559 2 2 2 2 1 1 PM01-E12 PM01 E12 Adenosine C-Source, carbohydrate C00212 3 2 3 3 2 2 PM01-F01 PM01 F01 Gly-Asp C-Source, amino acid C02871 6 6 3 3 2 3 PM01-F02 PM01 F02 Citric acid C-Source, carboxylic acid C00158 6 5 3 5 3 3 PM01-F03 PM01 F03 m-Inositol C-Source, carbohydrate C00137 5 6 3 2 1 2 PM01-F04 PM01 F04 D-Threonine C-Source, amino acid C00820 1 1 2 1 1 2 PM01-F05 PM01 F05 Fumaric acid C-Source, carboxylic acid C00122 6 4 3 6 3 3 PM01-F06 PM01 F06 Bromosuccinic acid C-Source, carboxylic acid 2 3 2 3 2 2 PM01-F07 PM01 F07 Propionic acid C-Source, carboxylic acid C00163 3 2 1 3 1 3 PM01-F08 PM01 F08 Mucic acid C-Source, carboxylic acid C01807 3 2 2 2 1 2 PM01-F09 PM01 F09 Glycolic acid C-Source, carboxylic acid C00160 3 2 2 2 1 3 PM01-F10 PM01 F10 Glyoxylic acid C-Source, carboxylic acid C00048 2 2 2 2 1 2 PM01-F11 PM01 F11 D-Cellobiose C-Source, carbohydrate C00185 8 9 4 9 5 4 PM01-F12 PM01 F12 Inosine C-Source, carbohydrate C00294 6 5 4 5 2 2 PM01-G01 PM01 G01 Gly-Glu C-Source, amino acid 8 6 3 6 2 3 PM01-G02 PM01 G02 Tricarballylic acid C-Source, carboxylic acid 3 2 2 2 1 0 PM01-G03 PM01 G03 L-Serine C-Source, amino acid C00065 6 3 3 5 3 4

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PM01-G04 PM01 G04 L-Threonine C-Source, amino acid C00188 5 6 2 3 1 1 PM01-G05 PM01 G05 L-Alanine C-Source, amino acid C00041 5 3 2 3 3 4 PM01-G06 PM01 G06 Ala-Gly C-Source, amino acid 6 2 3 5 2 4 PM01-G07 PM01 G07 Acetoacetic acid C-Source, carboxylic acid C00164 3 2 2 2 1 2 PM01-G08 PM01 G08 N-Acetyl-D-Mannosamine C-Source, carbohydrate C00645 4 2 2 2 1 2 PM01-G09 PM01 G09 Mono-Methylsuccinate C-Source, carboxylic acid 6 4 3 6 2 4 PM01-G10 PM01 G10 Methylpyruvate C-Source, ester 6 5 3 3 2 3 PM01-G11 PM01 G11 D-Malic acid C-Source, carboxylic acid C00497 3 3 3 3 1 2 PM01-G12 PM01 G12 L-Malic acid C-Source, carboxylic acid C00149 6 3 3 6 3 3 PM01-H01 PM01 H01 Gly-Pro C-Source, amino acid 5 3 6 5 2 4 PM01-H02 PM01 H02 p-Hydroxyphenyl Acetic acid C-Source, carboxylic acid C00642 2 3 2 2 1 2 PM01-H03 PM01 H03 m-Hydroxyphenyl Acetic acid C-Source, carboxylic acid C05593 5 3 2 3 2 2 PM01-H04 PM01 H04 Tyramine C-Source, amine C00483 2 1 2 2 1 2 PM01-H05 PM01 H05 D-Psicose C-Source, carbohydrate C06468 6 2 2 2 1 2 PM01-H06 PM01 H06 L-Lyxose C-Source, carbohydrate C01508 4 1 1 1 0 1 PM01-H07 PM01 H07 Glucuronamide C-Source, amide D01791 3 1 0 1 1 0 PM01-H08 PM01 H08 Pyruvic acid C-Source, carboxylic acid C00022 6 3 3 6 2 4 PM01-H09 PM01 H09 L-Galactonic acid-g-Lactone C-Source, carboxylic acid C01115 6 4 3 6 3 2 PM01-H10 PM01 H10 D-Galacturonic acid C-Source, carboxylic acid C00333 6 3 3 6 2 2 PM01-H11 PM01 H11 Phenylethylamine C-Source, amine C05332 2 1 1 1 1 1 PM01-H12 PM01 H12 2-Aminoethanol C-Source, alcohol C00189 5 2 2 6 3 2 PM02-A01 PM02 A01 Negative Control C-Source, negative control 2 1 1 2 1 2 PM02-A02 PM02 A02 Chondroitin Sulfate C C-Source, polymer C00635 2 1 7 2 1 2 PM02-A03 PM02 A03 a-Cyclodextrin C-Source, polymer 3 2 2 2 1 2 PM02-A04 PM02 A04 b-Cyclodextrin C-Source, polymer 2 1 2 2 1 2 PM02-A05 PM02 A05 g-Cyclodextrin C-Source, polymer 5 2 3 5 2 2 PM02-A06 PM02 A06 Dextrin C-Source, polymer C00721 9 5 5 6 6 3 PM02-A07 PM02 A07 Gelatin C-Source, polymer C01498 3 2 3 3 1 2 PM02-A08 PM02 A08 Glycogen C-Source, polymer C00182 7 7 2 7 3 2 PM02-A09 PM02 A09 Inulin C-Source, polymer C00368 5 2 2 6 5 2 PM02-A10 PM02 A10 Laminarin C-Source, polymer C00771 7 8 5 9 6 7 PM02-A11 PM02 A11 Mannan C-Source, polymer C00464 2 2 2 2 1 2 PM02-A12 PM02 A12 Pectin C-Source, polymer C00714 6 4 5 5 3 2 PM02-B01 PM02 B01 N-Acetyl-D-Galactosamine C-Source, carbohydrate C01074 2 2 2 2 1 2 PM02-B02 PM02 B02 N-Acetyl-Neuraminic acid C-Source, carboxylic acid C00270 6 5 3 3 1 2 PM02-B03 PM02 B03 b-D-Allose C-Source, carbohydrate C01487 2 5 2 2 1 1 PM02-B04 PM02 B04 Amygdalin C-Source, carbohydrate C08325 2 2 1 2 1 3 PM02-B05 PM02 B05 D-Arabinose C-Source, carbohydrate C00216 6 3 6 3 1 1 PM02-B06 PM02 B06 D-Arabitol C-Source, carbohydrate C01904 8 6 6 9 5 3 PM02-B07 PM02 B07 L-Arabitol C-Source, carbohydrate C00532 3 3 6 3 1 2 PM02-B08 PM02 B08 Arbutin C-Source, carbohydrate C06186 8 6 8 8 8 3 PM02-B09 PM02 B09 2-Deoxy-D-Ribose C-Source, carbohydrate C01801 1 2 1 1 0 1 PM02-B10 PM02 B10 i-Erythritol C-Source, carbohydrate C00503 3 6 6 3 2 2 PM02-B11 PM02 B11 D-Fucose C-Source, carbohydrate C01018 2 2 2 2 1 3 PM02-B12 PM02 B12 3-O-b-D-Galactopyranosyl-D-Arabinose C-Source, carbohydrate 1 7 1 0 0 0 PM02-C01 PM02 C01 Gentiobiose C-Source, carbohydrate C08240 8 4 6 8 6 7 PM02-C02 PM02 C02 L-Glucose C-Source, carbohydrate 2 2 2 2 1 1 PM02-C03 PM02 C03 D-Lactitol C-Source, carbohydrate 3 3 2 2 1 2 PM02-C04 PM02 C04 D-Melezitose C-Source, carbohydrate C08243 9 2 2 8 6 2 PM02-C05 PM02 C05 Maltitol C-Source, carbohydrate G00275 2 1 3 5 2 2 PM02-C06 PM02 C06 a-Methyl-D-Glucoside C-Source, carbohydrate 8 2 6 8 3 2 PM02-C07 PM02 C07 b-Methyl-D-Galactoside C-Source, carbohydrate C03619 8 7 5 8 5 2 PM02-C08 PM02 C08 3-Methylglucose C-Source, carbohydrate 2 2 2 3 1 2 PM02-C09 PM02 C09 b-Methyl-D-Glucuronic acid C-Source, carboxylic acid C08350 9 6 3 6 3 2 PM02-C10 PM02 C10 a-Methyl-D-Mannoside C-Source, carbohydrate 2 2 2 2 1 2 PM02-C11 PM02 C11 b-Methyl-D-Xyloside C-Source, carbohydrate 5 2 3 2 1 2 PM02-C12 PM02 C12 Palatinose C-Source, carbohydrate C01742 5 4 4 8 6 1 PM02-D01 PM02 D01 D-Raffinose C-Source, carbohydrate C00492 8 5 6 8 6 2 PM02-D02 PM02 D02 Salicin C-Source, carbohydrate C01451 6 2 3 5 2 4 PM02-D03 PM02 D03 Sedoheptulosan C-Source, carbohydrate 2 1 2 2 1 2 PM02-D04 PM02 D04 L-Sorbose C-Source, carbohydrate C00247 8 1 4 6 6 0 PM02-D05 PM02 D05 Stachyose C-Source, carbohydrate C01613 6 4 5 5 6 2 PM02-D06 PM02 D06 D-Tagatose C-Source, carbohydrate C00795 2 2 2 2 1 1 PM02-D07 PM02 D07 Turanose C-Source, carbohydrate G03588 9 3 3 8 6 2 PM02-D08 PM02 D08 Xylitol C-Source, carbohydrate C00379 4 2 6 3 3 1 PM02-D09 PM02 D09 N-Acetyl-D-Glucosaminitol C-Source, carbohydrate 2 2 2 2 1 0 PM02-D10 PM02 D10 g-Amino-N-Butyric acid C-Source, carboxylic acid C00334 5 5 3 5 3 3 PM02-D11 PM02 D11 d-Amino Valeric acid C-Source, carboxylic acid C00431 3 2 2 3 1 2 PM02-D12 PM02 D12 Butyric acid C-Source, carboxylic acid C00246 5 3 1 3 2 3 PM02-E01 PM02 E01 Capric acid C-Source, carboxylic acid C01571 0 0 0 0 0 0 PM02-E02 PM02 E02 Caproic acid C-Source, carboxylic acid C01585 1 1 0 1 1 0 PM02-E03 PM02 E03 Citraconic acid C-Source, carboxylic acid C02226 3 2 2 2 1 1 PM02-E04 PM02 E04 Citramalic acid C-Source, carboxylic acid C00815 3 1 2 2 1 1 PM02-E05 PM02 E05 D-Glucosamine C-Source, carbohydrate C00329 9 3 4 7 6 7 PM02-E06 PM02 E06 2-Hydroxybenzoic acid C-Source, carboxylic acid C00805 5 2 1 3 1 2 PM02-E07 PM02 E07 4-Hydroxybenzoic acid C-Source, carboxylic acid C00156 3 2 2 6 1 2

