The Texas Medical Center Library DigitalCommons@TMC

The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences

12-2017

MECHANISM OF CANDIDA ALBICANS AND VIRULENCE INHIBITION BY A BACTERIAL SECRETED FACTOR

Carrie Graham

Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations

Part of the Immunology and Infectious Disease Commons, Medicine and Health Sciences Commons, and the Pathogenic Microbiology Commons

Recommended Citation Graham, Carrie, "MECHANISM OF CANDIDA ALBICANS BIOFILM AND VIRULENCE INHIBITION BY A BACTERIAL SECRETED FACTOR" (2017). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 826. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/826

This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. MECHANISM OF CANDIDA ALBICANS BIOFILM AND VIRULENCE INHIBITION BY A

BACTERIAL SECRETED FACTOR

By

Carrie Elaine Graham, M.S.

APPROVED:

______Danielle A. Garsin, Ph.D. Advisory Professor

______Michael C. Lorenz, Ph.D. Advisory Professor

______Ambro van Hoof, Ph.D.

______Barrett R. Harvey , Ph.D.

______Michael C. Gustin, Ph.D.

APPROVED:

______Dean, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences

______Dean, The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences

MECHANISM OF CANDIDA ALBICANS BIOFILM AND VIRULENCE INHIBITION BY A

BACTERIAL SECRETED FACTOR

A

DISSERTATION

Presented to the Faculty of

The University of Texas

MD Anderson Cancer Center UTHealth

Graduate School of Biomedical Sciences

in Partial Fulfillment

of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

by

Carrie Elaine Graham, M.S. Houston, Texas

December 2017

Dedication

To my son, Emiliano Carlos Diaz-Graham, who is the light in my life and one of my greatest endeavors. I know you can break boundaries and accomplish all of your dreams. Never give up.

““If anything is worth doing, do it with all your heart.”

- Eknath Easwaran

iii Acknowledgement

First, I would like to thank my advisors Dr. Danielle Garsin and Dr. Michael Lorenz, who have consistently encouraged me and have offered endless support in both my academic and personal life. Pursing my Ph.D. has been one of the greater challenges in my life and has made me face many of my shortcomings. However, at every challenge both Danielle and Mike have offered words of advice and encouragement. They set a great example for me to follow and I hope to reach their level of expertise and scientific accomplishment. In spite of all the difficult challenges, pursing my Ph.D. has also been one of the greatest parts of my life and I have grown not only as a scientist, but also as a person. That would not have been possible without both Danielle and Mike. Many people have asked me how it is with two mentors and I will admit at time I did felt a little overwhelmed, but it was worth the challenge and I have been able to be part of two wonderful scientific communities working on such a unique and multifaceted project.

Thank you both so much for all of your advice and support throughout my time as your student it has been a great pleasure to have such high caliber scientist as mentors.

I would also like to acknowledge my committee members, Dr. Ambro van Hoof, Dr.

Barrett Harvey, and Dr. Mike Gustin, for their guidance and support throughout my Ph.D. and during my candidacy exam. Thank you all for your feedback, encouragement, and brilliant ideas that kept my project moving forward and helped optimize time. I have thoroughly enjoyed sharing my research with you all in an environment that fosters growth and love for science.

Next, I would like to thank all of the faculty, students, and administration in the

Microbiology and Molecular Genetics department. It has been a great pleasure to be a part of this wonderful department. I have also made many lifelong friends in the department that I would like to thank for their friendship and support. Yi Liu, who was one of my first friends in the department, has always shown me love and caring. Without you I might not have made it in so many different ways. I thank my good friend Norah Owiti for all of her advice and support, thank

iv you for always telling me the no nonsense, upfront truth, so helpful when I loose my way. I would also like to thank Pedro Miramon for his support and opinionated advice both about science and personal matters. There has not been a dull moment since you joined the Lorenz lab. Also, I would like to thank Karan Kaval for his friendship and technical advice; you are the best “Steve” in the world. I thank Elisa Vesely for her help and support; you will always be my sister in science. Last but definitely not least, I would like to thank Melissa Cruz for all of her support and for teaching me so many different scientific techniques. I hope to become as good at bacterial genetics as you. Thank you for laying the groundwork for this project and always being there to talk science and cats. It has been a truly wonderful and fruitful collaboration.

Thank you all so much you are now part of my family.

v

MECHANISM OF CANDIDA ALBICANS BIOFILM AND VIRULENCE INHIBITION BY A

BACTERIAL SECRETED FACTOR

Carrie Elaine Graham, M.S.

Advisory Professors: Danielle A. Garsin, Ph.D. and Michael C. Lorenz, Ph.D.

The human microbiome is a diverse polymicrobial population comprised of both fungi and . Perturbations of the normal microbiome can have a profound impact on health, including the development of infections. Exploitation of these polymicrobial interactions has the potential to provide novel treatment and prevention strategies for infectious diseases.

Enterococcus faecalis, a Gram-positive bacterium, and Candida albicans, a polymorphic fungus, occupy overlapping niches as ubiquitous constituents of the gastrointestinal and oral microbiome. Both species are also amongst the most important and problematic, opportunistic nosocomial pathogens and are often co-isolated during infection. Surprisingly, these two species antagonize each other’s virulence in both nematode infection and in vitro biofilm growth.

Herein we identify the E. faecalis secreted bacteriocin, EntV, as both necessary and sufficient for the reduction of C. albicans virulence and through the inhibition of hyphal formation, a critical virulence trait of C. albicans. Furthermore, we demonstrate that the EntV propeptide is proteolytically processed by the metaloprotease, GelE, in E. faecalis, resulting in production of a highly active of 68-amino acids, EntV68. The mature peptide effectively blocks biofilm development in multiple media conditions and disrupts pre-formed biofilms, which are resistant to current antifungal treatments. Moreover, EntV68 is efficacious against clinical strains of C. albicans, including azole resistant strains. Biofilm development of other vi pathogenic Candida species is also impeded by EntV68 treatment, demonstrating the target spectrum of EntV68 is not limited to C. albicans. EntV68 is protective in the murine macrophage and oropharyngeal candidiasis (OPC) infection models at nanomolar concentrations. Epithelial invasion, inflammation, and fungal burden in the OPC model were significantly reduced in response to treatment with EntV68. Collectively, C. albicans cells present in the hyphal form were greatly reduces in all models examined. Despite these profound effects, EntV68 has no effect on C. albicans viability, even in the presence of significant host-mimicking stresses.

EntV68 associates with cell surface of both and hyphal cells of C. albicans and deletion of genes involved in hyphal morphogenesis and composition abrogate the inhibitory activity in C. albicans biofilms. These findings demonstrate that EntV has potential as a novel antifungal agent that targets virulence rather than viability.

vii Table of Contents

Approval Sheet ...... i

Title Page ...... ii

Dedication ...... iii

Acknowledgement ...... iv

Abstract ...... vi

Table of Contents ...... viii

List of Figures ...... xi

List of Tables ...... xiii

CHAPTER 1: Background and Significance ...... 14

C. albicans as an Opportunistic Pathogen ...... 15

Clinical Importance of Biofilms ...... 16

Role of Hyphal Morphogenesis During Biofilm Formation ...... 18

Biofilm-Associated Genes ...... 22

Antifungal treatment strategies ...... 26

Antifungal Resistance in C. albicans Biofilms ...... 26

Bacterial and C. albicans Interactions and Health Related Prevalence ...... 28

Co-isolation of C. albicans and E. faecalis ...... 33

E. faecalis Pathogenesis ...... 37

E. faecalis Commensalism ...... 40

Bacteriocins Produced by Gram-positive Bacteria ...... 42

Bacteriocin Production in E. faecalis ...... 42

Bacteriocins as Treatment Strategies ...... 44

Bacteriocins Active Against Candida spp...... 45

viii Antimicrobial Active Against C. albicans ...... 46

CHAPTER 2: Materials and Methods ...... 49

Strains and Culture Conditions ...... 50

Strain Construction ...... 50

C. albicans Biofilm Assay ...... 51

Expression and Purification of Recombinant EntV ...... 52

Murine Macrophage Infection ...... 53

sEntV Toxicity Assay ...... 54

Oropharyngeal Candidiasis Model ...... 55

Proteolytic Cleavage of rEntV136 by GelE ...... 57

Homozygous Deletion Mutant Library Screens ...... 57

sEntV68-FITC localization on C. albicans ...... 58

CHAPTER 3: The faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans ...... 61

Introduction: ...... 62

Results: ...... 65

Discussion: ...... 76

CHAPTER 4: Post-Translational Processing of EntV influences the inhibition of hyphal

morphogenesis and biofilm formation of Candida albicans ...... 79

Introduction: ...... 80

Results: ...... 81

Discussion: ...... 91

CHAPTER 5: Assesssment of EntV Mechanism of Action against C. albicans ...... 79

Introduction ...... 93

Results: ...... 95

Discussion: ...... 110

ix Chapter 6: Discussion and Perspectives ...... 112

Summary: ...... 113

C. albicans-E. faecalis interactions during commensalism and pathogenesis ...... 117

Role of E. faecalis bacteriocins in the host environment ...... 118

Role of EntV in the host environment ...... 120

Therapeutic potential of bacteriocins and future application ...... 122

Future perspectives and directions ...... 126

Is sEntV68 efficacious in other mammalian infection models with C. albicans?...... 127

Is sEntV68 efficacious against other fungal pathogens not closely related to C. albicans? . 128

What is the role of the disulfide bridge and how is it formed? ...... 129

What role if any does SprE play in post-translational processing of sEntV68? ...... 129

What is the sEntV68 mode of action against C. albicans? ...... 130

Are the other class IIIb bacteriocins active against C. albicans? ...... 131

How does C. albicans gene expression change in the presence of E. faecalis? ...... 131

