Profiling of the Talaromyces (Penicillium) Marneffei Secretome

Profiling of the Talaromyces (Penicillium) marneffei Secretome

Brett C. Lomman

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Master of Science

in the

Biological Sciences

Program

YOUNGSTOWN STATE UNIVERSITY

August, 2020

Profiling of the Talaromyces (Penicillium) marneffei Secretome

Brett C. Lomman

I hereby release this thesis to the public. I understand that this thesis will be made available from the OhioLINK ETD Center and the Maag Library Circulation Desk for public access. I also authorize the University or other individuals to make copies of this thesis as needed for scholarly research.

Signature:

______Brett C. Lomman, Student Date

Approvals:

______Dr. Chester R. Cooper, Jr., Thesis Advisor Date

______Dr. David K. Asch, Committee Member Date

______Dr. Jonathan J. Caguiat, Committee Member Date

______Dr. Xiang Jia Min, Committee Member Date

______Dr. Gary R. Walker, Committee Member Date

______Dr. Salvatore Sanders, Dean of Graduate Studies Date

ABSTRACT

This study was focused on profiling the secretome of the thermally dimorphic fungus Talaromyces marneffei via SDS-PAGE with the intent to identify proteins which are differentially and/or preferentially expressed in either growth phase. SDS-PAGE analysis revealed several protein bands which showed differential expression between growth phases. Were the Coronavirus pandemic to not have impacted laboratory work, this study would be continued by sequencing the identified protein bands via tandem MS.

Additionally these amino acid sequences would be used to create primers for qRT-PCR analysis to quantify the levels of expression differences between growth phases.

Abbreviations: SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; tandem MS, tandem mass spectroscopy; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction

III

TABLE OF CONTENTS

ABSTRACT ...... III

TABLE OF CONTENTS ...... IV

LIST OF FIGURES ...... VI

CHAPTER 1: INTRODUCTION ...... 1

1.1 HISTORY ...... 1

1.2 LIFE CYCLE – MOLD VERSUS YEAST ...... 3

1.3 POTENTIAL VIRULENCE FACTOR RESEARCH...... 4

1.4 SELECTED PRIOR GENETIC RESEARCH ...... 5

1.5 SELECTED PRIOR PROTEOMIC RESEARCH ...... 9

1.6 ZEBRAFISH MODEL ...... 12

1.7 THE FUNGAL SECRETOME ...... 13

CHAPTER 2: SPECIFIC AIMS AND HYPOTHESIS ...... 16

2.1 SPECIFIC AIMS ...... 16

2.2 HYPOTHESIS ...... 16

CHAPTER 3: MATERIALS AND METHODS ...... 17

3.1 TALAROMYCES MARNEFFEI STRAIN AND FUNGAL CULTURING .... 17

3.2 BROTH CULTURE PREPARATION FOR PROTEIN ISOLATION ...... 17

3.3 PROTEIN ISOLATION AND PREPARATION METHOD ...... 18

3.4 BRADFORD ASSAY ...... 19

3.5 SDS-PAGE PREPARATION AND GEL ELECTROPHORESIS ...... 22

3.6 GEL ANALYSIS ...... 23

CHAPTER 4: RESULTS ...... 24

4.1 OVERVIEW ...... 24

4.2 INITIAL SDS-PAGE GEL RESULTS ...... 25

4.3 10 L LOAD VOLUME RESULTS...... 27

4.4 LARGER LOAD VOLUME RESULTS...... 29

4.5 DISRUPTION OF EXPERIMENTAL APPROACH DUE TO THE COVID- 19 PANDEMIC ...... 32

CHAPTER 5: DISCUSSION ...... 33

CHAPTER 6: REFERENCES ...... 35

LIST OF FIGURES

Figure Page

Table 1. Bradford Assay Spectrophotometry Results…………………………………...21

Figure 1. Results of Initial SDS-PAGE gel……………………………………………...25

Figure 2. Results of Repeat SDS-PAGE gel with diluted protein concentrations………26

Figure 3. Results of 10 L Load Volume SDS-PAGE gel of Replicates 1 and 2 ………27

Figure 4. Results of 10 L Load Volume SDS-PAGE gel of Replicate 3………………28

Figure 5. Results of Increased Load Volume SDS-PAGE gel for Replicate 1………….29

Figure 6. Results of Increased Load Volume SDS-PAGE gel for Replicate 2………….30

Figure 7. Results of Increased Load Volume SDS-PAGE gel for Replicate 3………….31

CHAPTER 1: INTRODUCTION

1.1 History

Talaromyces (Penicillium) marneffei is a thermally dimorphic human pathogenic fungus endemic to Southeast Asia.14-17,20, 49 Following its initial isolation in 1956 from the liver of a bamboo rat, Rhizomys sinensis, at the Pasteur Institute in Vietnam, T. marneffei has become a fungus of great importance in this region. 10,14-17,43,49 Much of the research conducted on T. marneffei in the early days centered on finding its ecological niche and reservoir, but to no avail – a fact that remains true today.12,14-17,22,39,40,49,52 Fourteen years after its first isolation, T. marneffei infected a human, making it a zoonotic organism.

Cases of T. marneffei infection to this point had been rare in organisms other than bamboo rats aside from a single accidental lab inoculation in 1959 which was promptly treated.40 In 1973 a 61-year old minister suffering from Hodgkin’s disease, who had traveled to Southeast Asia in 1970, underwent a routine splenectomy where it was found to be enlarged and partly infarcted.19 The necrotic tissue was cultured and T. marneffei was recovered. The clinical outcome of this diagnosis is unknown, but this case certainly had a positive outcome for T. marneffei research.