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PM02-E08 PM02 E08 b-Hydroxybutyric acid C-Source, carboxylic acid C01089 5 5 3 5 3 4 PM02-E09 PM02 E09 g-Hydroxybutyric acid C-Source, carboxylic acid C00989 6 5 3 6 2 2 PM02-E10 PM02 E10 a-Keto-Valeric acid C-Source, carboxylic acid C00567 6 3 2 3 2 3 PM02-E11 PM02 E11 Itaconic acid C-Source, carboxylic acid C00490 2 2 2 2 1 2 PM02-E12 PM02 E12 5-Keto-D-Gluconic acid C-Source, carboxylic acid C01062 6 3 3 3 3 2 PM02-F01 PM02 F01 D-Lactic acid Methyl Ester C-Source, ester 3 3 2 3 3 3 PM02-F02 PM02 F02 Malonic acid C-Source, carboxylic acid C00383 3 2 2 3 2 2 PM02-F03 PM02 F03 Melibionic acid C-Source, carbohydrate 9 2 2 9 3 2 PM02-F04 PM02 F04 Oxalic acid C-Source, carboxylic acid C00209 2 2 2 2 1 2 PM02-F05 PM02 F05 Oxalomalic acid C-Source, carboxylic acid C01990 3 2 2 2 1 2 PM02-F06 PM02 F06 Quinic acid C-Source, carboxylic acid C00296 5 3 2 5 2 2 PM02-F07 PM02 F07 D-Ribono-1,4-Lactone C-Source, carboxylic acid 3 2 2 2 1 2 PM02-F08 PM02 F08 Sebacic acid C-Source, carboxylic acid C08277 5 7 2 5 1 6 PM02-F09 PM02 F09 Sorbic acid C-Source, carboxylic acid 1 2 1 2 1 1 PM02-F10 PM02 F10 Succinamic acid C-Source, carboxylic acid 5 5 6 3 2 3 PM02-F11 PM02 F11 D-Tartaric acid C-Source, carboxylic acid C02107 6 2 1 3 1 1 PM02-F12 PM02 F12 L-Tartaric acid C-Source, carboxylic acid C00898 6 2 3 3 3 2 PM02-G01 PM02 G01 Acetamide C-Source, amide C06244 2 2 2 2 1 2 PM02-G02 PM02 G02 L-Alaninamide C-Source, amide 5 3 3 5 2 3 PM02-G03 PM02 G03 N-Acetyl-L-Glutamic acid C-Source, amino acid C00624 3 2 2 2 1 2 PM02-G04 PM02 G04 L-Arginine C-Source, amino acid C00062 6 3 3 5 2 4 PM02-G05 PM02 G05 Glycine C-Source, amino acid C00037 3 3 2 3 2 4 PM02-G06 PM02 G06 L-Histidine C-Source, amino acid C00135 1 1 1 1 1 1 PM02-G07 PM02 G07 L-Homoserine C-Source, amino acid C00263 3 2 2 2 1 2 PM02-G08 PM02 G08 Hydroxy-L-Proline C-Source, amino acid C01015 0 1 0 0 0 0 PM02-G09 PM02 G09 L-Isoleucine C-Source, amino acid C00407 5 6 3 5 2 3 PM02-G10 PM02 G10 L-Leucine C-Source, amino acid C00123 5 3 3 2 1 4 PM02-G11 PM02 G11 L-Lysine C-Source, amino acid C00047 5 3 3 3 2 1 PM02-G12 PM02 G12 L-Methionine C-Source, amino acid C00073 0 1 0 0 0 0 PM02-H01 PM02 H01 L-Ornithine C-Source, amino acid C00077 5 6 3 5 3 4 PM02-H02 PM02 H02 L-Phenylalanine C-Source, amino acid C00079 3 2 2 3 1 1 PM02-H03 PM02 H03 L-Pyroglutamic acid C-Source, amino acid C02238 5 5 3 5 3 4 PM02-H04 PM02 H04 L-Valine C-Source, amino acid C00183 3 6 3 3 2 4 PM02-H05 PM02 H05 D,L-Carnitine C-Source, carboxylic acid C00487 2 2 2 2 1 2 PM02-H06 PM02 H06 sec-Butylamine C-Source, amine 1 2 2 2 0 1 PM02-H07 PM02 H07 D,L-Octopamine C-Source, amine C04227 1 1 2 2 1 2 PM02-H08 PM02 H08 Putrescine C-Source, amine C00134 2 2 2 2 1 2 PM02-H09 PM02 H09 Dihydroxyacetone C-Source, alcohol C00184 3 3 3 5 2 3 PM02-H10 PM02 H10 2,3-Butanediol C-Source, alcohol C03044 3 1 2 2 1 2 PM02-H11 PM02 H11 2,3-Butanedione C-Source, alcohol C00741 3 2 2 2 1 2 PM02-H12 PM02 H12 3-Hydroxy-2-buta C-Source, alcohol C00466 2 2 2 2 1 2

Supplementary Table 1. Results of PM carbon source analysis at 13°C (without Biolog Dye). Activity indices (AIs) are given for each species in the 5 rightmost columns.