Bibliography: ...... 133

Vita ...... 166

x List of Figures

Figure 1.1: Stages of C. albicans biofilm development over time...... 17

Figure 1.2: C. albicans hyphal and biofilm development signaling cascade...... 20

Figure 1.3: The Candida albicans cell wall...... 23

Figure 3.1: E. faecalis-C. albicans interactions during infection of Caenorhabditis elegans. .... 64

Figure 3.2: E. faecalis supernatant inhibits C. albicans hyphal morphogenesis and biofilm

formation...... 66

Figure 3.3: EntV inhibition of hyphal morphogenesis and biofilm formation in C. albicans...... 68

Figure 3.4: Characterization of sEntV68 inhibitory activity...... 70

Figure 3.5: Protection of Murine Macrophages by sEntV68 during C. albicans infection...... 72

Figure 3.6: Activity of sEntV68 in mammalian cells...... 73

Figure 3.7: sEntV68 is protective in a murine OPC model...... 75

Figure 4.1:. Characterization of inhibitory activity from E. faecalis Supernatants...... 82

Figure 4.2: The E. faecalis Fsr system is involved in regulating the inhibitory activity...... 84

Figure 4.3: oxidation is important for inhibitory activity...... 86

Figure 4.4:. EntV is proteolystically processed by E. faecalis ...... 87

Figure 4.5: Comparative primary and secondary structures of class IIIb bacteriocins after

proteolytic processing...... 89

Figure 5.1: Deletion of hyphal morphogenesis and cell wall stress genes confers resistance to

E. faecalis supernatant...... 100

Figure 5.2: Deletion of the central stress response MAPK, HOG1, decreased C. albicans

sensitivity to E. faecalis supernatant...... 101

Figure 5.3: Impact of host relevant stresses on sEntV68 activity...... 103

Figure 5.4: sEntV68 activity on planktonic C. albicans yeast cells...... 104

Figure 5.5:. sEntV68 inhibitory activity in different media types...... 105

xi Figure 5.6: Deletion of ZCF21 decreased C. albicans sensitivity to E. faecalis supernatant and

sEntV68...... 107

Figure 5.7: Localization patterns of sEntV68 on the surface of C. albicans...... 108

Figure 5.8:. sEntV68 target spectrum...... 109

Figure 6.1: Summary of EntV processing and activity...... 114

Figure 6.2: Summary of homozygous deletion mutant functions...... 116

Figure 6.3: Model of C. albicans-E. Faecalis interactions in the GI tract...... 119

Figure 6.4: Predicted membrane topology of active EntV...... 123

xii List of Tables

Table 2.1: Strains and ...... 59

Table 4.1. Computation of physical and chemical properties of class IIIb bacteriocins...... 90

Table 5.1: Homozygous deletion mutants in non-essential transcription factors resistant to E.

faecalis supernatant...... 97

Table 5.2: Homozygous deletion mutants in cell wall resistant to E. faecalis

supernatant...... 98

xiii

CHAPTER 1:

Background and Significance

14 C. albicans as an Opportunistic Pathogen

Candida spp. rank 3rd among nosocomial pathogens, with C. albicans being the prevalent species and there is a 30% rate of morbidly in device-associated infections in the

U.S. (1-4). There are two-main types of infections caused by C. albicans that differ very drastically in severity: superficial infections of the mucosa, such as oral or vaginal candidiasis and life-threatening systemic infections that occur when C. albicans enters the blood stream and then infects organs throughout the body (5-7). C. albicans has specialized virulence and fitness attributes that allow it to survive and adapt in diverse environments in the host (5). The transition between yeast and hyphal morphotypes, expression of cell surface adhesins, and biofilm development are among these adaptive traits that are also considered important virulence factors (6).

As a commensal C. albicans can live in equilibrium with the normal bacterial flora on mucosal surfaces (3, 4, 8). C. albicans is transmitted at birth from mother to infant and no external environmental reservoirs exist, which suggests that C. albicans has coevolved with the mammalian host (9) However, these commensal populations are frequently the source of infection that arise from transmigration through the mucosal barrier or colonization of implant devices (10). Several factors have been identified that influence the switch from commensalism to pathogenesis in C. albicans such as: (1) changes in the normal microbial flora, (2) damaged epithelial barriers, and (3) immune dysfunction (4, 8, 11). After the onset of C. albicans superficial infections more severe systemic infections can occur (11-13). These blood-borne disseminated infections often have considerable mortality rates, near 40%. Severe Candida infections are most prevalent in immunocompromised individuals, such as those with HIV/AIDS or being treated with immunosuppressive therapy (14-17). Healthy individuals with implanted medical devices are also commonly plagued with Candida infections that stem from colonization of the medical devices (18, 19).

15 Clinical Importance of Biofilms

C. albicans has the ability to produce highly structured biofilms on abiotic and biotic surfaces, which promotes the high morbidity and mortality correlated with device-associated infections (20, 21). These highly structured biofilms are composed of different cell types that form a striated pattern with round, budding yeast-form cells and/or oval pseudohyphal cells at the bottom and elongated, cylindrical hyphal cells at the top encased in an extracellular matrix

(Fig. 1.1) (22-25). Of the bloodstream infections in the clinical setting 15% are caused by C. albicans or closely related species; however, C. albicans is the predominant fungal species found on medical devices (26-28). Different medical devices used throughout the body are susceptible to C. albicans biofilm, such as mechanical heart valves, pacemakers, central venous and urinary catheters, dentures and joint prosthetics to name a few (2, 29-31) .

Biofilms formed on medical devices have the potential to seed disseminated bloodstream infections that can lead to systemic infections of tissue and organs. Astonishingly,

50% of the over five million central venous catheters placed annually in the U.S. become infected with biofilms, both bacterial and fungal (2, 32). Routinely, once a medical device becomes infected it is removed and the patient will undergo antimicrobial therapy. Since fungal biofilms are resistant to traditional antifungal treatments, increased concentrations and longer durations of antifungal treatment are required (33-37). However, in some cases removal of the infected medical devices is costly or unsafe and the extremely elevated antifungal concentrations used to treat the infection can lead to further complications such as kidney or liver failure (15, 37, 38). Thus, due to the complicated nature of device-associated infections in the U.S. alone, $6.5 billion is spent annually to treat these types of infections (39). Not only do these infections have an immense economic impact, but also a significant health impact resulting in an estimated 100,000 deaths per year (39).

16

Figure 1.1: Stages of C. albicans biofilm development over time.

1) Adherence: yeast cell attach to an abiotic or biotic substrate. 2) Initiation: biofilm development starts with cell-cell adhesion and hyphal morphogenesis. 3) Maturation: hyphal cells form an extensive network encased in a secreted, extracellular matrix. 4) Dispersal: yeast cells bud off the biofilm and disperse and adhere at new locations. Modified from: Finkel JS, and Mitchell AP. Genetic control of Candida albicans biofilm development. Nature Reviews

Microbiology; 2010;9:109–18 (40-42). Permission to use this figure was granted by Copyright

Clearance Center (License #: 4242690280081).

17 Role of Hyphal Morphogenesis During Biofilm Formation

Biofilm formation can be divided into four distinct stages composed of different populations of yeast, psuedohyphal, and hyphal cells (Fig. 1.1) (23, 39, 40). C. albicans yeast- hyphal transition is essential for maximum virulence potential and the biofilm growth cycle (13,

20, 21, 43). The early stages of biofilm formation are adherence and initiation, which consist of yeast cell attachment to a substrate followed by development of psuedohyphae and then hyphae. During maturation of the biofilms, cells are encased in an extracellular matrix, biomass increases, and striated layers of yeast (bottom) and then hyphal cells (top) are observed. The final stage of biofilm formation is dispersal where yeast cells bud off and disperse, allowing them to form biofilms in other locations (Fig. 1.1) (23, 39, 40). The stages of biofilm growth are interdependent with regulation of hyphal morphogenesis and controlled by a complex signaling network.

Hyphal morphogenesis is regulated by intricate interconnected pathways in response to environmental signals that induce or repress hyphal morphogenesis, giving rise to the distinct morphologies that have been observed in C. albicans (13). Morphological variability allows C. albicans to adapt to different environmental changes. Intriguingly, many of the distinct morphologies can be observed during biofilm growth at distinct stages of development, indicating differences in gene expression of biofilm cells in response to the biofilm microenvironment. To form the striated biofilm structure described above cells must be able to regulate hyphal morphogenesis (40, 42, 44, 45).

Three major signaling cascades have been identified to date that regulate hyphal morphogenesis in C. albicans: (1) the cyclic AMP-protein kinase A pathway (cAMP-PKA), (2) mitogen-activated protein kinase pathway (Cph1-MAPK), and (3) pH pathway (Rim101) (46-

48). The cAMP-pKA pathway is the central signaling pathway that responds to an array of different conditions and regulates hyphal morphogenesis during biofilm growth via activation of

Ras1 (GTPase) by an unknown signal and receptor (49) (Fig. 2.1). Upon Ras1 activation,

18 cAMP is synthesized from ATP by adenylyl cyclase (Cyr1) (50, 51). cAMP then activates PKA, which consists of one of two redundant catalytic subunits, Tpk1 and Tpk2, and a regulatory subunit, Bcy1. PKA then phosphorylates the downstream transcription factor, Efg1, which regulates hyphal-specific genes (52, 53).

Efg1 is a key regulator of hyphal morphogenesis and has various functional roles in both the repression and the induction of hyphal morphogenesis. This multi-functionality is owed impart to the multiple functional domains of the protein (53). Efg1 is able to bind at least two different DNA binding motifs (E-box and MCB). It interacts with the terminal transcription factors of the other hyphal signaling cascades and modulates their functions. Post-translational modifications are also thought to modulate the regulatory behavior of Efg1 (53, 54). Efg1 is one of the core master regulators (Flo8, Rfx2, Gal4, Brg1, Bcr1, Rob1, Efg1, Ndt80, and Tec1) that positively regulate biofilm development and form an intricately regulated transcriptional network that controls biofilm-associated genes (Fig. 1.2) (23, 42, 55). Recently, an even larger set of genes has been identified that encompasses this subset of core master regulators to include a total of 51 transcription factors that regulate different aspects of biofilm development (56).