One of the many impacts the 1973 case had on T. marneffei research was correct laboratory identification. Talaromyces marneffei has many similar features to infection by Histoplasma capsulatum ssp. capsulatum and cannot be differentiated clinically.19

When cultured at 37°C, T. marneffei forms short hyphae which grow to a predetermined length and then gradually break apart at the septa between the cells until the majority of the culture contains these cells, termed arthroconidia. These arthroconidia continue to

reproduce using a fission mechanism rather than budding as in H. capsulatum. This reproductive distinction was a landmark for correct diagnoses. Following research rapidly increased the number of documented cases of peniclliosis marneffei (now commonly termed talaromycosis, though both are still used in the scientific literature) which may have been missed before finding this reproductive trait. The incidence rate steadily rose until a sharp spike came with the advent of the AIDS epidemic in the 1980s. In 1996 at least 155 cases, 80% of which were in immunocompromised patients, were reported.20

This number continued to climb to at least 550 as of 199716 and approximately 6709 between 1984 and 2004 in Thailand 49 and continues to rise in the endemic region. An area hit particularly hard was the Chiang Mai province of Thailand in 1992 when it was determined that 86 of 92 HIV-positive patients had contracted T. marneffei and a total of

1843 cases between 1990 and 2004.49

As the AIDS epidemic escalated, it brought with it the understanding that T. marneffei preferentially infects individuals with compromised immune systems, commonly those with HIV infection.14-17 In 1992 Thailand declared penicilliosis marneffei an AIDS-specific disease.43 Data from this time indicates that penicilliosis marneffei was also the third most common infection in AIDS patients.15 In addition to the prevalence in the endemic region, treatment varies widely. If T. marneffei infection is detected and diagnosed early it can usually be treated with long-term antifungals and eventual different prophylactic antifungals to prevent relapse.44,49,59 Talaromyces marneffei infection in immunocompromised hosts is fatal if no treatment is given due to late or incorrect diagnosis.25,49,52 Patients with some immunosuppression not related to HIV infection have seen positive outcomes,11,25,29,31,41,51,59 but those with HIV infection often do not fare as well

if not diagnosed in the early stages of disease.3,20,25,31,43,44,50,51 Diagnosis by different means yield different speeds of diagnosis – those which are diagnosed by lung aspirate, lung biopsy or lymph node aspirate have been seen to be more rapidly diagnosed.25 Data collected from clinical settings has shown that in addition to being so prevalent in the endemic region, diagnoses are not regularly made early enough to help patients recover.25

Treatments administered early enough usually bring about a promising prognosis. When diagnosing T. marneffei infection, many times initial treatment is given based upon the provider’s medical opinion. By performing susceptibility tests to various antifungals it has been seen that the original treatment is not the best choice for that patient. This can present an issue depending upon the patient’s condition. Changing treatment for a late- stage infection is often not successful even though the change can be considered medically sound.44 Patients who are treated in early stages of infection and have the treatment switched can see marked improvement.44,51,59 The treatment of T. marneffei infections is not adequate between the lack of actual “cures,” the low efficacy of treatments in late stage infection, and the need for long-term treatment to prevent relapse makes the case for the need to conduct more broad studies of T. marneffei.

1.2 Life Cycle – Mold versus Yeast

Newer research has been focused on studying the virulence of T. marneffei by attempting to elucidate the mechanisms involved in a phenomenon previously identified in the 1973 case – thermal dimorphism.1,17,19,26,49

When grown at 25-30°C, T. marneffei grows in a filamentous mold phase with hyphae which end in metulae, sterigmata, and conidia – characteristics consistent with the

Penicillium genus.14-17,19,49 As the fungus matures for 7-10 days, T. marneffei cultures increase in size, density, and wooliness while gradually changing color from white to green to eventually rose red and white while secreting a soluble red pigment into the surrounding medium.14-17,19,46,49 When cultured at 37°C the conidia grow as short hyphae which grow to a predetermined length and then gradually break apart at the septa between the cells until the majority of the culture consists of arthroconidia. The arthroconidia continue to reproduce by fission while not producing the soluble pigment.14-17,19,49 In addition to the identification of a dimorphic nature it was found that these phases are reversible. Cultures at 37°C can be transferred to 25-30°C which will cause the arthroconidia to revert back to forming hyphae, that begin to secrete the red pigment again, and eventually creating conidia.14-17,19,49

Although dimorphism is not unique to T. marneffei and is common in many pathogenic fungi, dimorphism, as with other fungal pathogens, can be linked to its virulence.1,14-17,26,49 From the cases of T. marneffei infection after 1973 the vast majority of isolates were found to be in the yeast phase in tissues yet still grew as a mold at 25-

30°C.14-17,19,49 This has prompted investigations into the link between the dimorphic switch and how it is linked to the virulence of T. marneffei.

1.3 Potential Virulence Factor Research

In the pursuit of understanding the mechanisms responsible for the dimorphic switch, which is putatively linked to virulence, many different approaches have been taken which range from genomic analyses using knockout mutants6,27 to protein profiling13 and host response studies using various cell lines.21,37,42,45 Most of the genetic

studies have focused on analyzing any differences in the expression levels of mRNA of various transcription factors, expression levels between phases, and some cell cycle control mechanisms.27,28,35,38 Most proteomic studies have followed similar paths while studying protein levels areas such as cell-to-cell interactions in host response experiments37 as well as looking for mechanism involved in the control of the dimorphic switch.2,6,13,27,28,34 A short review of selected prior genetic and proteomic studies follows.

1.4 Selected Prior Genetic Research

Much prior genetic research has focused on identifying homologs to genes from other fungal species, normally Aspergillus nidulans, A. fumigatus, and H. capsulatum.