238

#plate_id well_id chemical moa co_id 24MN13 23342-1 5NY8 Pverr WSF3629 Pd PM01 A01 Negative Control C-Source, negative control 3 1 2 1.5 1 2 PM01 A02 L-Arabinose C-Source, carbohydrate C00259 9 4 6 8.5 6 1.5 PM01 A03 N-Acetyl-D-Glucosamine C-Source, carbohydrate C00140 8 6 6 7.5 4 4.5 PM01 A04 D-Saccharic acid C-Source, carboxylic acid C00818 6 2 2 5 2 2 PM01 A05 Succinic acid C-Source, carboxylic acid C00042 5.5 2 5 5 4 2 PM01 A06 D-Galactose C-Source, carbohydrate C00124 8.5 4 6 8 6 2 PM01 A07 L-Aspartic acid C-Source, amino acid C00049 5.5 2 5.5 3 5 3 PM01 A08 L-Proline C-Source, amino acid C00148 7 4 5 7 5 5 PM01 A09 D-Alanine C-Source, amino acid C00133 2.5 0 1 1.5 5 1 PM01 A10 D-Trehalose C-Source, carbohydrate C01083 8 6 6 8 6 6 PM01 A11 D-Mannose C-Source, carbohydrate C00159 8.5 6 6 8.5 4 7.5 PM01 A12 Dulcitol C-Source, carbohydrate C01697 7.5 5 7 8 5 2 PM01 B01 D-Serine C-Source, amino acid C00740 1.5 0 1 1 0 0 PM01 B02 D-Sorbitol C-Source, carbohydrate C00794 8.5 5 6 7 7 2 PM01 B03 Glycerol C-Source, carbohydrate C00116 4 2 4 5 1 4.5 PM01 B04 L-Fucose C-Source, carbohydrate C01019 7 2 5 4 2 1 PM01 B05 D-Glucuronic acid C-Source, carboxylic acid C00191 6 2 5 4.5 5 1.5 PM01 B06 D-Gluconic acid C-Source, carboxylic acid C00257 9 5 5.5 6.5 6 2 PM01 B07 DL-a-Glycerol Phosphate C-Source, carbohydrate C00093 3 1 2 2 1 3 PM01 B08 D-Xylose C-Source, carbohydrate C00181 9 4 6 7 5 1 PM01 B09 L-Lactic acid C-Source, carboxylic acid C01432 4 1 3 3 2 3 PM01 B10 Formic acid C-Source, carboxylic acid C00058 3 2 3 3 1 2 PM01 B11 D-Mannitol C-Source, carbohydrate C00392 8.5 6 7 7.5 5 4 PM01 B12 L-Glutamic acid C-Source, amino acid C00025 7.5 4 6 6 5 4.5 PM01 C01 D-Glucose-6-Phosphate C-Source, carbohydrate C00092 4 1 2 2 1 2 PM01 C02 D-Galactonic acid-g-Lactone C-Source, carboxylic acid C03383 2 1 2 4 1 0 PM01 C03 DL-Malic acid C-Source, carboxylic acid C00497 5.5 4 4.5 5 2 4 PM01 C04 D-Ribose C-Source, carbohydrate C00121 9 2 6 8 5 2 PM01 C05 Tween 20 C-Source, fatty acid C11624 3 2 3.5 4 4 4.5 PM01 C06 L-Rhamnose C-Source, carbohydrate C00507 7 4 4 7 5 4.5 PM01 C07 D-Fructose C-Source, carbohydrate C00095 8 7 6 7.5 7 6 PM01 C08 Acetic acid C-Source, carboxylic acid C00033 4 2 3 4.5 2 4 PM01 C09 a-D-Glucose C-Source, carbohydrate C00031 8.5 6 6 8 7 6 PM01 C10 Maltose C-Source, carbohydrate C00208 9 6 6 8 7 3 PM01 C11 D-Melibiose C-Source, carbohydrate C05402 8 5 6 8.5 6 2 PM01 C12 Thymidine C-Source, carbohydrate C00214 3 1 2 2 1 2 PM01 D01 L-Asparagine C-Source, amino acid C00152 6.5 4 5 6.5 5 3 PM01 D02 D-Aspartic acid C-Source, amino acid C00402 4 1 3 2 1 2 PM01 D03 D-Glucosaminic acid C-Source, carboxylic acid C03752 2 1 2 2 1 2 PM01 D04 1,2-Propanediol C-Source, alcohol C00583 2 1 2 2 1 2 PM01 D05 Tween 40 C-Source, fatty acid 5.5 5 6.5 6 7 6 PM01 D06 a-Ketoglutaric acid C-Source, carboxylic acid C00026 5 2 4.5 3 2 2 PM01 D07 a-Ketobutyric acid C-Source, carboxylic acid C00109 4.5 2 4 4 2 2 PM01 D08 a-Methyl-D-Galactoside C-Source, carbohydrate C03619 3 1 2 3 2 2 PM01 D09 a-D-Lactose C-Source, carbohydrate C00243 7.5 4 7 9 2 2 PM01 D10 Lactulose C-Source, carbohydrate C07064 6.5 1 4.5 5 1 2 PM01 D11 Sucrose C-Source, carbohydrate C00089 8 6 6 7 7 2 PM01 D12 Uridine C-Source, carbohydrate C00299 4 1 4 3.5 2 2 PM01 E01 L-Glutamine C-Source, amino acid C00064 7 5 5 7 5 6 PM01 E02 m-Tartaric acid C-Source, carboxylic acid C00552 5.5 1 1 4 1 1 PM01 E03 D-Glucose-1-Phosphate C-Source, carbohydrate C00103 3 1 2 3 1 2 PM01 E04 D-Fructose-6-Phosphate C-Source, carbohydrate C00085 4 1 2 2 1 2 PM01 E05 Tween 80 C-Source, fatty acid C11625 6.5 5 5 6 7 5.5 PM01 E06 a-Hydroxyglutaric acid-g-Lactone C-Source, carboxylic acid 3 2 2 3 2 2 PM01 E07 a-Hydroxybutyric acid C-Source, carboxylic acid C05984 4.5 1 3 5 2 2 PM01 E08 b-Methyl-D-Glucoside C-Source, carbohydrate 8.5 6 6 8 5 4 PM01 E09 Adonitol C-Source, carbohydrate C00474 9 5 6 7.5 7 2 PM01 E10 Maltotriose C-Source, carbohydrate C01835 9 6 6.5 8 6 2 PM01 E11 2`-Deoxyadenosine C-Source, carbohydrate C00559 3 1 2 2 2 1 PM01 E12 Adenosine C-Source, carbohydrate C00212 4 1 5.5 3 2 4 PM01 F01 Gly-Asp C-Source, amino acid C02871 5.5 2 4 5 2 3 PM01 F02 Citric acid C-Source, carboxylic acid C00158 5 4 4.5 5 4 4.5 PM01 F03 m-Inositol C-Source, carbohydrate C00137 4.5 1 4.5 5 1 2 PM01 F04 D-Threonine C-Source, amino acid C00820 1.5 1 2 1 1 1 PM01 F05 Fumaric acid C-Source, carboxylic acid C00122 5.5 4 5 5.5 4 4 PM01 F06 Bromosuccinic acid C-Source, carboxylic acid 4 2 4 4.5 1 2 PM01 F07 Propionic acid C-Source, carboxylic acid C00163 4.5 2 1 4 1 3 PM01 F08 Mucic acid C-Source, carboxylic acid C01807 3 2 3 2 2 3 PM01 F09 Glycolic acid C-Source, carboxylic acid C00160 4.5 1 2 2 2 2 PM01 F10 Glyoxylic acid C-Source, carboxylic acid C00048 3 1 2 2 1 2 PM01 F11 D-Cellobiose C-Source, carbohydrate C00185 8 7 6 8 7 4 PM01 F12 Inosine C-Source, carbohydrate C00294 6 1 5.5 7 2 4 PM01 G01 Gly-Glu C-Source, amino acid 6 2 5 6 4 4 PM01 G02 Tricarballylic acid C-Source, carboxylic acid 3 1 3 2 2 0 PM01 G03 L-Serine C-Source, amino acid C00065 6 2 4 6 4 5