Additionally, other key transcriptional regulators of interest include the negative regulators Nrg1 and Rfg1 that with co-repressor Tup1 negatively regulate hyphal-specific genes and allow dispersal of yeast cells in the final stage of biofilm development (57). The positive regulator of hyphal-specific genes, Ume6, is important for hyphal extension by maintaining expression of hyphal-specific genes and is important for the later stages of biofilm growth (54, 57, 58). Downstream from every hyphal morphogenesis signal cascade, Ume6 is thought to be a global positive regulator of hyphal-specific genes and is negatively regulated by

Nrg1-Tup1 and to a lesser extent by Rfg1-Tup1 (59).

19

Figure 1.2: C. albicans hyphal and biofilm development signaling cascade.

Ras1-cAMP-PKA (purple) is responsible for transducing the environmental signal to the terminal transcription factor Efg1 (blue). Activated Efg1 is part of the biofilm core regulators and positively regulates the biofilm transcriptional network (green), hyphal specific and biofilm-associated genes. The positive regulator Ume6 regulates hyphal elongation (brown) and is important for developing mature biofilms. Repressors Nrg1 and Rfg1 with the co-repressor Tup1 (red) negatively regulate the signaling cascade and are important for the late biofilm stage, dispersal. Modified from Sudbery PE.

20 Growth of Candida albicans hyphae. Nature Reviews Microbiology; 2011;9:737-748 .

Permission to use this figure was granted by Copyright Clearance Center (License #:

4242730485058).

21 Biofilm-Associated Genes

Nobile et al. (42) identified a subset of genes that are differentially regulated during biofilm development. This subset of genes is regulated by different combinations of the six- biofilm master regulators and includes cell wall proteins, metabolic proteins, and transporter proteins. Functional analysis of these genes during biofilm growth revealed that cell wall proteins play a direct role in biofilm development (42). Considering that the cell wall interacts most directly with surface substrates and other cells it is no surprise that the cell wall and surface proteins play a major role in biofilm formation. The cell wall of C. albicans is comprised of primary and glycoproteins (8, 12). Chitin, β-1,3-glucan, and β-1,6-glucan comprise the inner wall just above the cell membrane, while cell wall proteins and mannans that are in the form of N- and O-linked glycosylation on the cell wall proteins constitute the outer wall (Fig. 1.3) (8, 13). Adhesins directly mediate adherence to a substrate or other cells.

Several different adhesins have been identified in C. albicans by deletion of the gene resulting in loss of adherence or due to homology to known adhesins (60). The content of the cell wall and surface proteins can affect adhesin activity that results in masking or exposing adhesins to the cell surface (8, 13, 60).

Two different classes of cell wall proteins have been identified in C. albicans: proteins glycophosphatidylinositol (GPI) anchored to β-1,6-glucan and proteins with internal repeats

(Pir) linked to β-1,3-glucan (60). The GPI anchored cell wall proteins are the most abundant and many are important for biofilm formation. One such protein is hyphal wall protein 1 (Hwp1), a well-characterized GPI-anchored cell wall protein that is covalently linked to β-glucan.

Deletion of hwp1 reduces virulence in a mouse infection model and results in biofilm defects

(42, 55, 61, 62). The ALS (agglutinin like sequence, Als1-7, 9) family of proteins is a family of

GPI-anchored cell wall proteins, of which several have been identified as being important for biofilm formation. Als1 and Als3 have both been shown to play important roles in hyphal

22

Figure 1.3: The Candida albicans cell wall.

Two distinct layers make up the cell wall. The outer layer composed of O- and N-linked mannans covalently linked to proteins to form glycoproteins. The inner layer is comprised of polysaccharides β-1,3-glucan, and β-1,6-glucan and chitin. Modified from Gow NA, Hube B.

Importance of the Candida albicans cell wall during commensalism and infection. Current

Opinion in Microbiology; 2012;15:406–12 (13). Permission to use this figure was granted by

Copyright Clearance Center (License #: 4242731391290).

23 morphogenesis and biofilm formation; deletion mutants have defects in biofilm growth (62, 63).

Over expression of the other ALS genes rescues the in vivo and in vitro biofilm growth defects of an als1Δ/Δ als3Δ/Δ mutant, indicating redundancy in the function of the Als family of proteins

(62).

Both HWP1 and ALS1 are regulatory targets of BCR1 and over expression of either rescues a bcr1 biofilm defect (42). However, overexpression of HWP1 does not rescue biofilm growth defects of an als1Δ/Δ als3Δ/Δ mutant suggesting they have distinct roles during biofilm formation (62). When biofilms of mixed mutant strains (als1Δ/Δ als3Δ/Δ and hwp1Δ/Δ) are grown together the biofilm defects are overcome indicating Hwp1 and Als1/Als3 have complementary functions (62). Heterologous expression of HWP1 in S. cerevisiae mediates adherence to wild-type C. albicans but not the als1Δ/Δ als3Δ/Δ mutant (62). Taken together these results suggest Hwp1 and Als1/Als3 have distinct complementary roles during biofilm growth by mediating cell-cell attachment via interactions together on the cells surface.

Additional cell wall associated proteins have been shown to play important roles in biofilm formation. Eap1 (enhanced adherence to polystyrene 1) is a GPI anchored protein with a similar structure to the Als family and is important for biofilm initial adherence. Deletion of eap1 causes a reduction in adherence to polystyrene and causes defects in both in vivo and in vitro biofilm growth (64, 65). Rbt1 (repressed by Tup1) is also a GPI anchored protein that belongs to the same family of adhesins as Hwp1 (60). Deletion of RBT1 results in mild biofilm defects in vitro although additional deletions of HWP1 and HWP2 have enhanced biofilm defects (66, 67). Expressed at high concentrations in C. albicans yeast cells, Ywp1 (yeast wall protein 1) is an adhesin-like cell wall protein that is thought to act as an anti-adhesin by an unknown mechanism (68, 69). Deletion of YWP1 results in loss of dispersal and increased adherence suggesting that Ywp1 is important for dispersal, the final stage of biofilm development (69). Most of the multiple families of adhesins are associated with hyphal growth and play a major role in biofilm development. However, a balance of cell-substrate and cell-cell adherence is important for biofilm integrity. Cabral et al. (70) found that overexpression of 24 certain cell wall adhesins, such as PGA22, disrupted biofilm growth. Furthermore, upregulation of cell adhesins had different consequences in mono-culture biofilms compared to polymicrobial. Thus, regulation of cell ahesions is dependent on the nature of the biofilm and substrate.

The secreted aspartyl , (Sap1-10), are secreted proteases that are differentially expressed in hyphal and yeast cells and important for adhesion, invasion and tissue degradation during infection (11, 71). During biofilm formation the SAP-encoding genes are significantly upregulated in both in vitro and in vivo growth conditions (42, 71). In denture stomatitis, inflammation of the palatal mucosa in denture wearers is induced by biofilm growth on the surface of the top of denture. Under these conditions, SAP8 expression is highly unregulated followed by SAP5, 6, 2 and 1. Overall a positive correlation between proteinase activity and biofilm formation associated with infection have been observed in C. albicans oral isolates (71, 72). Bloodstream isolates of C. albicans have upregulated gene expression of

SAP5 and 9 during in vitro biofilm growth compare to isolates from other sites (73). Deletion of

SAP5 and 6 causes in vivo and in vitro biofilm defects (74).

The Saps have been shown to be hyphae-specific genes important for induction of hyphae-infiltration (4, 75). However, the observations during biofilm growth suggest that the

Saps have functions distinct from hyphal invasion during infection (71-74). Defects in biofilm adhesion suggest that these proteases are required for proper adhesion during the initiation stage (74). Thus, the Saps may also play a role in both seeding new biofilm after dispersal and maintaining adhesion during biofilm maturation (71, 73, 74). The dynamic expression of the

SAP genes over time suggests they play different roles during the stages of biofilm development such as nutrient acquisition, cell-cell interactions (adhesion, quorum sensing), and extracellular matrix production and remodeling (42, 71-74, 76).

25 Antifungal treatment strategies

Traditional antifungal drug development is limited because humans and fungi are evolutionarily closely related resulting in a limited number of probable drug targets and greater potential for toxicity due to off target affects (77-79). Currently, three classes of traditional antifungals (polyenes, azoles, echinocandins) are commonly used to treat C. albicans infections (80). These antifungals have different advantages and limitations with toxicity and cross reactivity being the main challenges (78).

Polyenes were the first antifungals discovered and are natural derivatives of antifungals from Streptomyces spp. (77, 81). Polyenes generate pores in the cell membrane by binding ergosterol, leading to ion leakage and ultimately cell death (82, 83). The most successful polyene is Amphotericin B and is commonly used for serious fungal infections, but has severe toxic side affects (77, 84). Azoles disrupt the cell membrane fluidity by disrupting biosynthesis of ergosterol and are extensively used because of their low toxicity (85). Fluconazole, itraconazole, voriconazole, and posaconazole are the most commonly used azoles and are often used as a first measure against fungal infections (77). Azole resistance has increased with the widespread use of azoles in the clinical setting (83). Echinocandins are the newest class of antifungal and inhibit cell wall biosynthesis by inhibiting β-1,3-glucan synthase (Fks1)

(86). Caspofungin, anidulafungin, and micafungin are the most commonly used echinocandidns and have little to no side effects (87).

Antifungal Resistance in C. albicans Biofilms

The ability of C. albicans to form biofilms influences antifungal drug resistance due, in part, to the inability to disrupt or permeabilize the biofilms (88, 89). The inability to disrupt C. albicans biofilms correlates with the hypha-yeast ratio, where biofilms with more than 50% hyphae have a significant increase in compression strength thus increasing resistance to disruption (90). Therefore, the structure of the biofilm allows C. albicans to persist even in the

26 presence of antifungals (20, 21, 88). Biofilms of C. albicans are up to 1,000-fold more resistant to antifungals compared to planktonic cells (20, 88, 91, 92).