Most of these studies, especially older, seem to focus on genes specific to mold phase growth including conidiation and control of hyphal development. As reviewed by Cooper and Vanittanakom in 2008, several studies have found these type of homologous gene sequences and examined their presence and effects on T. marneffei.17

In addition to these studies, much work has been done to sequence the entire genome of T. marneffei for use in bioinformatics studies as well as using it for finding sequences for use in genetic studies as probes or primers. In 2003 Woo and colleagues completed the sequence for the entire mitochondrial genome and went on to sequence the entire genome of T. marneffei strain PM1 in 2011.55,56,58

Studying the genome of T. marneffei for homologous genes for cellular maintenance in other fungi has been used to determine how this fungus survives in its natural state as well as helping identify what mechanisms are used. Therefore research has been focused on understanding regular cell reproduction both in vitro and in vivo

which is usually simulated by cell line infection. In 2000, Borneman et al. studied the effects which might occur by introducing into the T. marneffei genome a homolog of the

Aspergillus nidulans abaA gene. abaA, an ATTS/TEA DNA-binding domain transcriptional regulator, was shown to be involved in both conidiophore development and dimorphic transition to single-celled yeasts in T. marneffei.2 Deletion blocked normal conidiophore development which caused the phialide cells to swell, multiply aberrantly, and not produce conidia. These observations were similar to those noted in A. nidulans abaA mutants. Deletion of abaA in T. marneffei also caused improper nuclei assortment within the arthroconidia filaments. Overexpression, however, caused swelling within hyphae which also contain large amounts of septa and multiple nuclei in a cluster.

Borneman and colleagues concluded that abaA has control over the cell cycle during both conidiation and dimorphic growth by synchronizing nuclear and cell division.

One of the main symptoms of T. marneffei infection is fever which can sometimes be long-term.28 Since dimorphism is temperature dependent in T. marneffei, the organism must have an innate mechanism to combat the stress of increased temperatures. The most commonly associated proteins responsible for this effect are heat shock proteins (Hsps).

Heat shock proteins are a family of proteins generally seen as chaperones which help refold proteins which have been slightly denatured due to an increase in temperature. To understand the mechanisms required for this fungus to survive febrile conditions,

Kummasook et al. studied the gene expression patterns of the heat shock protein hsp70. 28

By probing cultures of T. marneffei with an anti-H. capsulatum Hsp70 monoclonal antibody, a clone of the entire T. marneffei hsp70 gene was isolated. The nucleotide and amino acid sequences made from cDNA were compared to that of known fungi resulting

in homologies of 86% to H. capsulatum, 87% to P. brasiliensis and 88% to A. nidulans hsp70 genes. In addition the T. marneffei hsp70 gene contained several amino acid motifs homologous across 32 other fungal Hsp70s specific to the cytosol. This homology suggests that T. marneffei Hsp70 is most likely not a secreted protein and therefore not part of the secretome. To assess when the hsp70 gene expression changes, mRNA was taken from cultures at 25°C 37°C and from a culture at 25°C transferred to 37°C. Levels of hsp70 expression at either 25°C or 37°C remained consistent over time suggesting that it is constitutively activated and acts as a housekeeping gene. Expression in the culture switching from mycelium to yeast showed a marked increase in hsp70 expression within the first 12 hours after the temperature switch. This is consistent with the timeline required for mycelial cells to break into arthroconidia and begin fission. Additional testing of the heat shock response was conducted at 39°C to simulate human fever. The levels of hsp70 expression increased rapidly and reached maximum expression within 30 minutes of the heat shock. This suggests that hsp70 may play a role in virulence by restoring internal protein structure during the initial phases of infection, but due to their cytosol-specific nature and lack of shuttling capacity are likely not proteins of the fungal secretome.

The isolation of a catalase-peroxidase gene was done by Pongpom and colleagues which selected for the gene sequence of a catalase-peroxidase enzyme. 38 In organisms which are intracellular pathogens, such as T. marneffei, their response to host immune defenses must be substantial to overcome these destructive forces. Many of these defenses may use exposure to superoxides, hydrogen peroxide, or other oxygen radicals.

A catalase-peroxidase enzyme can use H2O2 or break it down depending upon the

circumstances which makes it a versatile enzyme in a stress response. The existence and analysis of a catalase-peroxidase gene was examined through the construction of a T. marneffei cDNA library. The sequences were probed with purified IgG pooled from sera of infected patients and identified through chemiluminescence which were subsequently transformed into plasmids where the sequence could later be amplified for use as a probe.

Amplification of these clones resulted in the identification of one gene cpeA which was found to code for a catalase-peroxidase, the first of such which could potentially be used as a diagnostic tool for identifying T. marneffei infection. More work with cpeA is needed, but this catalase-peroxidase is potentially linked to virulence by fighting the host immune response. Since not much work has been done with this enzyme, it is not currently known if this protein could be part of the secretome since the position within the cell has not yet been determined.

In a study conducted to examine the effect of polyamines on the growth of T. marneffei Kummasook and colleagues created a knockout mutant of the sadA gene. 27

Through the course of their investigation the investigators determined that spermidine is important in fungal growth and initiation of dimorphism. The sadA mutant was found to be defective in conidial germination, growth, and dimorphism. SadA is the enzyme A- adenosylmethionine decarboxylase which is critical in the final steps of polyamine synthesis in the cell. Mutants without SadA can have the dimorphic switch restored with addition of spermidine. This suggests that spermidine is a compound that is used as an intermediate in a pathway or used as a signaling molecule in the dimorphic switch.

In studies which focus on the transition of T. marneffei within macrophages, the more predominant type of research presently, there have been some genes postulated to

be involved in virulence. A study by Nimmanee and colleagues examined the ability of T. marneffei to transition to the yeast form within macrophages. 35 Most of these experiments used a mutant of the sakA gene, a homolog of the C. albicans hog1 gene which mutants of show decreased virulence and increased phagocytosis in murine models. The T. marneffei sakA mutant exhibited similar characteristics in these experiments. The sakA mutant was unable to use the nutrients in BHI and SDB medium to transition to the yeast form. Additionally the mutant was shown to have decreased survival in macrophages.

Some of these mutants even germinated as hyphal cells. Based upon previous research in the C. albicans hog1 gene the researchers concluded that sakA is likely involved in the survival of T. marneffei conidia within macrophages which could be linked to virulence.

1.5 Selected Prior Proteomic Research

Prior research on the proteome of T. marneffei has focused on identifying and controlling the proteins coded for in genomic explorations. Some of these proteins could potentially be part of the secretome, but further investigation into the cellular niche of these proteins is necessary.