239

PM01 G04 L-Threonine C-Source, amino acid C00188 5.5 1 4 4 2 2 PM01 G05 L-Alanine C-Source, amino acid C00041 5 5 3.5 5 4 5 PM01 G06 Ala-Gly C-Source, amino acid 6.5 2 5 7 4 4.5 PM01 G07 Acetoacetic acid C-Source, carboxylic acid C00164 4 1 3 4 2 0.5 PM01 G08 N-Acetyl-D-Mannosamine C-Source, carbohydrate C00645 3 1 2 3 2 2 PM01 G09 Mono-Methylsuccinate C-Source, carboxylic acid 6 2 4.5 6 4 5 PM01 G10 Methylpyruvate C-Source, ester 4.5 2 4.5 5 2 4 PM01 G11 D-Malic acid C-Source, carboxylic acid C00497 4.5 2 4.5 4.5 2 2 PM01 G12 L-Malic acid C-Source, carboxylic acid C00149 5.5 2 5 5 5 4 PM01 H01 Gly-Pro C-Source, amino acid 6.5 2 5 7 4 4.5 PM01 H02 p-Hydroxyphenyl Acetic acid C-Source, carboxylic acid C00642 4 2 4 3 1 1 PM01 H03 m-Hydroxyphenyl Acetic acid C-Source, carboxylic acid C05593 7 2 4.5 5 4 2 PM01 H04 Tyramine C-Source, amine C00483 3 1 3 3 1 1 PM01 H05 D-Psicose C-Source, carbohydrate C06468 3 1 3 3 2 1.5 PM01 H06 L-Lyxose C-Source, carbohydrate C01508 5.5 1 1 3.5 0 1 PM01 H07 Glucuronamide C-Source, amide D01791 4 1 0 1 1 0 PM01 H08 Pyruvic acid C-Source, carboxylic acid C00022 5 2 5 5 2 3 PM01 H09 L-Galactonic acid-g-Lactone C-Source, carboxylic acid C01115 5.5 5 4 5.5 4 2 PM01 H10 D-Galacturonic acid C-Source, carboxylic acid C00333 6.5 2 5 6 4 2 PM01 H11 Phenylethylamine C-Source, amine C05332 2 1 1.5 1 2 1 PM01 H12 2-Aminoethanol C-Source, alcohol C00189 5 1 4.5 5 4 2 PM02 A01 Negative Control C-Source, negative control 3 1 1.5 1.5 1 1.5 PM02 A02 Chondroitin Sulfate C C-Source, polymer C00635 3 1 2 2 2 2 PM02 A03 a-Cyclodextrin C-Source, polymer 2 1 1.5 3 1 1.5 PM02 A04 b-Cyclodextrin C-Source, polymer 1 1 1 1.5 1 2 PM02 A05 g-Cyclodextrin C-Source, polymer 6 5 5 5 4 2 PM02 A06 Dextrin C-Source, polymer C00721 9 6 6 6.5 5 4 PM02 A07 Gelatin C-Source, polymer C01498 3 2 4.5 3 2 4 PM02 A08 Glycogen C-Source, polymer C00182 7.5 4 2 6 6 4 PM02 A09 Inulin C-Source, polymer C00368 6 1 2 6 5 2 PM02 A10 Laminarin C-Source, polymer C00771 9 4 6.5 7.5 6 6 PM02 A11 Mannan C-Source, polymer C00464 3 1 2 2 2 3 PM02 A12 Pectin C-Source, polymer C00714 6 2 5 5 4 3 PM02 B01 N-Acetyl-D-Galactosamine C-Source, carbohydrate C01074 3 1 2 2 1 4 PM02 B02 N-Acetyl-Neuraminic acid C-Source, carboxylic acid C00270 4.5 4 4.5 3 1 2 PM02 B03 b-D-Allose C-Source, carbohydrate C01487 2 1 2 2 1 1 PM02 B04 Amygdalin C-Source, carbohydrate C08325 3 1 1 2 1 4.5 PM02 B05 D-Arabinose C-Source, carbohydrate C00216 5 1 5 4 2 1 PM02 B06 D-Arabitol C-Source, carbohydrate C01904 9 7 6.5 7.5 7 4 PM02 B07 L-Arabitol C-Source, carbohydrate C00532 4 1 5 3 2 2 PM02 B08 Arbutin C-Source, carbohydrate C06186 9 2 8 8 8 1 PM02 B09 2-Deoxy-D-Ribose C-Source, carbohydrate C01801 0.5 1 2.5 1.5 0 1.5 PM02 B10 i-Erythritol C-Source, carbohydrate C00503 4 1 5.5 3 2 2 PM02 B11 D-Fucose C-Source, carbohydrate C01018 2 1 2 2 2 4 PM02 B12 3-O-b-D-Galactopyranosyl-D-Arabinose C-Source, carbohydrate 1.5 0 1 0 0 0 PM02 C01 Gentiobiose C-Source, carbohydrate C08240 8.5 3 6 7.5 4 5.5 PM02 C02 L-Glucose C-Source, carbohydrate 4 1 1 2 2 1 PM02 C03 D-Lactitol C-Source, carbohydrate 3 1 2 2 1 2 PM02 C04 D-Melezitose C-Source, carbohydrate C08243 9 5 2 7.5 6 2 PM02 C05 Maltitol C-Source, carbohydrate G00275 3 2 4.5 4.5 4 2 PM02 C06 a-Methyl-D-Glucoside C-Source, carbohydrate 7 2 5.5 7 5 2 PM02 C07 b-Methyl-D-Galactoside C-Source, carbohydrate C03619 8 4 6.5 7.5 6 2 PM02 C08 3-Methylglucose C-Source, carbohydrate 3 2 2 4 1 2 PM02 C09 b-Methyl-D-Glucuronic acid C-Source, carboxylic acid C08350 6.5 4 6 5 5 2 PM02 C10 a-Methyl-D-Mannoside C-Source, carbohydrate 2 1 2 2 1 2 PM02 C11 b-Methyl-D-Xyloside C-Source, carbohydrate 5 1 4 2 1 2 PM02 C12 Palatinose C-Source, carbohydrate C01742 7 4 6 7.5 6 2 PM02 D01 D-Raffinose C-Source, carbohydrate C00492 7.5 6 6 6.5 6 2 PM02 D02 Salicin C-Source, carbohydrate C01451 5.5 4 4.5 5 2 3 PM02 D03 Sedoheptulosan C-Source, carbohydrate 4 1 2 2 1 1.5 PM02 D04 L-Sorbose C-Source, carbohydrate C00247 8 4 6 6 4 0 PM02 D05 Stachyose C-Source, carbohydrate C01613 5 5 7 6 6 2 PM02 D06 D-Tagatose C-Source, carbohydrate C00795 2 1 2 2 2 1.5 PM02 D07 Turanose C-Source, carbohydrate G03588 9 4 5 7 4 2 PM02 D08 Xylitol C-Source, carbohydrate C00379 5 1 5.5 4 4 3 PM02 D09 N-Acetyl-D-Glucosaminitol C-Source, carbohydrate 2 1 2 2 1 0 PM02 D10 g-Amino-N-Butyric acid C-Source, carboxylic acid C00334 7 2 5 7 5 2.5 PM02 D11 d-Amino Valeric acid C-Source, carboxylic acid C00431 4.5 1 2 4 2 2 PM02 D12 Butyric acid C-Source, carboxylic acid C00246 5.5 4 2 4 2 2 PM02 E01 Capric acid C-Source, carboxylic acid C01571 0 0 0 0 0 0 PM02 E02 Caproic acid C-Source, carboxylic acid C01585 1 0 0 1 1 0 PM02 E03 Citraconic acid C-Source, carboxylic acid C02226 3 1 4 2 1 2 PM02 E04 Citramalic acid C-Source, carboxylic acid C00815 3 1 2 1.5 1 0 PM02 E05 D-Glucosamine C-Source, carbohydrate C00329 9 4 6 7.5 5 3.5 PM02 E06 2-Hydroxybenzoic acid C-Source, carboxylic acid C00805 5 1 2 4 2 1 PM02 E07 4-Hydroxybenzoic acid C-Source, carboxylic acid C00156 5 1 3 4 2 1.5