One key characteristic of biofilm growth is the encasement of the biofilm in the protective extracellular matrix that protects the biofilm integrity and acts as a physical barrier against antifungal agents. The extracellular matrix of C. albicans biofilms is comprised of proteins, polysaccharides, , and nucleic acid. Although each of the extracellular matrix components augment the architecture and integrity of the biofilm, only polysaccharides and nucleic acid have been shown to enhance antifungal resistance (93-97).

The extracellular matrix also contains polysaccharides, mostly mannan-glucan complexes (98). The β-1,3-glucan synthase gene, FKS1, produces cell wall β-1,3-glucan that is deposited into the ECM of C. albicans biofilms. Nett et al. (20, 99) demonstrated that β-1,3- glucan sequesters antifungals, impeding diffusion of the antifungal and conferring resistance of the biofilm to antifungals. Heterozygous deletion of FKS1 (GSC1) reduced β-1,3-glucan synthesis and deposition in the extracellular matrix, which increased biofilm susceptibility to fluconazole (99). Moreover, depletion of β-1,3-glucan by β-1,3-glucanase treatment increased sensitivity to fluconazole. Whereas, addition of exogenous β-1,3-glucans increases resistance to fluconazole in planktonic cells (20).

Another component of the extracellular matrix that contributes to antifungal resistance is extracellular DNA (eDNA). DNase treatment enhances caspofungin and amphotericin B disruptive activity of mature biofilms (100). eDNA contributes to the maintenance and stability of biofilms and is not required for the early biofilm stages (adherence and initiation). DNase treatment of mature biofilms disrupts biofilm integrity, but does not affect establishment of biofilm (101). Moreover, addition of eDNA to biofilms increases biofilm biomass and does not affect adherence (101). eDNA is also present, to a lesser extent, in the supernatant of planktonic cells and can be enhanced by addition of chitinase in both planktonic and biofilm cells suggesting that the eDNA source is from cell lysis (97, 100, 101).

27 Efflux pumps also contribute to the intrinsic resistance of C. albicans biofilms. There are two major classes of efflux pumps in C. albicans: ATP-binding cassette transporters (CDR1 and CDR2) and major facilitator class pumps (MDR1) (80, 102, 103). In the presence of antifungal drugs these efflux pumps are upregulated in planktonic cells (80, 102). However, during biofilm growth these efflux pumps are upregulated in response to surface adherence, which further influences resistance to antifungal agents (42, 104, 105). Independent of efflux pumps, persister cells contribute to antifungal resistance in biofilms (88). Biofilms are metabolically heterogeneous, with cells at the base more dormant than those at the surface.

Persister cells are metabolically dormant yeast cells found in biofilms of C. albicans that are exceedingly resistant to antifungals and can survive very high concentrations of antifungal drugs to seed new biofilms once antifungal treatment ceases (88, 106). Most antifungal are fungistatic and not fungicidal and are more effective against actively growing cells, which explains the resistance observed in persister cells.

Due to the rise in antifungal resistance the discovery of novel antimicrobial agents is imperative. The rise in resistance to traditional antimicrobials is due impart to targeting essential processes that increase the selective pressure for mutations that confer resistance (3,

91, 92). One promising alternative is the use of antimicrobials that target virulence factors, such as hyphal morphogenesis and the biofilm matrix in C. albicans (3, 4, 92). Although immense advances have been made recently in understanding the molecular details of hyphal morphogenesis and biofilm formation, clinical application of virulence targeting therapeutics has remained underexplored.

Bacterial and C. albicans Interactions and Health Related Prevalence

In the host C. albicans encounters hostile environments with antagonism from the host and commensal bacterial flora. As a result C. albicans has developed a multitude of strategies to overcome these challenges, making C. albicans a resilient pathogen (107). However, in a

28 healthy host C. albicans is for the most part benign. Although the switch from commensal to pathogen is largely dependent on the status of the host, the microflora and presence of other pathogens also play a role in this switch. As both a commensal and pathogen of the human host C. albicans encounters numerous bacterial species, which include both pathogenic and commensal species (11, 108-111). Complex interactions have been previously documented that include both synergistic and antagonist interactions where bacteria and C. albicans can have direct or indirect interactions (108, 110). These interactions can influence the growth and physiology of both microbes, often modulating the virulence of C. albicans (108, 112, 113).

Physical interactions where bacteria adhere to C. albicans has been demonstrated with specific affinity for hyphal cell wall adhesins (112-114). Chemical exchange between C. albicans and bacteria that involves crosstalk between quorum sensing molecules has also been demonstrated in previous studies (114, 115).

Apart from the studies described here, there are only three bacterial species whose interactions with C. albicans have been significantly characterized, ,

Staphylococcus aureus, and Streptococcus gordonii. As the complexity of these types of interactions are being uncovered, two main types of interactions have been observed: antagonism between pathogens (P. aeruginosa−C. albicans) and synergism between pathogens (S. aureus−C. albicans, S. gordonii−C. albicans) (110, 113, 116).

Antagonistic Interactions

Antagonistic interactions between P. aeruginosa and C. albicans have been the most extensively examined and reveal a complex dynamic between two pathogens that are commonly co-isolated from the lungs of cystic fibrosis (CF) patients (117-119). A Gram- negative bacterium, P. aeruginosa is an opportunistic pathogen with a multitude of virulence factors (120). In clinical cases where C. albicans is found during pulmonary infection, such as pneumonia and cystic fibrosis-related infections, it is usually co-isolated with bacteria (121,

122). Furthermore, C. albicans and P. aeruginosa are commonly co-isolated from burn wounds.

Burn victims often have primary infections of P. aeruginosa that are followed by severe life- 29 threatening candidemia (108). Similarly, increased virulence was observed in a murine pseudomonal burn wound infection model during co-infection with P. aeruginosa (123). In vitro investigations in rats found that pre-colonization with C. albicans in lung tissue increased rates of P. aeruginosa associated pneumonia (122). This suggests that C. albicans mucosal biofilms promotes secondary bacterial infections, which has also been supported by a clinical study of ventilator patients who had an increased rate of bacterial infection with prior C. albicans colonization (124).

Conversely, in vitro examination of C. albicans and P. aeruginosa demonstrated antagonistic interaction in a wide range of environmental conditions. During biofilm growth P. aeruginosa preferentially adheres to C. albicans hyphal cell and forms biofilms on the hyphae, which leads to killing of C. albicans. This hyphal specific killing is due to the production of pseudomonal virulence factors, secreted haemolytic phospholipase and phenazines (125-127).

Furthermore, P. aeruginosa inhibits the yeast-hyphal transition by production of a 3-oxo-C12 homoserine lactone, a bacterial quorum sensing signaling molecule that mimics farnesol (126,

128). Farnesol is a quorum sensing signal molecule produced by C. albicans, which represses hyphal morphogenesis and plays an important role during biofilm dispersal (40, 129). Farnesol and 3-oxo-C12 homoserine lactone inactivate the Ras1-cAMP-PKA pathway and increase

TUP1 expression, thus blocking expression of hyphal-specific and biofilm genes that are positively regulated by Efg1 (129-133). Homoserine lactones and similar compounds are produced by other Gram-positive bacteria that similarly repress hyphal morphogenesis in C. albicans, suggesting cross-talk between bacteria and C. albicans might be important in polymicrobial interactions (134, 135).

In addition to the effects P. aeruginosa has on C. albicans, C. albicans does exert changes in P. aeruginosa. Farnesol inhibits the Pseudomonas quinolone signal (PQS), which responds to the production of 3-oxo-C12 homoserine lactone in P. aeruginosa and controls the production of phenezine products (136). During co-culture of P. aeruginosa and C. albicans, a reduction in PQS and thus phenezine products, suggests that the production of farnesol from 30 C. albicans is great enough to have an influence on P. aeruginosa (136). Taken together these results suggest an antagonistic relationship exists. However, these pathogens are frequently found during co-isolation in the clinical setting suggesting that a synergistic relationship might exist in certain settings. It is possible that the antagonistic behaviors observed in vitro allow P. aeruginosa to use C. albicans biofilms as a sort of scaffold or foundation, thus increasing the survival and colonization rates. There is apparent antagonism in vitro, but synergism in vivo, similar to our observation with E. faecalis and C. albicans.

Antagonist interactions between C. albicans and Streptococcus mutans have also been documented. S. mutans are Gram-positive bacteria found in the oral cavity and are associated with dental caries. They are resistant to highly acidic environments and produce acids that are associated with dissolution of the tooth surface, which leads to dental caries (137, 138). S. mutans and C. albicans are commonly co-isolated from early childhood dental caries and denture plaques of healthy individuals (139, 140). Similar to P. aeruginosa, S. mutans produces quorum sensing signaling molecules that inhibit C. albicans hyphae development.

The competence-stimulating peptide (CSP) was found to inhibit hyphal development in

C. albicans (141). CSP is necessary for natural competence of S. mutans. Additionally, trans-2- decenoic acid, a diffusible signaling molecule inhibits hyphal development of C. albicans (142).

SigX, an alternative sigma factor in S. mutans, is the master regulator of quorum sensing (143).

Strong induction of sigX is observed in S. mutans biofilms grown with C. albicans compared to monoculture biofilms suggesting that C. albicans stimulates quorum sensing signaling in S. mutans (144).

Synergistic Interactions

Adhesion is an important factor for C. albicans colonization, pathogenesis and polymicrobial interactions. The ALS proteins in particular play an important role in the biofilm lifecycle and polymicrobial interactions. Many of the clinical conditions where C. albicans is co- isolated with bacteria are considered biofilm-associated and adherence and/or colonization to a surface are a requirement for infection (145). The ALS (agglutinin-like sequence) genes encode 31 eight different glycoproteins that contain an N-terminal domain with adhesive function that are found diffusely over the cell surface of C. albicans (146).