A study conducted in 2006 by Srinoulprasert and colleagues expanded on previous research which showed that for T. marneffei to infect a host there needs to be attachment to elements of the extracellular matrix (ECM) or to the actual cells of the host.42 Their work looked at the adherence of T. marneffei conidia to various ECM materials by treatment with glycosaminoglycans (GAGs), chondroitin sulfate, and various heparin compounds. They found that the GAGs may play a role in adherence to the ECM which in turn leads to better attachment to the host cells materials, using an

assay which simulated lung epithelium by observing the adherence to fibronectin and laminin. They concluded that GAGs may help adherence to a host ECM but heparin and several chondroitin sulfate compounds inhibited adhesion. This study shows that there are elements specific to the ECM of T. marneffei as the specific interactions with host cells which are very important to study.

For many years research has been focused looking for quicker diagnostic tests for

A. fumigatus in clinical settings. The University of Hong Kong led much of this research which was targeted towards a cell wall mannoprotein termed Afmp1p. An ELISA was developed for the rapid detection of Afmp1p for use in serodiagnosis of aspergillosis patients which yielded promising results.53 Around the same time a similar effort was made looking at a similar protein for rapid diagnosis of T. marneffei infection. In 1998

Cao and colleagues cloned and characterized the MP1 gene in T. marneffei which codes for a 462 amino acid mannoprotein, termed Mp1p, with a sequence unique to T. marneffei with no homologous sequences in BLAST searches at the time.7 This mannoprotein contains a signal peptide at the C-terminal end similar to those of S. cerevisiae which are hypothesized to direct the protein to the correct place in the cell wall.48 Due to the homology with S. cerevisiae and A. fumigatus cell wall proteins it was presumed at the time that MP1 was associated with the cell wall but its exact function was not yet known.

Additionally it was determined that this mannoprotein can be found in conidia, hyphae, and predominantly in the yeast phase.9 Additional tests of Mp1p showed it was easily removed from the cell wall, had antigenic properties, and antibodies to Mp1p were consistently present across patient sera8 which lent itself to use in rapid serodiagnosis of

T. marneffei infection.9

To further understand the impact of Mp1p on the virulence of T. marneffei, Woo and colleagues examined survival rates of mice and macrophages challenged with an

MP1 deficient mutant. 54 The results were interesting since mice challenged with the mutant had survival rates near 100%. In the macrophage model the mutant showed very low survival rates which suggests that Mp1p is used as a defense mechanism within macrophages. The conflicting results led the team to conclude that indeed Mp1p is a virulence factor of T. marneffei though its exact mechanisms were not expanded upon in this study. To answer these questions, the research group went on to examine the internal interactions of Mp1p with macrophage defense mechanisms.45 It had previously been found that the Mp1p protein has a fatty acid binding protein domain which can bind palmitic acid.32 The action of this ligand binding domain was used to examine the cellular mechanisms and interactions within macrophages. This lipid binding domain was found to bind a molecule similar to palmitic acid, arachidonic acid (AA), which is found within macrophages and associated with the inflammatory response cascade pathway against pathogen invasion. By examining the cellular levels of free AA in pull-down experiments it was determined that Mp1p binds to AA with great potency, sometimes holding two molecules at once. To assess the impact of Mp1p on virulence, macrophages were challenged with mutant MP1 conidia which were shown to haves a larger amount of cellular AA than wild-type conidia. By having a larger free AA level it was assumed that the binding ability of Mp1p was lost in the knockout mutant. These results suggested that due to the constant release of Mp1p the AA of the macrophage response was reduced.

This mechanism may indicate how T. marneffei conidia seem to survive better in macrophages – by suppressing the inflammatory response and its cascade.45

An interesting caveat of Mp1p research is the exact location of this mannoprotein.

Cao claimed that it is specifically located in the cell walls of the yeast, hyphae and conidia.7 In 1999 the same group claimed that it is an abundant cell wall protein with a secretion signal peptide.9 In 2010 this group claimed that based upon the levels of Mp1p is sera that it is abundantly secreted but did not say if that was from the cell wall or from the cytoplasm.32 In 2016 the same group then claimed it to be an immunogenic surface and secretory protein.54 This large discrepancy is of interest because determining the exact niche of Mp1p may help better explain some of its characteristics and role in virulence. Part of the current study is to elucidate which proteins found in culture supernatants are associated with the secretome and which are found as artifacts from procedural processes. Mp1p will be a protein of interest to find and hopefully place in a specific niche along with identifying other proteins which may be specific to the secretome.

1.6 Zebrafish model

In an interesting alteration to studying the intracellular interactions within macrophages Ellett and colleagues developed a unique infection model.21 They showed that in temperatures slightly different from standard culture conditions within the bodies of zebrafish T. marneffei infection can be modeled similar to that of murine models. The team examined the in vivo response of zebrafish macrophages by challenge with T. marneffei conidia. It has been seen that within macrophages T. marneffei conidia tend to have a better survival rate due to protection from leukocyte-independent fungicidal activity of other parts of the host immune response. To this end they studied the effects

on the activity of neutrophils and macrophages. By increasing the neutrophil population, in their case two-fold, that the relative concentrations of these leukocytes did not interfere with the phagocytosis levels of macrophages. As a compliment to that experiment they dropped the neutrophil population and found that the macrophages did not react to this loss of these other leukocytes which indicates that macrophages alone can phagocytize and subsequently shelter conidia. In contrast, by decreasing the level of only macrophages clearance of conidia by neutrophils was greatly increased. This suggests that the different fungicidal mechanisms of neutrophils are not readily overcome by T. marneffei conidia. Additional tests which assessed the efficacy of a zebrafish model showed possible evidence that the dimorphic switch characteristic of initial T. marneffei infection may not be entirely thermally regulated. At 33°C the conidia grew filamentously in neutrophils and tissues while being predominantly in the yeast phase within macrophages. They concluded that this may be evidence that the dimorphic switch may be such that with environment within macrophages during initial infection may override the normal thermal regulation. This is a new model and will need extensive testing to confirm the validity of these claims, but if zebrafish infection proves to be an effective model then the idea that dimorphism may not always be thermally regulated but dependent upon internal macrophage environment is intriguing and exciting.