240

PM02 E08 b-Hydroxybutyric acid C-Source, carboxylic acid C01089 6 1 4.5 5 4 4 PM02 E09 g-Hydroxybutyric acid C-Source, carboxylic acid C00989 5 2 4.5 5 4 3 PM02 E10 a-Keto-Valeric acid C-Source, carboxylic acid C00567 4.5 1 4.5 5 2 1.5 PM02 E11 Itaconic acid C-Source, carboxylic acid C00490 3 1 1 2 1 2 PM02 E12 5-Keto-D-Gluconic acid C-Source, carboxylic acid C01062 5 2 5 4 5 2 PM02 F01 D-Lactic acid Methyl Ester C-Source, ester 4.5 1 4 4 4 4 PM02 F02 Malonic acid C-Source, carboxylic acid C00383 4 1 4 3 2 2 PM02 F03 Melibionic acid C-Source, carbohydrate 9 4 3 9 5 1 PM02 F04 Oxalic acid C-Source, carboxylic acid C00209 3 1 2 2 1 2 PM02 F05 Oxalomalic acid C-Source, carboxylic acid C01990 3 1 2 2 1 1.5 PM02 F06 Quinic acid C-Source, carboxylic acid C00296 5.5 2 3 5 1 2 PM02 F07 D-Ribono-1,4-Lactone C-Source, carboxylic acid 3 1 2 2 2 2 PM02 F08 Sebacic acid C-Source, carboxylic acid C08277 6 2 4 5 1 4 PM02 F09 Sorbic acid C-Source, carboxylic acid 1 1 0.5 1 1 0 PM02 F10 Succinamic acid C-Source, carboxylic acid 6.5 1 5 5 2 3 PM02 F11 D-Tartaric acid C-Source, carboxylic acid C02107 6 1 1 4 1 0.5 PM02 F12 L-Tartaric acid C-Source, carboxylic acid C00898 6 2 4.5 4.5 4 2 PM02 G01 Acetamide C-Source, amide C06244 3 1 2 2 1 2 PM02 G02 L-Alaninamide C-Source, amide 6.5 2 4 5 4 4.5 PM02 G03 N-Acetyl-L-Glutamic acid C-Source, amino acid C00624 4 1 2 2 1 2 PM02 G04 L-Arginine C-Source, amino acid C00062 5.5 2 5 6 4 3 PM02 G05 Glycine C-Source, amino acid C00037 4.5 1 4 4.5 2 4.5 PM02 G06 L-Histidine C-Source, amino acid C00135 3.5 1 1 1 1 1 PM02 G07 L-Homoserine C-Source, amino acid C00263 3 1 2 3 1 1.5 PM02 G08 Hydroxy-L-Proline C-Source, amino acid C01015 0 0 0 0.5 0 0 PM02 G09 L-Isoleucine C-Source, amino acid C00407 6 1 5 6 4 3 PM02 G10 L-Leucine C-Source, amino acid C00123 5 2 4 4 4 1.5 PM02 G11 L-Lysine C-Source, amino acid C00047 5 2 4.5 5 4 1 PM02 G12 L-Methionine C-Source, amino acid C00073 0 1 0 0.5 0 0 PM02 H01 L-Ornithine C-Source, amino acid C00077 7 4 5 6 4 3.5 PM02 H02 L-Phenylalanine C-Source, amino acid C00079 3.5 1 2.5 4 2 1 PM02 H03 L-Pyroglutamic acid C-Source, amino acid C02238 7 4 5 6 5 4.5 PM02 H04 L-Valine C-Source, amino acid C00183 4.5 1 4.5 4 2 3 PM02 H05 D,L-Carnitine C-Source, carboxylic acid C00487 3 1 2 1.5 1 2 PM02 H06 sec-Butylamine C-Source, amine 1.5 0 2 0.5 0 2 PM02 H07 D,L-Octopamine C-Source, amine C04227 2 1 2 2 1 1.5 PM02 H08 Putrescine C-Source, amine C00134 3 1 2 2 1 1.5 PM02 H09 Dihydroxyacetone C-Source, alcohol C00184 4.5 2 4 4 4 4 PM02 H10 2,3-Butanediol C-Source, alcohol C03044 3 1 2 2 2 2 PM02 H11 2,3-Butanedione C-Source, alcohol C00741 4 1 2 4 2 1 PM02 H12 3-Hydroxy-2-buta C-Source, alcohol C00466 3 1 2 2 2 2

Supplementary Table 2. Results of PM carbon source analysis at 18°C (without Biolog Dye). Activity indices (AIs) are given for each species in the 5 rightmost columns.

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#plate_id well_id chemical moa co_id 24MN13 3VT5 5NY8 Pverr WSF3629 Pd PM01 A01 Negative Control C-Source, negative control 1 3 3 1 1 3 PM01 A02 L-Arabinose C-Source, carbohydrate C00259 7 7 6 7 6 1 PM01 A03 N-Acetyl-D-Glucosamine C-Source, carbohydrate C00140 7 7 7 7 6 8 PM01 A04 D-Saccharic acid C-Source, carboxylic acid C00818 4 3 3 3 4 3 PM01 A05 Succinic acid C-Source, carboxylic acid C00042 4 5 5 3 3 4 PM01 A06 D-Galactose C-Source, carbohydrate C00124 7 7 6 6 4 5 PM01 A07 L-Aspartic acid C-Source, amino acid C00049 4 5 5 3 3 4 PM01 A08 L-Proline C-Source, amino acid C00148 5 5 5 5 3 7 PM01 A09 D-Alanine C-Source, amino acid C00133 1 3 1 1 0 1 PM01 A10 D-Trehalose C-Source, carbohydrate C01083 7 7 7 7 6 9 PM01 A11 D-Mannose C-Source, carbohydrate C00159 7 8 6 6 6 9 PM01 A12 Dulcitol C-Source, carbohydrate C01697 5 5 5 5 1 3 PM01 B01 D-Serine C-Source, amino acid C00740 1 3 1 1 0 0 PM01 B02 D-Sorbitol C-Source, carbohydrate C00794 7 5 6 7 5 4 PM01 B03 Glycerol C-Source, carbohydrate C00116 3 5 3 4 1 7 PM01 B04 L-Fucose C-Source, carbohydrate C01019 5 3 3 1 1 1 PM01 B05 D-Glucuronic acid C-Source, carboxylic acid C00191 6 4 4 4 4 3 PM01 B06 D-Gluconic acid C-Source, carboxylic acid C00257 6 6 6 6 6 4 PM01 B07 DL-a-Glycerol Phosphate C-Source, carbohydrate C00093 3 2 3 1 1 3 PM01 B08 D-Xylose C-Source, carbohydrate C00181 8 8 7 8 6 3 PM01 B09 L-Lactic acid C-Source, carboxylic acid C01432 1 5 3 4 4 8 PM01 B10 Formic acid C-Source, carboxylic acid C00058 1 3 3 1 1 3 PM01 B11 D-Mannitol C-Source, carbohydrate C00392 5 5 5 6 1 6 PM01 B12 L-Glutamic acid C-Source, amino acid C00025 6 5 5 5 3 8 PM01 C01 D-Glucose-6-Phosphate C-Source, carbohydrate C00092 1 3 3 4 1 3 PM01 C02 D-Galactonic acid-g-Lactone C-Source, carboxylic acid C03383 1 1 3 1 0 0 PM01 C03 DL-Malic acid C-Source, carboxylic acid C00497 5 5 3 4 3 6 PM01 C04 D-Ribose C-Source, carbohydrate C00121 9 7 8 7 8 4 PM01 C05 Tween 20 C-Source, fatty acid C11624 3 5 3 3 3 8 PM01 C06 L-Rhamnose C-Source, carbohydrate C00507 5 7 5 5 6 8 PM01 C07 D-Fructose C-Source, carbohydrate C00095 7 7 6 6 6 9 PM01 C08 Acetic acid C-Source, carboxylic acid C00033 3 5 3 3 4 8 PM01 C09 a-D-Glucose C-Source, carbohydrate C00031 6 6 7 7 6 9 PM01 C10 Maltose C-Source, carbohydrate C00208 6 7 5 7 6 4 PM01 C11 D-Melibiose C-Source, carbohydrate C05402 7 7 5 7 6 3 PM01 C12 Thymidine C-Source, carbohydrate C00214 1 2 3 1 1 3 PM01 D01 L-Asparagine C-Source, amino acid C00152 6 5 5 6 3 7 PM01 D02 D-Aspartic acid C-Source, amino acid C00402 1 3 3 1 1 1 PM01 D03 D-Glucosaminic acid C-Source, carboxylic acid C03752 1 3 3 1 1 4 PM01 D04 1,2-Propanediol C-Source, alcohol C00583 1 2 3 1 1 4 PM01 D05 Tween 40 C-Source, fatty acid 4 6 6 6 6 9 PM01 D06 a-Ketoglutaric acid C-Source, carboxylic acid C00026 3 5 3 3 3 3 PM01 D07 a-Ketobutyric acid C-Source, carboxylic acid C00109 3 5 3 3 1 3 PM01 D08 a-Methyl-D-Galactoside C-Source, carbohydrate C03619 1 3 3 1 1 4 PM01 D09 a-D-Lactose C-Source, carbohydrate C00243 7 6 5 6 1 3 PM01 D10 Lactulose C-Source, carbohydrate C07064 4 8 3 3 1 3 PM01 D11 Sucrose C-Source, carbohydrate C00089 7 5 7 6 6 3 PM01 D12 Uridine C-Source, carbohydrate C00299 1 3 3 1 1 3 PM01 E01 L-Glutamine C-Source, amino acid C00064 5 5 6 7 3 8 PM01 E02 m-Tartaric acid C-Source, carboxylic acid C00552 4 3 1 1 1 1 PM01 E03 D-Glucose-1-Phosphate C-Source, carbohydrate C00103 1 3 3 1 1 3 PM01 E04 D-Fructose-6-Phosphate C-Source, carbohydrate C00085 3 5 3 4 1 3 PM01 E05 Tween 80 C-Source, fatty acid C11625 6 7 5 6 6 9 PM01 E06 a-Hydroxyglutaric acid-g-Lactone C-Source, carboxylic acid 1 3 3 1 1 4 PM01 E07 a-Hydroxybutyric acid C-Source, carboxylic acid C05984 3 5 3 4 3 5 PM01 E08 b-Methyl-D-Glucoside C-Source, carbohydrate 7 7 6 6 6 4 PM01 E09 Adonitol C-Source, carbohydrate C00474 6 7 7 6 4 3 PM01 E10 Maltotriose C-Source, carbohydrate C01835 7 7 5 7 6 3 PM01 E11 2`-Deoxyadenosine C-Source, carbohydrate C00559 1 5 3 1 1 1 PM01 E12 Adenosine C-Source, carbohydrate C00212 1 5 6 1 1 4 PM01 F01 Gly-Asp C-Source, amino acid C02871 5 5 5 3 1 4 PM01 F02 Citric acid C-Source, carboxylic acid C00158 4 5 5 4 3 8 PM01 F03 m-Inositol C-Source, carbohydrate C00137 3 5 3 1 1 1 PM01 F04 D-Threonine C-Source, amino acid C00820 1 1 3 1 1 1 PM01 F05 Fumaric acid C-Source, carboxylic acid C00122 4 7 3 4 4 6 PM01 F06 Bromosuccinic acid C-Source, carboxylic acid 1 5 3 3 1 4 PM01 F07 Propionic acid C-Source, carboxylic acid C00163 3 3 3 3 2 8 PM01 F08 Mucic acid C-Source, carboxylic acid C01807 1 3 3 1 1 3 PM01 F09 Glycolic acid C-Source, carboxylic acid C00160 1 5 3 1 6 5 PM01 F10 Glyoxylic acid C-Source, carboxylic acid C00048 1 5 3 1 1 3 PM01 F11 D-Cellobiose C-Source, carbohydrate C00185 7 7 7 7 6 8 PM01 F12 Inosine C-Source, carbohydrate C00294 5 5 9 5 1 4 PM01 G01 Gly-Glu C-Source, amino acid 6 5 5 6 3 6 PM01 G02 Tricarballylic acid C-Source, carboxylic acid 3 3 5 1 1 0 PM01 G03 L-Serine C-Source, amino acid C00065 4 5 3 5 3 8