A vital component in inter-kingdom interactions, coaggregation is essential for polymicrobial communities found in the oral cavity and facilitates the complex architecture found in dental plaques. C. albicans is capable of adhering to various oral streptococcus species (S. mutans, S. salivarius and S. gordonii). The ALS proteins mediate these interactions with streptococci during polymicrobial biofilm development. Specifically, Als3 in C. albicans is necessary for biofilm formation with S. gordonii on a salivary pellicle (147). Furthermore, S. gordonii was able to bind Saccharomyces cerevisiae heterologously expressing C. albicans

Als3p (147). Als1 was also found to bind to the surface of S. gordonii; the Als proteins have similar structures, and thus redundant function is feasible. The presence of S. gordonii enhanced hyphal morphogenesis and biofilm formation in human saliva mediated by SspA and

SspB bacterial surface proteins that interact with Als1 and Als3 (146, 148). Additionally, quorum sensing signaling via luxS regulation of autoinducer-2 (A-II) is necessary for enhanced hyphal morphogenesis and biofilm formation (149).

Synergistic interactions between and C. albicans have been observed. S. aureus has an affinity for binding to hyphal cells of C. albicans that is mediated by

Als3 on the surface of C. albicans (113). S. aureus, a Gram-positive opportunistic pathogen found commonly on the skin, possesses a series of virulence factors that contribute to its significant prevalence in nosocomial infections in immunocompromised individuals (150). S. aureus was unable to bind to the adhesin-deficient mutant strains of C. albicans ALS3 (113). In addition, S. aureus was able to bind the recombinant purified N-terminal domain of Als3 and

Saccharomyces cerevisiae heterologously expressing C. albicans Als3p (113). In an ex vivo murine co-infection model, tongues excised from mice where inoculated with C. albicans and S. aureus in a 12-well plate, S. aureus was found in the outer layers of the tissue when tongues were infected with S. aureus only or with the ALS3 mutant strain. Conversely, tongues infected

32 with S. aureus and wild-type C. albicans had S. aureus in epithelial tissue with invasive hyphae

(113).

The interactions between C. albicans and S. aureus have also been examined extensively in multiple animal models that demonstrate a significant increase in virulence of both microorganisms. During intraperitoneal infections of mice with C. albicans and S. aureus, mice have increased mortality rates and both microorganisms are found together in the spleen, pancreas, and esophagus. This demonstrates synergistic behavior during dissemination from the intraperitoneal site of infection, similar to in vitro observations (151-153). Co-infection with

C. albicans and S. aureus in a peritonitis murine model significantly increased mortality compared to mono-infection and increased fungal and bacterial burden was observed during co-infection. Increased proinflammatory cytokines (IL-6 and G-CSF) and neutrophil infiltration was observed during co-infection. Treatment with nonsteroidal anti-inflammatory drugs

(NSAIDs) increased mouse survival and lowered fungal and bacterial burdens in the kidneys and spleen (154).

C. albicans has complex interactions with both pathogens and commensal bacteria found in the human host in various niches. These complex interactions can be physical interactions where bacteria adhere with specific affinity to hyphal cells, as has been demonstrated to occur with C. albicans hyphal cell wall ahesions (112, 113). Chemical exchange between C. albicans and bacteria that involves crosstalk between quorum sensing molecules has also been observed (133, 155). Furthermore, a combination of these types of interactions can transpire between C. albicans and bacterial species, alluding to the complexity of the inter-kingdom interactions that occur.

Co-isolation of C. albicans and E. faecalis

The most well studied niches of C. albicans are the gastrointestinal (GI) and urogenital tracts and it is well documented to come into contact with the bacterial flora, such as

33 Enterococcus faecalis. Interestingly, co-isolation of C. albicans and E. faecalis is common, of which the frequency is likely due to their biological similarities, specifically colonization of overlapping niches (156-158). Both are opportunistic pathogens causing serious infection in the presence of immunological dysfunction.

The prevalence of C. albicans and E. faecalis co-isolation from sites throughout the human host is effectively demonstrated by a large-scale retrospective examination of 67,765 different clinical samples (109). The samples were collected at a large teaching hospital in

Germany over a two-year period. Candida spp. were detectable in about 7.7% of the samples that included blood, CSF, feces, sputum and skin. The frequency of E. faecalis isolation increased 2-fold in the Candida-positive samples compared to Candida-negative samples. This trend was further increased in Candida-positive samples from blood and ICU patients, demonstrating the prominence of co-infection in more severe infections and individuals with pre-existing medical conditions (109).

Commonly co-isolated from the oral cavity of individuals with secondary root canal infections, C. albicans and E. faecalis are typically resistant to antimicrobials, a probable cause for failed root canal treatment strategies (159). During root canal treatment, the infected root canals are mechanically removed and irritants are chemically removed followed by blocking the root canal system with dental filling (gold, porcelain, silver amalgam, polymethyl methacrylate resin, PMMA) to eliminate microbial activity thus preventing reinfection. However, absolute removal of all microorganisms from the root canal is not always achieved and the oral cavity is abundant with microorganisms that can colonize microspaces between the dental filling acrylic and tooth surface (160-162).

Multifaceted microbial communities that consist of anaerobic bacteria (strict and facultative) and fungi reside in the root canals of infected teeth (163, 164). Of these microbes,

E. faecalis and C. albicans are equipped to resist various endodontic treatment strategies that allow them to survive and cause secondary root canal infections. (165-167).

34 Many studies demonstrate the importance of C. albicans and E. faecalis separately during secondary root canal infection. Yet, only a handful of studies investigate the co-isolation of these microorganisms. Given their pathological similarities during periodontal infections it is plausible that the occurrence of co-infection rates is underrepresented. A study investigating single-rooted teeth with pulp necrosis, found that samples collected after root canal treatment had increased populations of enterococci co-isolated with Candida spp. and/or enteric bacteria

(168). However, the majority of samples tested had enterococci mono-infections, suggesting that interactions with other microorganisms are not necessary for this type of periodontal infection (168).

Although secondary root canal infections are treatable with additional surgical removal and debridement of the infected root canal and filling, if left untreated can become a more severe systemic infection. C. albicans and E. faecalis that survive the root canal system can spread to the periodontal tissues and invade the bloodstream, which can lead to bloodstream infections (BSI) and followed by development of endocarditis. BSIs with at least two different microorganisms are considered polymicrobial BSI and are on the rise with rates ranging from

5% to 32% of all BSI reports (169-172). With increased mortality rates (21%-63%) compared to mono-microbial mortality rates, polymicrobial BSIs are a growing concern (169, 170).

Of more than 8000 reported incidences of candidemia (from 1965 to 2007) 23% were polymicrobial, suggesting that high rates of polymicrobial BSI occur with candidemia (157). An extensive study examining patients with candidemia found that of 372 patients, 141 patients from 8 Veterans Affairs hospitals and 231 patients from a tertiary care hospital, 27% had polymicrobial BSI (24% with synchronous bacteremia and 3% with more than one Candida spp.) (157). Of the patients with synchronous bacteremia (88 patients) 27% where infected with

Enterococcus spp. Furthermore, the most frequent source of polymicrobial BSI is from intra- abdominal infections that are frequently the result of loss of GI integrity allowing

35 microorganisms, such as C. albicans and E. faecalis, from the GI tract to infect the peritoneal cavity (170, 173-175).

Most populations of C. albicans and E. faecalis are located in the GI tract where they act as commensals and are for the most part benign. However, if the host becomes immunocompromised these microbes can become pathogenic and the populations in the GI tract are a common reservoir for more severe types of infections. Intra-abdominal infections

(IAI) are commonly caused by infection from the GI tract microbiota and include many pathological conditions that can range from uncomplicated, where only one organ is infected and the infection does not progress to the peritoneum, to complicated, where the infection progresses past the organ to the peritoneum causing localized or diffuse peritonitis (176,

177).

Peritonitis is further classified into primary, secondary or tertiary peritonitis (176).

Primary peritonitis is a disseminated infection with no loss of GI tract integrity and mainly occurs in pediatric patients. The most common form, secondary peritonitis is an acute peritoneal infection that results from loss of GI integrity due to perforation of the GI tract or from intra-abdominal viscera from infected organs (e.g. gangrenous appendicitis). Recurrent infections of the peritoneal cavity that follow primary or secondary peritonitis are classified as tertiary infections. These types of infections can be acquired from the community or healthcare setting. The latter is associated with increased mortality due to the status of the host and multi drug resistance of the infections (178). Additionally, increased mortality rates (~%30) are associated with severe sepsis or toxic shock in secondary infections (179-181).

A retrospective study of 163 adult patients with intra-abdominal candidiasis (IAC) compared to 161 patients with candidemia found increased rates of bacterial co-infections in patients with IAC (67%) compared to polymicrobial BSI (6%) (182). Different types of IAC were evaluated: intra-abdominal abscesses (55%), secondary peritonitis (33%), primary peritonitis

(5%), infected pancreatic necrosis (5%), and cholecystitis/cholangitis (3%). The most common

36 Candida spp. isolated were C. albicans (56%) and C. glabrata (24%). Consistent with previous studies, enteric pathogens (Enterococcus and Enterobacteriaceae) were co-isolated with

Candida spp (183, 184). Of the 110 patients with bacterial co-infection, enterococci were most abundant (29%), followed by (19%) and Klebsiella spp. (15%) (182). However, the mortality rates for IAC mono-infection and IAC bacterial co-infections are comparable in these investigations, suggesting that Candida spp. are capable pathogens and not significantly modulated by co-infecting bacteria in IAI (182, 185).