1.7 The Fungal Secretome

The purpose of this thesis is to conduct a focused analysis of the T. marneffei secretome. The secretome has previously been defined as all secreted proteins and the associated secretory machinery.4,47 Additionally it has been restated that the secretome

can be subdivided into freely released proteins, proteins associated with the outer cell walls, and their secretion machinery.4 These secretion mechanisms would likely include intracellular transport mechanisms and possibly those involved in exocytosis, which is beyond the focus of this study. This study aims to profile only the secreted proteins NOT the transport mechanisms. More specifically, this study is focusing on isolating the proteins secreted outside of the cell into the culture medium.

Previous research on fungal secretomes, initially in plant pathogens, has found that proteins included in this extracellular domain may be a large attribute of that organism’s virulence.4,24,47 By creating a defense against host defenses, the fungus can better invade the host. Thermal dimorphism has been linked to the ability of T. marneffei to become an intracellular pathogen.14-17,49 It then stands to reason that there must be an extracellular mechanism, likely associated with the secretome, which allows these conidia to evade host defenses until the arthroconidia have been formed and dispersed.

That is why this investigation will be focused on those secreted proteins specific to the extracellular domain to assess their role in the virulence of T. marneffei.

Bugeja and colleagues studied the role of GATA-type zinc finger proteins in nitrogen use mechanisms of T. marneffei.6 One of the experiments performed was an extracellular protease production test. The researchers created a mutant deficient in the

GATA-type protein AreA which lacked production of extracellular proteases. A complemented strain restored secretion of these proteases. The researchers concluded that areA may be related to virulence for its role in the release or production of extracellular proteases. While this study showed that T. marneffei does secrete extracellular proteins, it did not examine the identity of those extracellular proteases which is the purpose of this

thesis. This study did show however that there are genes in T. marneffei which could be linked to virulence and that these genes can be manipulated to interrupt the secretion of extracellular proteins. It is likely that areA can be considered part of the secretome but potentially as part of the secretory machinery which is outside the scope of the definition used here.

One of the biggest hurdles when studying the secretome will be determining which proteins are truly specific to the extracellular space or are part of the transport mechanisms.4,47 The intent of this study is to differentiate those proteins which are truly part of the secretome and those that are artifacts through the use of SDS-PAGE, 2D-GE, and MS/MS analysis. The proteins will be isolated from the extracellular matrix of T. marneffei cultures, in this case the supernatant of liquid cultures, and screened for proteins which may show different expression patterns over time or between growth phases. Once these differences have been established, MS/MS data will be used to compare the amino acid sequences of the proteins of interest to those predicted to be secreted proteins by the FunSecKB database.33 Once the protein identity is found and confirmed as part of the secretome, this protein class can be examined for its role in virulence in other experiments. The current investigation will use the identities of the collected proteins showing differential expression or phase specificity to construct primers for use with qRT-PCR to quantify the differences previously seen in the SDS-

PAGE and 2D-GE analyses.

CHAPTER 2: SPECIFIC AIMS AND HYPOTHESIS

2.1 Specific Aims

This investigation is interested in defining the secretome of the pathogenic fungus

Talaromyces (Penicillium) marneffei using SDS-PAGE, 2D-GE, MS/MS, and qRT-PCR analyses. In this study the definition of the secretome of interest is the extracellular growth medium of a T. marneffei broth culture. The proteins which are isolated from this growth method are those assumed to be part of the extracellular niche and are potentially involved in influencing the virulence of T. marneffei. The isolated proteins will be identified and compared between the growth phases in an attempt to identify any which may be preferentially and/or differentially expressed between the two growth phases. In an attempt to begin to better understand the role in virulence of the isolated proteins, their amino acid sequences will be compared to those which have been identified as presumptive secreted proteins in the FunSecKB database.33

2.2 Hypothesis

I hypothesized that a number of secreted proteins would be found to have significant expression differences between the mycelial and yeast phases. Previous research has reported over 20 proteins being differentially expressed between phases or preferentially expressed in a single phase.57 Some of the target proteins, including Mp1p,7-

9,45,53,54 aspartyl proteases described by Payne et al.,37 AreA described by Bugeja et al.,6 and the extracellular proteases previously described by Moon et al.34 are sought out in this study, thereby serving to confirm the experimental approach described herein.

CHAPTER 3: MATERIALS AND METHODS

3.1 Talaromyces marneffei Strain and Fungal Culturing

This study utilized the F4 strain of T. marneffei which was originally isolated from a Chiang Mai AIDS patient (wild-type; CBS 119456).38 Cultures were grown on

Potato Dextrose Agar (PDA) (Part No. 213200, Difco) in 75 cm2 Nunc EasYFlasks (Part

No. 156499, Thermo Scientific) by inoculating with conidia from a previous culture and allowed to grow for 10 days at 25°C. At day 10 the conidia were collected as previously described.23 The resulting suspension of ~1 mL was counted on a hemocytometer to calculate the volume required for each experimental flask to contain 1 x 107 conidia/mL.

3.2 Broth Culture Preparation for Protein Isolation

Conditions for protein isolation were prepared by making 600 mL of Sabouraud

Dextrose Broth (SDB) (Part No. 238230, Difco) and aliquoting 25 mL into 8 500-mL

Erlenmeyer screw-cap flasks. Each flask was placed in a shaking water bath of the appropriate condition prior to being inoculated to allow the medium to acclimate and reach the condition temperature.

Each condition was inoculated with the appropriate volume of suspension of

1x107 conidia/mL and set to shake at 120 RPM for the designated time point. Each sample was removed from the shaking water bath at the prescribed time and two steps followed. The first was a removal of 1 mL of culture for microscopic examination by fixing in 4% paraformaldehyde. From the 1 mL aliquot, cells were collected using a microfuge tube subjected to microcentrifugation at 12,000 RPM for 2 minutes, decanting the resulting supernatant, resuspending that pellet in 1 mL of 4% paraformaldehyde, and

storing the cells at -20°C. The remainder of the culture (24 mL) was transferred to a pre- chilled 50 mL conical centrifuge tube (Part No. 06-443-20, Fisher Scientific) and centrifuged at 10,000 RPM for 15 minutes at 4°C to pellet the culture and create the supernatant which contained the proteins of interest.