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PM01 G04 L-Threonine C-Source, amino acid C00188 3 5 3 3 1 3 PM01 G05 L-Alanine C-Source, amino acid C00041 5 5 4 5 3 8 PM01 G06 Ala-Gly C-Source, amino acid 5 5 5 5 4 6 PM01 G07 Acetoacetic acid C-Source, carboxylic acid C00164 1 5 3 3 1 2 PM01 G08 N-Acetyl-D-Mannosamine C-Source, carbohydrate C00645 1 3 3 3 1 3 PM01 G09 Mono-Methylsuccinate C-Source, carboxylic acid 4 5 5 3 4 8 PM01 G10 Methylpyruvate C-Source, ester 3 5 5 4 4 8 PM01 G11 D-Malic acid C-Source, carboxylic acid C00497 1 7 3 3 1 4 PM01 G12 L-Malic acid C-Source, carboxylic acid C00149 5 7 3 4 4 6 PM01 H01 Gly-Pro C-Source, amino acid 5 5 5 5 1 6 PM01 H02 p-Hydroxyphenyl Acetic acid C-Source, carboxylic acid C00642 1 3 3 1 1 1 PM01 H03 m-Hydroxyphenyl Acetic acid C-Source, carboxylic acid C05593 5 7 3 3 3 3 PM01 H04 Tyramine C-Source, amine C00483 1 3 3 1 1 3 PM01 H05 D-Psicose C-Source, carbohydrate C06468 1 3 3 1 1 3 PM01 H06 L-Lyxose C-Source, carbohydrate C01508 7 4 3 3 3 3 PM01 H07 Glucuronamide C-Source, amide D01791 4 3 1 1 1 0 PM01 H08 Pyruvic acid C-Source, carboxylic acid C00022 6 7 5 5 4 8 PM01 H09 L-Galactonic acid-g-Lactone C-Source, carboxylic acid C01115 4 7 7 5 5 3 PM01 H10 D-Galacturonic acid C-Source, carboxylic acid C00333 4 5 5 4 4 3 PM01 H11 Phenylethylamine C-Source, amine C05332 1 1 1 1 1 1 PM01 H12 2-Aminoethanol C-Source, alcohol C00189 5 5 3 4 1 3 PM02 A01 Negative Control C-Source, negative control 1 1 3 1 1 5 PM02 A02 Chondroitin Sulfate C C-Source, polymer C00635 1 1 1 1 1 3 PM02 A03 a-Cyclodextrin C-Source, polymer 1 1 1 1 1 1 PM02 A04 b-Cyclodextrin C-Source, polymer 1 1 3 1 1 3 PM02 A05 g-Cyclodextrin C-Source, polymer 3 3 3 3 1 1 PM02 A06 Dextrin C-Source, polymer C00721 6 6 7 9 6 6 PM02 A07 Gelatin C-Source, polymer C01498 1 3 3 3 1 4 PM02 A08 Glycogen C-Source, polymer C00182 6 7 1 6 4 3 PM02 A09 Inulin C-Source, polymer C00368 5 1 3 6 3 3 PM02 A10 Laminarin C-Source, polymer C00771 6 5 7 6 6 9 PM02 A11 Mannan C-Source, polymer C00464 1 1 3 1 1 3 PM02 A12 Pectin C-Source, polymer C00714 4 4 4 4 4 3 PM02 B01 N-Acetyl-D-Galactosamine C-Source, carbohydrate C01074 1 3 3 1 1 4 PM02 B02 N-Acetyl-Neuraminic acid C-Source, carboxylic acid C00270 5 7 5 5 1 3 PM02 B03 b-D-Allose C-Source, carbohydrate C01487 1 7 3 1 1 1 PM02 B04 Amygdalin C-Source, carbohydrate C08325 3 3 1 1 1 8 PM02 B05 D-Arabinose C-Source, carbohydrate C00216 6 6 6 4 3 1 PM02 B06 D-Arabitol C-Source, carbohydrate C01904 6 7 5 6 4 4 PM02 B07 L-Arabitol C-Source, carbohydrate C00532 1 3 5 3 1 4 PM02 B08 Arbutin C-Source, carbohydrate C06186 8 8 7 7 8 6 PM02 B09 2-Deoxy-D-Ribose C-Source, carbohydrate C01801 1 3 3 3 1 3 PM02 B10 i-Erythritol C-Source, carbohydrate C00503 3 7 5 3 1 4 PM02 B11 D-Fucose C-Source, carbohydrate C01018 1 1 3 1 1 4 PM02 B12 3-O-b-D-Galactopyranosyl-D-Arabinose C-Source, carbohydrate 1 6 1 0 0 0 PM02 C01 Gentiobiose C-Source, carbohydrate C08240 7 7 6 7 6 9 PM02 C02 L-Glucose C-Source, carbohydrate 1 1 3 1 1 1 PM02 C03 D-Lactitol C-Source, carbohydrate 1 3 3 1 1 4 PM02 C04 D-Melezitose C-Source, carbohydrate C08243 7 1 3 7 4 3 PM02 C05 Maltitol C-Source, carbohydrate G00275 1 1 3 3 1 4 PM02 C06 a-Methyl-D-Glucoside C-Source, carbohydrate 5 3 5 5 4 4 PM02 C07 b-Methyl-D-Galactoside C-Source, carbohydrate C03619 5 6 4 7 4 4 PM02 C08 3-Methylglucose C-Source, carbohydrate 1 1 1 3 1 4 PM02 C09 b-Methyl-D-Glucuronic acid C-Source, carboxylic acid C08350 5 5 5 5 3 4 PM02 C10 a-Methyl-D-Mannoside C-Source, carbohydrate 1 1 1 1 1 4 PM02 C11 b-Methyl-D-Xyloside C-Source, carbohydrate 1 1 3 1 1 4 PM02 C12 Palatinose C-Source, carbohydrate C01742 5 6 3 5 4 3 PM02 D01 D-Raffinose C-Source, carbohydrate C00492 5 5 7 7 4 5 PM02 D02 Salicin C-Source, carbohydrate C01451 6 3 4 4 4 6 PM02 D03 Sedoheptulosan C-Source, carbohydrate 1 1 1 1 1 4 PM02 D04 L-Sorbose C-Source, carbohydrate C00247 5 1 5 6 4 0 PM02 D05 Stachyose C-Source, carbohydrate C01613 5 3 5 5 4 4 PM02 D06 D-Tagatose C-Source, carbohydrate C00795 3 3 3 1 1 1 PM02 D07 Turanose C-Source, carbohydrate G03588 6 3 3 5 6 6 PM02 D08 Xylitol C-Source, carbohydrate C00379 3 1 5 3 3 1 PM02 D09 N-Acetyl-D-Glucosaminitol C-Source, carbohydrate 1 3 1 1 1 1 PM02 D10 g-Amino-N-Butyric acid C-Source, carboxylic acid C00334 5 5 5 5 3 4 PM02 D11 d-Amino Valeric acid C-Source, carboxylic acid C00431 1 1 3 1 1 3 PM02 D12 Butyric acid C-Source, carboxylic acid C00246 3 5 3 3 1 8 PM02 E01 Capric acid C-Source, carboxylic acid C01571 0 0 0 0 0 0 PM02 E02 Caproic acid C-Source, carboxylic acid C01585 1 1 1 1 0 0 PM02 E03 Citraconic acid C-Source, carboxylic acid C02226 1 1 3 1 1 3 PM02 E04 Citramalic acid C-Source, carboxylic acid C00815 1 1 3 1 1 0 PM02 E05 D-Glucosamine C-Source, carbohydrate C00329 8 6 7 8 8 8 PM02 E06 2-Hydroxybenzoic acid C-Source, carboxylic acid C00805 5 3 1 1 1 1 PM02 E07 4-Hydroxybenzoic acid C-Source, carboxylic acid C00156 3 3 3 3 1 1