E. faecalis Pathogenesis

Enterococci are the initial colonizers of the GI tract in infants and were once only considered as commensal colonizers with nominal clinical relevance. However, over the last four decades, with increased use of antibiotics, enterococci have become prominent opportunistic pathogens responsible for causing numerous types of infections: urinary tract infections (UTI), IAI, bacteremia, and endocarditis (186). Antibiotic resistance is directly related to the emergence of enterococci as a clinically important pathogen and the most clinically significant species are Enterococcus faecalis and Enterococcus faecium (186, 187). Since enterococci are intrinsically resistant to many antibiotics, have the ability to acquire resistance genes, and are tolerant to antibiotics that target the cell wall, treatment of enterococcal infections is often problematic (187, 188). Thus, the use of combinational therapy is required to synergistically acquire bactericidal effects (189).

As part of the microflora enterococci colonize the GI tract of mammals (including humans) and insects. Although both E. faecalis and E. faecium colonize humans, E. faecalis colonizes the GI tract at a higher density (107 CFU/μg) than E. faecium [191]. E. faecalis is also the predominant species of enterococci found in the urogenital tract. The endogenous microflora was previously thought to be the predominant source of enterococcal infections.

Nevertheless with increased investigations in the clinical setting, transmission of pathogenic

37 enterococci from nosocomial sources (e.g. hands of healthcare provider) has been extensively documented (190, 191). The ability of enterococci to persist for extended periods on hands (60 minutes) and inanimate objects (4 months) creates a reservoir for continual transmission in the hospital setting (192). Necessary for the numerous types of infections they cause, E. faecalis has a multitude of virulence factors, such as: cytolysin, gelatinase, serine , aggregation substances, Esp (enterococcal surface protein), Ace (adhesin to collagen of E. faecalis), EfaA (E. faecalis antigen A), EbpABC (endocarditis and biofilm-associated pili), and enterococcal capsule (193). Acquisition of extrachromosomal elements plays an important role in adaptation and survival of enterococci (194). Extrachromosomal elements are usually acquired by conjugation, where conjugative plasmids are transferred from a donor to recipient controlled by peptide sex pheromones (194). Antibiotic resistance, elevated resistance to UV light, virulence factors and bacteriocins are frequently -determined traits. Thus, significant genetic diversity in response to environmental selection pressure is observed in enterococci (195, 196).

Part of the core genome, the proteases, gelatinase (GelE, a matrix metalloprotease) and a (SprE), in E. faecalis are important for degradation and damage of host tissues during infection (197). These proteases affect a broad range of host functions by degradation of host connective tissues and proteins, and interference with host cell signaling and immune response (198, 199). Included in a single operon, GelE and SprE gene expression is regulated by the fsr (E. faecalis regulator) genes that encode a canonical two-component system, a homolog to the agr (accessory gene regulator) operon in S. aureus (200, 201).

The response regulator (FsrA) positively regulates the fsr genes (fsrABCD) and downstream virulence factors (gelE, sprE). A quorum sensing two-component system, Fsr responds to increased concentrations of a peptide pheromone encoded by fsrD, gelatinase biosynthesis-activating pheromone (GBAP) that is secreted and processed by the FsrB. FsrA is constitutively expressed at basal levels, when threshold

38 concentrations of extracellular GABP have accumulated the histidine kinase, FsrC is phosphorylated followed by phosphorylation of the response regulator, FsrA. This signal transduction via the Fsr system results in upregulated expression of the fsr genes and downstream virulence factors, gelE, sprE (197, 202).

Although both GelE and SprE, to a lesser extent, are important virulence factors, only

GelE activity and role in virulence has been extensively examined. The gelE gene encodes a

509- protein of which 29-amino acids at the N-terminus serve as a Sec secretion signal sequence. This signal sequence is cleaved upon secretion resulting in release of the proenzyme of 480-amino acids. After secretion the proenzyme is further proteolytically processed at the N-terminus and C-terminus by an autocatalytic process resulting in the mature protein of 33kDa (203, 204).

GelE is a member of the M4 family of proteases and is similar to from

Bacillus thermoproteolyticus and from Staphylococcus aureus (205, 206). With broad substrate specificity, GelE has the ability to hydrolyze gelatin, cleave enterococcal conjugative sex pheromones, and has several host-derived substrates important during infection (insulin B chain, endothelin, hemoglobin, fibrinogen, fibronectin, collagen, and laminin)

(207, 208). Furthermore, GelE interferes with the innate immune response by inactivating human LL-37 and β-defensins and is pertinent for E. faecalis dissemination (209-211).

A serine glutamyl of 25kDa in size, SprE is similar to staphylococcal glutamyl V8 and GluSE (212, 213). Similar to GelE, insulin, fibrinogen, and casein have been observed as substrates of SprE and disruption of sprE results in decreased virulence in different infection models, mouse peritonitis, Caenorhabditis elegans, and a rabbit endophthalmitis (201, 214-217). SprE enzymatic activity is modified in the presence of GelE similar to reports in S. aureus, where the GelE homolog, aureolysin, inhibits autocatalytic processing of the SprE homolog, SspA (V8), by proteolytically processing the N-terminus of

39 SspA by aureolysin (218, 219). Purification of SprE proenzyme from gelE mutant supernatant resulted in purification of several forms of SprE that were proteolytically processed at both the

N-terminus and C-terminus (218, 220). Three different mature forms of SprE were purified that had enzymatic activity (Leu1-Ala237, Ser-1-Glu227, Leu1-Glu227). However, only one form of SprE

(25kDa) was purified from wild-type supernatant (218, 220). Although GelE processes SprE, the reverse is not apparent because a sprE insertion mutant produces gelatinase (201).

Considering the interconnected gene regulation and proteolytic processing of GelE and

SprE, it no surprise that they function in combination. While GelE and SprE play independent roles in different infection models (mouse peritonitis, Caenorhabditis elegans, rabbit endophthalmitis) disruption of both proteases had an additive effect (201, 214-217). Moreover, roles for both GelE and SprE have been observed in biofilm development and regulating autolysis in E. faecalis (221). Deletion of gelE resulted in a significant reduction in biofilm biomass compared to wild-type biofilms. Whereas deletion of sprE resulted in accelerated biofilm development and increased autolysis, suggesting that interplay between these proteases controls biofilm development in E. faecalis (221).

E. faecalis Commensalism

Enterococci colonize the GI tracts of mammals, reptiles, birds and insects, which suggests that enterococci have been part of the GI microflora since the early Devonian period,

(~412 MYA), making them one of the earliest colonizers of the GI tract (222-224). In the human

GI tract, enterococci are predominantly located in the small and large intestine (jejunal, ileal, cecal, and recto-sigmoidal sections) (225). E. faecalis and E. faecium are the predominant enterococci found in human feces, and E. faecalis is most common species found in the GI tracts of mammals and insects (226, 227).

E. faecalis is a member of the lactic acid group of bacteria (LAB), which are low-GC gram-positive bacteria that produce lactic acid as a major end product of

40 fermentation [232,233]. Many of the LAB are used as probiotics, specifically Lactobacilli spp., which have been shown to benefit GI health (228, 229). In spite of the fact that enterococci are as abundant in the human GI tract as lactobacilli, they are commonly associated with being a multidrug resistant (MDR) pathogen (228). Although commensal enterococci can act as opportunistic pathogens by translocating across the mucosal barrier causing systemic infections in immune-compromised individuals, more commonly infections stem from colonization, overgrowth, and translocation of hospital-adapted strains that have enhanced pathogenicity and antibiotic-resistance (230, 231).

Enterococci are commonly used in food fermentation and preservation, traditionally used in cheeses, fermented and vegetables manufactured in Mediterranean countries

(232-234). E. faecium and E. faecalis are the most commonly used and impact the ripening and aroma development in these food products (229, 233). Enterococci are not purposely added as starter cultures to these types of food. However, they occur in high numbers and their natural habitat is probably not fermented food, but an outside environmental source that has yet to be identified (235).

Enterococci as probiotics are used in countries (European Union) other than the US

(229, 236, 237). E. faecium and E. faecalis are the species most commonly used as probiotics.

The E. faecium strain SF68 probiotic, was demonstrated to be clinically effective in children for treatment of diarrhea and prevention of diarrhea due to antibiotic treatment (238). The efficacy of E. faecium SF68 in treatment of intestinal infections is likely due to the strain being part of the natural intestinal microflora and having a short doubling time in optimal conditions. Growth inhibition of E. coli, serovars, Shigella spp. and Enterobacter spp by E. faecium

SF68 was demonstrated in vitro and shown to tolerate the extreme conditions of the GI tract

(239). Other probiotic preparations containing E. faecium have been shown to reduce irritable bowel syndrome, dysbiosis as a result of foodborne pathogens, and lowering of serum cholesterol (240, 241).

41

Bacteriocins Produced by Gram-positive Bacteria

Bacteriocins are produced by Gram-positive and Gram-negative bacteria and are ribosomally synthesized proteinaceous secreted compounds that have antimicrobial activity against other bacteria, typically those that are closely related to the bacteriocin producing species. Production of bacteriocins in Gram-positive bacteria, especially the

(LAB), has been increasingly studied with the growing interest of probiotic LAB. Bacteriocins produced by Gram-positive bacteria can be organized into four major groups: (1) ,

(2) small non-modified peptides, (3) large proteins, and (4) cyclic peptides. The classification of bacteriocins from Gram-positive bacteria is continually modified with the discovery of new types of bacteriocins. However, Heng et. al. (242) have formulated a systematic classification plan based on previous literature that encompasses recently characterized bacteriocins (243-245).