3.3 Protein Isolation and Preparation Method

The Amicon Ultra-15 centrifugal filter (Part No. UFC9000324, Merck Millipore) was selected as the protein isolation vessel for this study. Once the broth cultures described above had been centrifuged to create the supernatant they were transferred to two Amicon filter units in volumes up to 12 mL in each filter unit. The volume added was variable at times due to the cell pellet resuspending into the supernatant when trying to remove the supernatant and only the amount which could be reasonably certain contained only the supernatant was used. Each sample was then centrifuged at 5,000 x g for 1 hour at 4°C. This time was determined to be the best for this study after consulting the manufacturer’s recommendations and testing the total collection volume in 15-minute increments.

The top part of both filter units for each growth condition were then emptied into a new pre-chilled (on ice) Amicon filter unit by pouring off as much as possible and using long acrylamide gel loading tips to remove and transfer the remainder. The viscosity of the samples required the use of this transfer method. The new Amicon filters were then filled with 12 mL of phosphate buffered saline (PBS) (Part No. E404-200,

Amresco) to resuspend the sample for washing. These newly resuspended samples were then centrifuged again as before to collect the washed proteins. After centrifugation, the

samples were transferred to fresh microfuge tubes and stored at -20°C until further analysis.

For the trial gel and methodology verification the concentration of conidia suspension used was 4.7 x 107 conidia/mL, which was split in half and inoculated into 25 mL of SDB and grown for 48 hours at both temperatures. Additionally this sample was initially centrifuged to create the cell pellet and supernatant using a Sorvall high speed centrifuge for 15 minutes at 4°C before being subjected to Amicon filter protein extraction for a total of 35 minutes. Some alterations happened when processing this initial sample which resulted in the following: the Amicon speed at 3,000 x g initially but reset to 5,000 x g and the filter units properly oriented to create the best possible flow rate and recentrifuged for 15 minutes resulting in a total of 65 minutes of centrifugation. This protein extraction set was then centrifuged in a microfuge tube at 12,000 RPM for 2 minutes and decanting the supernatant. These samples were then washed as above with

PBS for a total of 1 hour. These samples were then recentrifuged in the microfuge and had the supernatant removed as before and placed at -20°C for further analysis. This removal of final liquid of the protein samples after each step in an Amicon filter unit was removed from the protocol due to these samples being much too viscous to adequately process in subsequent steps.

3.4 Bradford Assay

In an attempt to quantify the amount of protein present in the collected samples a

Bradford assay5 was used. The standards used for this assay were created by using Bio-

Rad Protein Assay Standard II (Part No. 500-0007, BioRad). The concentrated Bovine

Serum Albumin was rehydrated with 20 mL of ultrapure molecular water to give a concentration of 1.37 mg/mL. Five ten-fold dilutions were made to produce the standard for assay comparison.

The Bradford assay was completed using the “Standard Procedure for Microtiter

Plates” provided by BioRad for the dye concentrate (Part No. 5000006, Lit33C, BioRad).

In a microtiter plate, 10 L of each standard or sample and 200 L of 1:4 dilute concentrate were mixed and allowed to incubate for 5 minutes. Absorbance was measured on a Nanodrop 2000C Spectrophotometer (Part No. ND2000C, Thermo Fisher

Scientific) using the default Bradford Assay settings. The data is shown in Table 1.

Table 1. Absorption values in a Bradford assay for samples from three replicates. These values are used to try to determine the load volumes for the best chance for band separation. Note the number of “Out of range (low)” readings.

Replicate 1 Replicate 2 Replicate 3 Protein Protein Protein

Concentration Concentration Concentration (Condition, (Condition, (Condition, (g/mL) (g/mL) (g/mL) Time Point) Time Point) Time Point) 4.108 3.952 3.857 25°C, 24h 25°C, 24h 25°C, 24h Out of range Out of range Out of range 25°C, 48h 25°C, 48h 25°C, 48h (low) (low) (low)

Out of range Out of range Out of range 25°C, 72h 25°C, 72h 25°C, 72h (low) (low) (low)

Out of range Out of range Out of range 25°C, 96h 25°C, 96h 25°C, 96h (low) (low) (low)

3.234 4.303 2.971 37°C, 24h 37°C, 24h 37°C, 24h 2.504 3.055 3.484 37°C, 48h 37°C, 48h 37°C, 48h 2.277 3.457 3.179 37°C, 72h 37°C, 72h 37°C, 72h Out of range 2.439 3.769 37°C, 96h 37°C, 96h 37°C, 96h (low)

3.5 SDS-PAGE Preparation and Gel Electrophoresis

Due to the viscosity of the samples and poor result acquisition in the Bradford assay it was decided to use the first few gels to determine the best load volume and concentration for optimal band resolution and validation of the protein isolation methods.

Each protein sample was prepared for SDS-PAGE30 by adding equal parts 2X

Laemmli buffer (Part No. 1703125, BioRad) to equal parts sample, and then adding 5%

β-mercaptoethanol (Part No. 97064, VWR Scientific). Each sample was then mixed and boiled for 5 minutes to better distribute the SDS in each sample.

All gels produced were loaded with 10 L of PageRuler (Part No. 26616,

ThermoFisher Scientific) as a protein standard. The first gel used was a 4-15% Criterion

TGX acrylamide gel (Part No. 5671044, BioRad). Each lane was loaded with 10 L of fully concentrated samples. The gel was electrophoresed at a constant 40 mA for 1 hour and stained with Coomassie Blue (Part No. 161-0436, BioRad) and imaged using the

PharosFX Plus System gel imager (Part No. 170-9450, BioRad). The staining solution was prepared by adding 0.5g of Coomassie Blue to 250 mL of 95% ethanol, 100 mL of acetic acid, and adding 450 mL of water and sterilized.