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PM02 E08 b-Hydroxybutyric acid C-Source, carboxylic acid C01089 4 5 3 5 1 8 PM02 E09 g-Hydroxybutyric acid C-Source, carboxylic acid C00989 4 4 3 3 1 4 PM02 E10 a-Keto-Valeric acid C-Source, carboxylic acid C00567 6 5 2 6 5 7 PM02 E11 Itaconic acid C-Source, carboxylic acid C00490 1 1 1 1 1 8 PM02 E12 5-Keto-D-Gluconic acid C-Source, carboxylic acid C01062 6 6 6 6 4 4 PM02 F01 D-Lactic acid Methyl Ester C-Source, ester 1 4 3 3 3 8 PM02 F02 Malonic acid C-Source, carboxylic acid C00383 3 3 3 3 4 3 PM02 F03 Melibionic acid C-Source, carbohydrate 6 1 3 6 3 4 PM02 F04 Oxalic acid C-Source, carboxylic acid C00209 1 1 1 1 1 3 PM02 F05 Oxalomalic acid C-Source, carboxylic acid C01990 5 3 5 3 4 3 PM02 F06 Quinic acid C-Source, carboxylic acid C00296 3 5 3 5 1 3 PM02 F07 D-Ribono-1,4-Lactone C-Source, carboxylic acid 3 1 1 1 1 6 PM02 F08 Sebacic acid C-Source, carboxylic acid C08277 5 5 3 5 4 8 PM02 F09 Sorbic acid C-Source, carboxylic acid 3 2 1 3 1 0 PM02 F10 Succinamic acid C-Source, carboxylic acid 4 5 3 3 1 4 PM02 F11 D-Tartaric acid C-Source, carboxylic acid C02107 4 1 1 4 1 0 PM02 F12 L-Tartaric acid C-Source, carboxylic acid C00898 3 1 4 3 3 3 PM02 G01 Acetamide C-Source, amide C06244 1 1 3 1 1 4 PM02 G02 L-Alaninamide C-Source, amide 4 3 3 5 1 8 PM02 G03 N-Acetyl-L-Glutamic acid C-Source, amino acid C00624 1 1 3 1 1 4 PM02 G04 L-Arginine C-Source, amino acid C00062 5 5 3 7 4 5 PM02 G05 Glycine C-Source, amino acid C00037 3 3 3 3 4 6 PM02 G06 L-Histidine C-Source, amino acid C00135 7 1 1 1 2 1 PM02 G07 L-Homoserine C-Source, amino acid C00263 1 1 3 1 0 3 PM02 G08 Hydroxy-L-Proline C-Source, amino acid C01015 0 1 1 0 0 0 PM02 G09 L-Isoleucine C-Source, amino acid C00407 3 5 3 3 1 5 PM02 G10 L-Leucine C-Source, amino acid C00123 3 8 3 3 1 5 PM02 G11 L-Lysine C-Source, amino acid C00047 3 3 3 4 1 1 PM02 G12 L-Methionine C-Source, amino acid C00073 0 1 1 0 0 0 PM02 H01 L-Ornithine C-Source, amino acid C00077 5 5 5 5 3 7 PM02 H02 L-Phenylalanine C-Source, amino acid C00079 3 3 3 3 2 1 PM02 H03 L-Pyroglutamic acid C-Source, amino acid C02238 5 5 3 7 3 8 PM02 H04 L-Valine C-Source, amino acid C00183 3 5 3 3 1 5 PM02 H05 D,L-Carnitine C-Source, carboxylic acid C00487 1 1 3 1 1 4 PM02 H06 sec-Butylamine C-Source, amine 1 1 1 0 0 1 PM02 H07 D,L-Octopamine C-Source, amine C04227 3 1 3 1 1 1 PM02 H08 Putrescine C-Source, amine C00134 1 1 3 1 1 6 PM02 H09 Dihydroxyacetone C-Source, alcohol C00184 6 8 7 8 8 8 PM02 H10 2,3-Butanediol C-Source, alcohol C03044 1 1 3 1 1 3 PM02 H11 2,3-Butanedione C-Source, alcohol C00741 3 3 3 1 1 1 PM02 H12 3-Hydroxy-2-buta C-Source, alcohol C00466 1 1 3 1 1 4

Supplementary Table 3. Results of PM carbon source analysis at 13°C with Biolog Dye present. Activity indices (AIs) are given for each species in the 5 rightmost columns.

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Appendix E: Genetic evaluation of a putative LxrA homolog in Pseudogymnoascus sp.

I performed all experiments described in this appendix with the following exception:

Eleanor Kim performed agrobacterium mediated transformations of P. destructans

for introduction of the 3VT5-LxrA-consruct1 plasmid shown in Figure 1.

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Results

To test whether disruption of the putative LxrA ortholog in P. destructans can

explain its inability to use arabinose and galactose pathway substrates, I introduced the

corresponding gene from related Pseudogymnoascus species into the P. destructans

genome using an established agrobacterium-mediated transformation (ATMT) method. 1

While this ATMT method has a low efficiency of homologous recombination, in our hands

it is effective for the stable, random introduction of DNA into the P. destructans genome. I

designed 6 different expression constructs with promoter/terminator sequences fused to

open reading frames (ORFs) of the putative LxrA orthologs from Pseudogymnoascus sp.

03VT05 and 24MN13 (Figure 1), both of which exhibited robust growth on arabinose and galactose. Three of these constructs were successfully generated, cloned into the ATMT pRF-HU2 binary plasmid1 and transformed into P. destructans cells. To examine whether

the presence of these DNA constructs impacted overall C-source metabolism, spores from

transformed P. destructans clones were used to inoculate Biolog PM plates alongside

untransformed controls. As the putative LxrA expression constructs were introduced by

random genomic integration, 2 independent clones from each transformation were

evaluated to account for differences due to the location of the integrated construct. The

presence of any of these three DNA constructs did not have a major impact on overall

carbon utilization. Some of the transformed strains showed slight increases the ability to

utilize galactose and arabinose within the PM panel (Figure 2A) however this increased

growth was not seen on solid medium containing the same carbon sources (Figure 2B).

The observed growth differences between the liquid PM medium and solid medium may

reflect real biological differences. However, the elevated growth in PM medium +

galactose and arabinose was subtle and inconsistent across transformed strains.

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Additionally, the transformed strains showed no increased growth on L-arabitol or

galactitol, which are also upstream of the LxrA-catalyzed step in each pathway. This experiment will need to be repeated in order to assess whether the observed differences are real or simply a result of assay variability.

These inconsistent findings may indicate that the putative LxrA ortholog identified here has a divergent function in the Pseudogymnoascus genus and that the polymorphism observed in P. destructans is coincidental to its inability to use arabinose and galactose.

However biological and technical limitations could also explain this negative result.

Successful expression of the described DNA constructs has not been validated and future experiments will include quantitative reverse-transcriptase PCR (qPCR)-based analysis of their expression levels. Though the Tef1 promoter/terminator sequence has enabled successful gene expression in other filamentous fungi, genetic transformation has not been extensively studied in P. destructans and the effectiveness of this promoter sequence here is unknown. Additional experimentation may be required to identify effective promoter/terminator sequences and other factors that enable robust heterologous gene expression in P. destructans. Additionally, low expression levels of other Ara/gal pathway enzymes in P. destructans may negate the impact of this gene. The inability of P. destructans to utilize substrates of later steps in the ara/gal pathways supports this hypothesis. Accordingly, restoration of these pathways in P. destructans may therefore require the simultaneous expression of multiple enzymes. Currently such an approach is not feasible in P. destructans. However further development of genetic transformation methods and identification of additional selection markers may enable the introduction of multiple gene constructs in future experiments.