Class I bacteriocins are the lantibiotic bacteriocins that contain modified amino acids that are not genetically encoded (246). Lantibiotics are produced ribosomally as precursor peptides that undergo post-translational modifications to produce the non-genetically encoded amino acids (Lan), 3-methyllanthionine (MeLan), dehydroalanine (Dha), and dehydrobutyrine (Dhb) (247). Class II bacteriocins are small (<10 kDa), unmodified (non- lantibiotic and non-cyclic) peptides that encompass bacteriocins from diverse origins making it the largest class of characterized bacteriocins (248). Although most characterized bacteriocins from Gram-positive bacteria are small (<10 kDa), class III contains larger (>10 kDa) bacteriocins that are typically heat liable, but not always (243, 249). The most distinctive bacteriocins produced by Gram-positive are the cyclic bacteriocins of class IV, which are post- translationally modified to produce the mature cyclic peptide (250).

Bacteriocin Production in E. faecalis

42 Enterococci that produce bacteriocins have been isolated form a wide-range of environments. Bacteriocin-producing enterococci are commonly found in cheeses, fermented vegetables and meats, thus, have been implicated in (229, 233, 250). The high frequency of bacteriocin-producing enterococci isolated from healthy individuals stool samples suggests that they are important part of the healthy microflora and may benefit the host and microbe (251). Although bacteriocin production in LAB is commonly associated with probiotics and benefiting the health of the GI tract, enterococcal bacteriocins have also been associated with infection (252, 253).

In E. faecalis bacteriocin encoding genes are often carried on mobile genetic elements, such as conjugative plasmids, suggesting that efficient dissemination of bacteriocin-producing traits may be important for survival and virulence. Cytolysin is a secreted lantibiotic class of bacteriocins with cytotoxic effects on prokaryotic and eukaryotic cells (252, 253).

Epidemiological data support a role for the cytolysin as a in human infection. Enrichment for cytolysin expressing E. faecalis has been observed in the hospital-acquired populations compared to community acquired populations of isolates derived from infection (254). These findings suggest that selection pressure for cytolysin activity is prevalent in the hospital environment, which corresponds to experimental and clinical evidence that cytolysin activity plays an important role during infection (255).

On the other hand, the heat-stable circular bacteriocin AS-48 produced by E. faecalis strains that are commonly isolated from cheese, milk, and fermented food products has potential as food biopreservative (250, 256). This bacteriocin is encoded on a plasmid that contains ten biosynthetic genes (257). Arranged into five alpha helices giving it a compact globular structure, this 70 amino acids bacteriocin is circularized head to tail by a peptide bond.

AS-48 has broad inhibitory spectrum that includes both Gram-positive and Gram-negative species (250). AS-48 is active against foodborne pathogens, toxigenic bacteria and bacteria associated with food spoilage (258).

43 The role of bacteriocins production in natural environments is debatable. However, one logical and common idea is that bacteriocins provide the producing organism with a fitness benefit that allows them an ecological advantage over competitors (259). The occurrence of bacteriocins as a beneficial probiotic trait in the human GI tract and as a virulence trait during infection, suggests that bacteriocin production allows enterococci the ability to adapt to different environmental conditions. Moreover, bacteriocins enriched during infection associated with antibiotic resistance are different from bacteriocins associated with commensal enterococci from healthy individuals (260).

Bacteriocins as Treatment Strategies

Bacteriocins produced by LAB have mainly been used and extensively studied in . However, more recently the interest for using bacteriocins for biomedical applications has increased with investigations for the use of bacteriocins against pathogenic microorganisms still gaining significance (261). Studies examining biomedical application of

LAB bacteriocins include efficacy analyses against systemic infections caused by S. aureus and P. aeruginosa, oral infections caused by Streptococci, and vaginal infections caused by

Gardnerella vaginalis (262, 263).

One of the most extensively characterized bacteriocins is the lantibiotic that is produced by LAB (Lactococci and Streptococci) (264, 265). First characterized in Lactococcus lactis isolated from fermented milk cultures, nisin is approved by the Joint Food and Agriculture

Organization/ World Health Organization (FAO/WHO) and the Food and Drug Administration

(FDA) for use as biopreservative (244, 265). Nisin A from Lactococcus lactis is a small (34 amino acids) cationic peptide that contains five lanthionine rings that are formed by dehydration of precursor serines and threonines that react with neighboring cysteines to give lanthionine or

3-methyllanthionine that contain a thioether group (266).

44 Nisin A targets II, which is essential for bacterial cell wall biosynthesis and growth,

(267, 268). The high affinity for binding lipid II initiates pore formation in the membrane leading to depletion of the membrane potential due to leakage of cellular content followed by cell death.

Nisin A has broad spectrum of activity against Gram-positive bacterial pathogens (foodborne and non-food related) and Gram-negative bacteria when used in conjunction with antibiotics

(269, 270). Other natural variants of nisin have been identified from different Lactococcus spp. and Streptococcus spp (270). Although, these natural variant share similar structure, slight changes in the amino acid sequence alter the efficacy and target spectrum of these bacteriocins, as seen with Nisin Z (271, 272). Moreover, bioengineered nisin variants have enhanced activity against Gram-negative pathogenic bacteria, demonstrating the potential of bacteriocins based therapeutic strategies (273).

Bacteriocins Active Against Candida spp.

Bacteriocins have traditionally only been tested for their antibacterial properties.

However, the prominence of nosocomial fungal infections resistant to traditional antifungals has amplified the need for alternative treatment strategies, such as bacteriocin-based therapeutics.

A similar natural-variant of nisin A, nisin Z is produced by L. lactis NIZO22186 and significantly inhibits in vitro growth and hyphal morphogenesis of C. albicans (271, 274). Additionally, nisin

Z inhibits hyphal morphogenesis and adhesion of C. albicans to oral gingival cell in an ex vivo oropharyngeal candidiasis (OPC) model (275). Although nisin A and Z have similar antimicrobial properties, there is a single residue change (H27N) that confers a superior diffusion rate and increases solubility at neutral pH (272). This suggests that subtle changes in bacteriocins can yield increased efficacy against fungal pathogens. However, other natural or bioengineered variants of nisin have not been reported to have activity against C. albicans.

Lactobacillus plantarum, another LAB associated with fermented food, produces multiple bacteriocins (class II) that comprise the plantaricin C11 complex, components of which

45 have been reported to have fungicidal activity against C. albicans (276, 277). The plantaricin

C11 locus includes five operons: plnABCD, the three-component signal transduction system; plnEFI, two bacteriocins (PlnE and F) and immunity factor; plnJKLR, two bacteriocins (PlnJ and

K) and immunity factors; plnMNOP, unknown function; plnGHSTUV, bacteriocin export machinery (278). The mechanism of action for plantaricin C11 is similar to other bacteriocins of its class, PlnEF and PlnJK target the membrane, create pores that disrupt the membrane potential and lead to bacterial cell death (269, 279). Given the difference in cell wall anatomy of

Gram-positive bacteria and C. albicans the mechanism of action could vary. Treatment of C. albicans with the different bacteriocins disrupted the membrane potential, however, PlnJ had the greatest effect and caused release of potassium ions (K+) from cells. Additionally, all plantaricin peptides increased reactive oxygen species (ROS) in C. albicans, suggesting that plantaricins induce apoptotic cell death resulting in toxin-induced necrosis (277).

Antimicrobial Peptides Active Against C. albicans

Part of the innate immune response, antimicrobial peptides (AMPs) are differentially expressed in response to pathogen-associated molecular patterns (PAMPs) that are recognized by pattern recognition receptors (PRRs) present on host cells. AMPs from the major classes, including cathelicidins, histatins, and defensins are demonstrated to be active against

C. albicans. Interestingly, AMPs have common characteristic found in bacteriocins (cationic, amphipathic, positive net charge, structure stabilized by thiols), suggesting mechanisms of action may overlap between AMPs and bacteriocins (266, 280).

The antimicrobial activity of histatin-5 (Hst-5) against C. albicans is the most extensively examined of the AMPs that target C. albicans (281-283). Hst-5 is a cationic peptide found in saliva providing the first line of defense against oral pathogens such as C. albicans. The mechanism of action against C. albicans involves binding cell wall β-glycans and the cell wall protein, Ssa1 and 2 (Stress-Seventy subfamily A), that allow for translocation of Hst-5 into the

46 cell via polyamide transporters (Dur3 and 31, Degradation of URea) (284-287). Inside the C. albicans cell, Hst-5 has multiple intracellular targets that lead to disruption of mitochondrial function, production of ROS, osmotic stress due to loss of potassium ion (K+), and then ultimately cell death (288, 289).

LL-37 is a human cathelicidin that is derived by of the CAP18 protein C- terminus and is produced by macrophages, neutrophils, keratinocytes, and mucosal epithelial cells (290-292). Shown to have fungicidal activity against C. albicans, LL-37 associates with the cell wall and/or membrane and binds to the cell wall protein Xog1 (β-1,3-exoglucanase) that causes remodeling of the cell wall of C. albicans (293). Increased concentrations of LL-37 disrupt the cell membrane causing segregation of the membrane into small vesicles disrupting the membrane potential due to loss of intracellular contents. The mechanism suggests that LL-

37 is able to remodel the membrane via Xog1 binding, thereby allowing for interaction with the cell membrane and eventually disrupting the cell membrane and causing cell death (293-295).

Defensins are small (3.5 to 6 kDa) with three disulfide bridges and are grouped in two classes, α-defensins and β-defensins (296, 297). Found in neutrophil granulocytes, α-defensin

(HNP-1) is active against C. albicans by non-lytic ATP efflux that depletes intracellular ATP leading to cell death (298). β-defensins are larger and share little primary structure similarity, but have very similar tertiary structures (297). Expressed in environmentally exposed epithelia,

β-defensin (hBD-3) has candidacidal activity against C. albicans by binding Xog1 which causes cell wall remodeling similar to LL-37, without depletion of ATP (295).

AMPs are generally positively charged (+2 to +9), small peptides of 10 to 50 amino acids that are frequently hydrophobic residues (≥30%). The structure of AMPs is important for antimicrobial activity and many are amphipathic helices that form pores on bacterial membranes (299). As reported, the mechanisms of action against C. albicans of these AMPs are different and often require remodeling or transient interactions with the cell wall of

C.albicans. However, the structural conformation of these AMPs is important for interactions 47 with the cells wall and membrane that initiate the mechanism of action (294, 296, 300).