Upon visual analysis of the first gel, the best choice was to make 10-fold dilutions for several of the samples and try to separate them on a single-percentage gel for better band separation due to high sample viscosity. These samples were created with 10 L of sample mixed with 90 L of 1X Laemmli buffer (equal parts 2X buffer and sterile molecular water). This sample set was loaded into a 12% Criterion TGX gel (Part No.

5671044, BioRad) and electrophoresed at a constant 40 mA for 1 hour and stained with the previously described Coomassie Blue staining solution.

Successive gels were produced using the same preparation methods as for the trial gels except that they were loaded into 4-20% Criterion TGX gels (Part No. 5671094,

BioRad) and stained with 1X Sypro Ruby (Part No. 1703125, BioRad). Figures 3 and 4 represent a loading volume of 10 L while Figures 5-7 show gels loaded with 15 and 20

L loads for better band visualization.

3.6 Gel Analysis

Using the Pharos FX imager (Part No. 170-9450, BioRad), each sample was scanned in using the Quantity One software (BioRad) using the most appropriate setting for the stain type used. In the pair of test gels the Coomassie Blue setting was chosen using an image quality of 50 micron. The latter gels were stained with SYPRO Ruby therefore the SYPRO Ruby setting was chosen again using the 50 micron image quality.

In order to obtain the best visualization there were several images taken using each of the intensity settings available in Quantity One (high, medium, low sample intensity).

After image capture analysis was conducted with both the Quantity One software as well as comparing them to one another. I decided that there were indeed several bands which showed differential expression between the two phases and would be bands to excise and sequence.

The gels used for band excision, shown in Figures 5 and 6, were those which were loaded with 15 L sample volumes. These samples were excised with a scalpel on a UV light box and placed into microfuge tubes and stored at -80°C until they could be sent off for mass spectroscopy analysis.

CHAPTER 4: RESULTS

4.1 Overview

After protein collection with the Amicon-15 filter units, the prepared proteins were separated on polyacrylamide gels and stained with either a colorimetric or fluorescent dye, visualized and imaged. The bands which showed a distinct difference in intensity were excised and stored until further analysis with mass spectroscopy could be completed.

4.2 Initial SDS-PAGE Gel Results

An initial SDS-PAGE was performed to assess the appropriate conditions for protein separation. Figure 1 shows the results of band separation from the method verification SDS-PAGE gels. This 4-15% gel showed that separation was possible with these gels but the viscosity of the prepared samples caused excessive smearing in at least six samples.

180

130

100

40 - 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Figure 1. 4-15% TGX SDS-PAGE gel of recovered supernatant proteins from 48h cultures at 25 and 37°C, stained with Coomassie Blue. Lane 1 PAGERuler. Lanes 2-5 respectively, 2,4,6,10 L load of 10x dilute 25°C sample. Lanes 6-9 respectively, 2,4, 6, 10 L load of concentrated 25°C sample. Lanes 10-13 respectively, 2, 4, 6, 10 L load of 10-fold dilute 37°C sample. Lanes 14-17 respectively, 2,4,6,10 L load of concentrated 37°C sample. Lane 18, culture broth plus 5% β-mercaptoethanol as a control. Size markers are in kilobases.

In an attempt to achieve better band separation and combat sample viscosity dilutions were made of the samples which showed the worst smearing. These samples were diluted 10-fold and subjected to SDS-PAGE on a 12% gel to give the results in

Figure 2. This gel showed that better band separation was possible in the diluted samples.

180 130

100

70 55

40 -

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 2. 12% TGX SDS-PAGE gel of samples chosen by best resolution on previous SDS-PAGE, stained with Coomassie Blue. Lanes 1 and 14, PAGERuler. Lanes 2 and 3 respectively, 4,6 L of concentrated 25°C sample. Lanes 4-6, respectively, 10,15,20 L of 10x dilute 25°C sample. Lanes 7-9, respectively, 4,6,10 L load of 10-fold dilute 37°C sample. Lanes 10-13, respectively, 2,4,6,10 L load of 1:1 dilution of culture broth plus 5% β-mercaptoethanol as a control. Size markers are in kilobases.

4.3 10 L Load Volume Results

Once the verification of the methodology was confirmed, three replicates of protein collection were subjected to SDS-PAGE in various load volumes and stained with

Sypro Ruby fluorescent stain. Figure 3 shows the band separation from Replicates 1 and

2 and Figure 4 is of Replicate 3. Replicates 2 and 3 show the best differences in band separation between the growth phases. This verified that distinctly different proteins were present between the 25° and 37°C growth phases.

Replicate 1 Replicate 2

180 130 100 70 55 40

35 25

15 Only at 25°C 10 Only at 37°C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Figure 3. 4-20% Criterion TGX gel of 10 L load volume of Replicates 1 and 2. Lanes 1 and 18, PageRuler. Lanes 2-5 and 10-13 were loaded with supernatant samples from 25°C cultures incubated for 24h, 48h, 72h, and 96h, respectively. Lanes 6-9 and 14-17 were loaded with supernatant samples from 37°C cultures incubated for 24h, 48h, 72h, and 96h, respectively. Lane 10 contains 10 L of culture broth plus 5% β- mercaptoethanol as a control. Arrows indicate the bands which showed differential expression between growth phases. Size markers are in kilobases.

180 130 100 70 55 40 35 25

15 Only at 25°C Only at 37°C

1 2 3 4 5 6 7 8 9 10

Figure 4. 4-20% Criterion TGX gel of 10 L load volume of Replicate 3. Lane 1, PageRuler. Lanes 2-5 were loaded with supernatant samples from 25°C cultures incubated for 24h, 48h, 72h, and 96h, respectively. Lanes 6-9 were loaded with supernatant samples from 37°C cultures incubated for 24h, 48h, 72h, and 96h, respectively. Lane 10, 10 L of culture broth plus 5% β-mercaptoethanol as a control. Arrows indicate the bands which showed differential expression between growth phases. Size markers are in kilobases.