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Methods

Generation of “LxrA ortholog” expression constructs

RNA and gDNA were isolated from Pseudogymnoascus strains grown on solid medium

with MasterPureTM Yeast DNA/RNA purification kits (Illumina). cDNA was generated from

RNA samples using the SuperScriptTM IV first-strand synthesis kit (Thermo). All DNA fragments were amplified from isolated cDNA/gDNA with Phusion high-fidelity polymerase

(Thermo). Promoter and terminator sequences were generated using gDNA templates to amplify 500-100 bp regions 5’ and 3’ (respectively) of the corresponding coding region.

Open reading frame (ORF) sequences were amplified from cDNA. All PCR-generated

fragments were amplified with primers containing >= 20 bp of overlapping sequence with

adjacent DNA fragment/vector sequences to facilitate Gibson-based assembly2 (See

Table 1 for primer sequences). Corresponding promoter, terminator and ORF fragments

were simultaneously assembled into the pRF-HU2 ATMT vector linearized with AfeI

restriction enzyme (NEB) using the Gibson cloning method. Briefly, PCR fragments and

Vector combined at ratios between 2:1 and 6:1 in a total volume of 5 µL were added to 15

µL Gibson Reaction mix and incubated at 50°C for 1.5 hours. 12 µL of each reaction was

transformed into chemically competent JM109 E. coli cells (Promega) using standard

procedures and successful generation of ATMT constructs was confirmed by DNA

sequencing and restriction digests of plasmid DNA isolated from resulting colonies.

Transformation of Agrobacterium tumefaciens

DNA plasmids (24MN13-LxrA construct 1, 3VT5-LxrA construct 2 and 3VT5-LxrA construct 3) were first transformed into Agrobacterium tumefaciens strain AGL-1 by

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electroporation following established methods. Briefly, ~50 ng of each DNA plasmid was

mixed with 80 µL of electrocompetent A. tumefaciens cells in a MicroPulser 0.2 cm

cuvette (Biorad) on ice. Cuvettes were then pulsed once using a GenePulser

electroporator (Biorad) at 2.4kV (5 ms time constant) and 1 mL cold yeast-peptone (YP)

liquid was immediately added. Electroporated cells were transferred to a culture tube,

allowed to recover at 28°C for 2-3 hours then plated on LB agar + kanamycin. PCR

checks were used to confirm the presence of the corresponding DNA construct in

transformed AGL-1 cells.

ATMT transformation of P. destructans

ATMT mediated transformation of P. destructans was carried out according to the method developed by Zhang et al.1 Firstly, transformed AGL-1 strains were cultured in

YEB medium + kanamycin and carbenicillin at 28°C for 48 hours. AGL-1 cultures were then washed in induction medium containing acetosyringone (IM+AS) once, resuspended again in IM+AS at 0.3 OD600 and incubated overnight @ 28°C to ~0.5-0.7

OD600 and concentrated 10-fold in IM+AS . P. destructans spores were collected,

washed once in IM+AS and resuspended again at 1x107 spores per mL. For each

transformation 100 µL of spores were mixed with an equal volume of concentrated AGL-

1 cells, spread onto a nitrocellulose membrane over IM+AS agar and incubated at 15°C

for 72 hours. Nitrocellulose membranes were then transferred to Potato-Dextrose agar +

hygromycin for selection and incubated for ~ 2weeks. Transformed P. destructans

colonies were re- patched to fresh PDA + hyg, allowed to grow up and PCR checks were

conducted to confirm the presence of the corresponding DNA construct.

Solid medium carbon-source growth assay

For evaluation of growth on different Arabinose/Galactose pathway substrates spores

from each P. destructans strain were collected and adjusted to 1 x 107/mL in sterile

249 water and spotted (2 µL) 3 times per plate onto solid Vogel’s minimal media containing each individual carbon source at 0.5% (mass/volume).

Figures and Tables

Figure 1. Cloning strategy for expression of putative LxrA orthologs in P. destructans.

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PCR products of promoter/terminator regions and open reading frames (ORFs) were generated from cDNA/gDNA from corresponding species (top) and used to assemble 3 different “LxrA” expression cassettes (middle). Each expression cassette was cloned into the binary prF-HU2 Vector1 to generate ATMT constructs (bottom) for integration into the P. destructans genome.

Figure 2. Growth of P. destructans strains transformed with LxrA ortholog constructs.

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(A) Growth curves from individual wells of PM carbon source panel. Each line represents a single biological replicate with 2 independently isolated transformant strains used for each DNA construct. (B) Growth of transformed strains on solid (Vogel’s minimal) medium supplemented with indicated carbon source. Each strain was spotted in triplicate and incubated at 13°C for 16 days.

Sequence Primer name PCR product Construct cgccacttcgggctcatgagcTAGGCGGATGTCCATGGTTTGG Gib-24MNPro-F 24MN13 "LxrA" promoter 24MN13-LxrA-Construct 1 CCAAAGTTGGAGCCATCGTGTAGATATCAAGTTGCACCTTG Gib-24MNorfOL-24MNPro-R 24MN13 "LxrA" promoter 24MN13-LxrA-Construct 1 CTTGATATCTACACGATGGCTCCAACTTTGGTTCCTAG Gib-24MNProOL-24MNorf-F 24MN13 "LxrA" ORF 24MN13-LxrA-Construct 1 CTATCTACCACGCCTCAACCAGTACCAAACGGCG Gib-24MNTermOL-24MNorf-R 24MN13 "LxrA" ORF 24MN13-LxrA-Construct 1 CGTTTGGTACTGGTTGAGGCGTGGTAGATAGTTGGGTATATATG Gib-24MNorfOL-24MNterm-F 24MN13 "LxrA" terminator 24MN13-LxrA-Construct 1 ccatacccacgccgaaacaagcACACCTTCGTCGCCGCCTC Gib-24MNTerm-R 24MN13 "LxrA" terminator 24MN13-LxrA-Construct 1 cgccacttcgggctcatgagcTGGGATGGTGCCGTCCTGTG Gib-Pd-LxrAPro-F P. destructans "LxrA" promoter 03VT05-LxrA-Construct 2 CCAAAGTTGGAGCCATGGGGAGGAGGGGACCGCG Gib-3VT5OL-Pd-LxrAPro-R P. destructans "LxrA" promoter 03VT05-LxrA-Construct 2 CTCCCCATGGCTCCAACTTTGGTACCC Gib-PdProOL-3VT5orf-F 03VT05 "LxrA" ORF 03VT05-LxrA-Construct 2 GGAACTGGTTGATGTACGAAGGATGCGGGGTG Gib-3VT5OL-PdTerm-F P. destructans "LxrA" terminator 03VT05-LxrA-Construct 2 GCATCCTTCGTACATCAACCAGTTCCAAACCTCATCC Gib-PdTermOL-3VT5orf-R 03VT05 "LxrA" ORF 03VT05-LxrA-Construct 2 ccatacccacgccgaaacaagcTTGCCTTGCGAGGTGGGGA Gib-Pd-LxrATerm-R P. destructans "LxrA" terminator 03VT05-LxrA-Construct 2 cgccacttcgggctcatgagcCTGAATGGACAGGTTCAGGCTTG Gib-PdTef1Pro-F P. destructans "TEF1" promoter 03VT05-LxrA-Construct 3 CCAAAGTTGGAGCCATTTGCGGTTGTGTGAGCTGGAAATG Gib-3VT5orfOL-PdTEF1Pro-R P. destructans "TEF1" promoter 03VT05-LxrA-Construct 3 CACAACCGCAAATGGCTCCAACTTTGGTACCC Gib-PdTEF1ProOL-3VT5orf-F 03VT05 "LxrA" ORF 03VT05-LxrA-Construct 3 CCAGTCAAATGAGAATTTATCAACCAGTTCCAAACCTCATCCC Gib-PdTEF1Term-3VT5orf-R 03VT05 "LxrA" ORF 03VT05-LxrA-Construct 3 GGTTTGGAACTGGTTGATAAATTCTCATTTGACTGGAAAACGG Gib-3VT5orfOL-PdTEF1Term-F P. destructans "TEF1" terminator 03VT05-LxrA-Construct 3 ccatacccacgccgaaacaagcGGAGTGCACAAATCCATTCCGAG Gib-PdTEF1Term-R P. destructans "TEF1" promoter 03VT05-LxrA-Construct 3

Table 1. Primers used in generation of “LxrA” expression constructs

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References

1 Zhang, T., Ren, P., Chaturvedi, V. & Chaturvedi, S. Development of an Agrobacterium-mediated transformation system for the cold-adapted fungi Pseudogymnoascus destructans and P. pannorum. Fungal Genet Biol 81, 73-81, doi:10.1016/j.fgb.2015.05.009 (2015).

2 Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6, 343-345, doi:10.1038/nmeth.1318 (2009).

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