4.4 LARGER LOAD VOLUME RESULTS

In an attempt to achieve better band separation and identify more distinct banding differences, I made the decision to rerun the samples using a larger load volume. Figure 5 shows the results of Replicate 1 using 15 and 20 L volumes, Figure 6 is of Replicate 2, and Figure 7 is of Replicate 3. Figures 5 and 6 have boxes around the bands which showed distinct band patterning between the growth phases. These bands became the bands of interest which were excised for sequence analyses via mass spectroscopy.

15 L load volume 20 L load volume

100 70 55

40 - 35 25

15 Only at 25°C Only at 25°C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Figure 5. 4-20% Criterion TGX gel of Replicate 1. Lane 1, PageRuler. Lanes 2-5 and 11-14 were loaded with supernatant samples from 25°C cultures incubated for 24h, 48h, 72h, and 96h, respectively. Lanes 6-9 and 15-18 were loaded with supernatant samples from 37°C cultures incubated for 24h, 48h, 72h, and 96h, respectively. Lane 10 contains 10 L of culture broth plus 5% β-mercaptoethanol as a control as a control. Boxed bands were excised for sequence analyses. Size markers are in kilobases.

When analyzing these results several bands were noted which appeared more often in one growth phase or another. Figure 6 has arrows indicating the bands which show up in only the 37°C growth phase with great band intensity. These bands were excised from the results for Replicate 1 (Figure 5) but also appeared in the results for

Replicate 2.

15 L load volume 20 L load volume

70 55

40 - 35

15 Only at 37°C Only at 37°C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Figure 6. 4-20% Criterion TGX gel of Replicate 2. Lanes 1, PageRuler. Lanes 2-5 and 11-14 were loaded with supernatant samples from 25°C cultures incubated for 24h, 48h, 72h, and 96h, respectively. Lanes 6-9 and 15-18 were loaded with supernatant samples from 37°C cultures incubated for 24h, 48h, 72h, and 96h, respectively. Lane 10 contains 15 L of culture broth plus 5% β-mercaptoethanol as a control as a control. Boxed bands were excised for sequence analyses. Size markers are in kilobases.

15 L load volume 20 L load volume

180 130 100 70 55 40

35 25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Figure 7. 4-20% Criterion TGX gel of Replicate 3. Lane 1, PageRuler. Lanes 2-5 and 11-14 were loaded with supernatant samples from 25°C cultures incubated for 24h, 48h, 72h, and 96h, respectively. Lanes 6-9 and 15-18 were loaded with supernatant samples from 37°C cultures incubated for 24h, 48h, 72h, and 96h, respectively. Lane 10 contains 20 L of culture broth plus 5% β-mercaptoethanol as a control as a control. Size markers are in kilobases.

4.5 Disruption of Experimental Approach Due to the Covid-19

Pandemic

The next steps in my experimental approach was to be the identification of selected protein samples taken from the above gels using MS/MS analysis. However, the abrupt rise in Covid-19 infections across the country, including the State of Ohio, forced the University to cease all face-to-face interactions. This included closing all research laboratories on campus. This was especially disruptive to this project given that differential protein expression had been shown from culture supernatants at 25°C and

37°C.

Had my project not been curtailed, the initial step would have been the submission of gel samples for protein analysis. The data from those analyses would have been used to determine proteins of interest produced by both the mycelial (25°C) and yeast (37°C) cultures that were differentially secreted. Subsequently, this differential expression would have been confirmed using the protein identities to design primers which would then be employed in quantitative assays, specifically qRT-PCR.

CHAPTER 5: DISCUSSION

The results shown here are suggestive of the differences in proteins present in the secretome of Talaromyces marneffei broth cultures. Distinct bands were seen around 55 kD in 3 of the 25°C time points as shown in Figure 6 which did not appear in the 37°C samples. Additionally, several distinct bands were found at ~40 kD in all 4 of the 37°C time points as shown in Figure 5 which were not found in the 25°C samples. Each of these bands were excised for amino acid sequence analysis via mass spectroscopy.

Due to the global Coronavirus pandemic and various issues with access to mass spectroscopy, this is all the further that this study was able to proceed. If the pandemic had not happened, the intention of this study would be to determine the amino acid sequences of the excised bands and determine their identities. These identities would be compared to those in the FunSecKB of presumptively identified secretome proteins.

Additionally the identities would be compared to those previously identified as proteins relevant to changes in the physiology of T. marneffei.6, 7, 37,57At this point the amino acid sequences would have been used to create PCR primers for qRT-PCR to quantify the differences in expression of secretome proteins between the growth phases.

Part of the findings of this study is that there are indeed proteins which seem to be differentially, and potentially preferentially, expressed in a specific growth phase. As previously described, MP1 has been shown to be ~43-47.5 kD7 and several bands were observed in the results for Replicate 2 (Figure 3) in the 25°C samples in this range which did not appear in the 37°C samples for that same replicate. Additionally in all three replicates at around 70 kD there are bands present for all conditions and time points which is in the range of 2 proteins previously identified as specific to T. marneffei (60-82

kD).7 It is also of note that the proteinases identified by Moon et al.34 are approximately

24 kD as identified in their investigation. There are several wide bands in this region in several of the gel images, but there is not substantial evidence that a protein of that size exists as part of these results. Finally, as part of my hypothesis I had intended to look for

AreA identified by Bugeja et al.6 but this protein is hypothetically found around 97 kD or larger which was not a range which this SDS-PAGE protocol could readily and reliably separate.

Much of the results produced by this report are speculative and presumptive since sequencing could not be obtained, but it is reasonable that the proteins identified in my hypothesis statement as well as in the FunSecKB database list could be found in the secretome of T. marneffei. With further analysis these proteins identified herein could be separated from those which were collected aberrantly and are not presumed to be part of the secretome which would result in a fuller understanding of the types of proteins secreted by T. marneffei. It is also important to note that these proteins identified here are those which were present in a laboratory setting and may not reflect those found in an infected organism so more in vitro and in vivo testing is needed. Part of the goal of studying the secretome is to identify protein types and proteins of interest which could play a direct role in the virulence of this fungus. By identifying these proteins and their impact on host environments it may be possible in the future to find new targets for use in new therapeutic approaches and ultimately maybe be part of a cure against talaromycosis.

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