Interestingly, sub-lethal concentrations of human LL-37, hBD-3 and Hst-5 reduce adhesion of

C. albicans to polystyrene (293, 295, 301, 302). LL-37 and hBD-3 also inhibit hyphal morphogenesis at sub-lethal concentrations, suggesting that these AMPs could potentially have clinical relevance in biofilm prevention (295, 302).

48

CHAPTER 2:

Materials and Methods

Portions of this chapter are based on my first author publication. I have received permission to reproduce data or text from this article. I have contributed significantly to this publication. The article is listed for reference: Graham, C. E., M. R. Cruz, D. A. Garsin, and M. C. Lorenz. 2017.

Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans. Proc. Natl. Acad. Sci. U.S.A. 114: 4507–4512 (303).

49

Strains and Culture Conditions

Bacterial and fungal strains used in this study are listed in Table 2.1. Media was purchased from DIFCO and chemicals from Sigma, unless otherwise stated. Escherichia coli strains were cultured in Luria Bertani broth overnight at 37°C with antibiotics where appropriate using the following concentrations (µg/ml): kanamycin, 50; spectinomycin, 100; erythromycin

300. E. faecalis strains were cultured overnight at 37°C in Brain Heart Infusion (BHI) medium or

M9HY (304) with 100 μM glucose. Addition of antibiotics where applicable were at the following concentrations (µg/ml): erythromycin, 50; and rifampicin, 100. Fungal strains were propagated in yeast extract-peptone-dextrose (YPD) (305). All C. albicans biofilms were grown in artificial saliva (YNB-AS) modified from Wong et al. (306) (0.17 % YNB (w/v), 0.5 % casamino acids

(w/v), 0.25 % mucin (w/v), 14 mM potassium chloride, 8 mM sodium chloride, 100 μM choline chloride, 50 μM sodium citrate, 5 μM ascorbate).

Strain Construction

The entV mutant strain was constructed as previously described to create an in-frame markerless deletion using a P-pheS* counterselection system (307). Briefly, sequences flanking the 5’ and 3’ ends of entV were amplified by PCR. The 2 products were fused together by PCR with primer-introduced NotI and Pst1 sites. The resulting fragment was ligated into

NotI/PstI digested pCJK47 vector, resulting in pCEG1. pCEG1 was electroporated into CK111 cells, and counterselection was completed to create strain CEG1F. Deletion of entV was confirmed by PCR and sequencing. The entV complement strain was constructed using a previously described strategy in which the wild type locus is regenerated with a silent mutation in the gene to distinguish it from the original wild type strain (308). To generate entV*, a mutation (CTT) was introduced into the leucine codon TTA corresponding to amino acid position 50. The hyphal specific reporter strain (HWP1p::GFP) was transformed with plasmid

50 pADH1-mcherry (ADH1p::mcherry) to construct a C. albicans strain expressing both hyphal specific GFP and constitutive mcherry (309).

C. albicans Biofilm Assay

The C. albicans biofilm assay was developed using a similar technique as described previously with the following modifications (42, 44, 310). C. albicans strains were grown overnight at 30°C in YPD and washed three times with phosphate-buffered saline (PBS), and

7 the final OD600 was adjusted to 0.2 (~1 x 10 cell/ml) in PBS. Tissue culture treated 96 well polystyrene plates (Corner) or chambered polystyrene slides (Ibidi) were inoculated with C. albicans in PBS and incubated at 37°C for 90 minutes. Inoculated plates and slides were then gently washed with PBS. After removal of PBS, YNB-AS was added with 50% bacterial supernatant or sterile M9HY and incubated at 37°C for 24 to 48 hours. The supernatant of E. faecalis (OG1RF, ΔfsrB, ΔentV, ΔentV/entV) was collected as previously described (115).

Biofilms grown with rEntV136 or sEntV68 were grown similarly with the peptide added to the

YNB-AS medium at the appropriate concentrations and compared to HEPES or DMSO vehicle controls, respectively. After 24 to 48 hours of growth biofilms were washed three times with

PBS and hyphal morphogenesis and biofilm biomass were assessed.

Hyphal morphogenesis was quantified in C. albicans (HWP1::pGFP) biofilms grown in

96 well microplates by measuring GFP fluorescence, fluorescence intensity (excitation 488 nm, emission 520 nm) was determined using a fluorescence microplate reader (SynergyMX,

Biotek). The relative fluorescence was calculated per well as a ration of GFP fluorescence to cell density (OD600) and normalized to control treated biofilms (sterile medium, HEPES, or

DMSO). Biofilm biomass was determined by resazurin staining of C. albicans biofilms as previously described (311), using 0.015mg/ml of resazurin for up to 1 hour in the dark.

For microscopic imaging, C. albicans biofilms were gently washed in PBS, as stated above. Wild-type C. albicans (SC5341) were stained using 35 g/ml of calcofluor white for 5

51 minutes in the dark and imaged using 4',6-diamidino-2-phenylindole (DAPI), Fluorescein isothiocyanate (FITC), and differential interference contrast (DIC) filters on an Olympus IX81 automated inverted microscope with Slidebook software (version 6.0) or Nikon A1R Confocal

Laser automated inverted microscope system with NIS Elements (version 4.5).

All biofilm assays were done in triplicate for three independent experiments. The different treatment types per experiment were compared by pairwise by Student’s t-test or One-

Way ANOVA with a P value of <0.05 considered statistically significant using GraphPad Prism

(version 6.0).

Expression and Purification of Recombinant EntV

Hexahistidine-tagged EntV136 was expressed as described previously (312) with the following modifications. The Coding regions of EntV (residues 35 to 171) were cloned in the pET28a vector and transformed in to E. coli BL21 (DE3) cells. Cells were lysed in lysis buffer

(25 mM TrisHCl pH 8.0, 0.1 M sodium chloride, 1% (v/v) NP40, 10% (v/v) glycerol and 1 mM

PMSF) by sonication for 10 minutes on an ice water bath followed by centrifugation at 10,000 x g at 4°C for 20 minutes.

The clarified lysate was then loaded on to a pre-equilibrated TALON affinity resin

(Clontech) and washed three times with 10 column volumes each of wash buffer (50 mM sodium phosphate, 100 mM sodium chloride, 10 mM imidazole). The hexahistidine-tagged protein was eluted in elution buffer (50 mM sodium phosphate, 100 mM sodium chloride, 150 mM imidazole) and elution fractions were collected. Samples with purified protein were combined and dialyzed against buffer containing 5 mM HEPES pH 7.0. The collected fractions were then subjected to Tricine-SDS-PAGE and analyzed by western blot using anti-His antibiodies (THETM, GenScript).

52 Murine Macrophage Infection

The activity of sEntV68 against C. albicans was evaluated in the murine macrophage infection model using the end point dilution assay (313, 314). Briefly, RAW264.7 murine macrophages were seeded in a 96-well plate with RPMI 1640 with phenol red supplemented with 10% fetal bovine serum (RPMI-10) at a concentration of 2.5 x 105 cells/ml per well and incubated overnight at 37°C with 5% CO2. After overnight growth at 30°C in YPD, C. albicans cultures were washed twice with PBS and resuspended at a concentration of 5 x 105 cells/ml in

RPMI minus phenol with 100 nM of sEntV68 or DMSO and incubated at room temperature (RT) with shaking for 1.5 hours. The macrophage seeded 96-well plate was inoculated with serial dilutions of the pretreated C. albicans cell suspension. Media only controls were prepared in the same manner. The plates were incubated at 37°C with 5% CO2 for 15 hours. Fungal biomass was determined by quantification of C. albicans microcolonies using the XTT assay as previously described (315). Absorbance was read at 451 nm in a microplate reader

(SynergyMX, Biotek). Assays were performed with replicates in three independent experiments.

Protection of macrophages by sEntV68 was examined by monitoring cytotoxicity of C. albicans on macrophages using the CytoTox96 Non-Radioactive Cytotoxicity assay (Promega) that measures release of lactate dehydrogenase (LDH) release (316). RAW264.7 murine macrophages in RPMI-10 were seeded in a 96-well plate at a concentration of 2.5 x 105 cells/ml and incubated overnight at 37°C with 5% CO2. C. albicans cell suspensions were prepared at 1.5 x 106 cells/ml in RPMI minus phenol with 100 nM of sEntV68 or DMSO and incubated at RT with shaking for 1.5 hours. Media only controls were also prepared in the same manner. The macrophage seeded plates were inoculated with the pretreated C. albicans cell suspension and incubated at 37°C with 5% CO2 for 5 hours. LDH release was normalized to chemically lysed macrophages and assays were performed with replicates in three independent experiments.

53 Hyphal morphogenesis of C. albicans in phagocytes was quantified after infection of

RAW264.7 murine macrophages as described above with the following modifications.

Macrophages were seeded on glass coverslips in 12-well plates at a concentration of 2.5 x 105 cells/ml and incubated overnight at 37°C with 5% CO2. Overnight cultures of CEG1C

(HWP1p::GFP; ADH1p::mcherry) were subcultured in YPD for 5 h at 30°C. Cells were collected and washed with PBS, and diluted in RPMI minus phenol with 100 nM of sEntV68 or DMSO to a concentration of 1.5 x 106 cells/ml and incubated at RT with shaking for 1.5 hours. Media only controls were also prepared in the same manner. The macrophage seeded glass coverslips were inoculated with the pretreated C. albicans cell suspension and incubated at 37°C with 5%

CO2 for 2 hours. The media was aspirated and cells were fixed in 2.7% paraformaldehyde (pH

7.5) for 15 minutes at 37 °C followed by washing twice with PBS. Images were captured using

Tetramethylrhodamine (TRITC), Fluorescein isothiocyanate (FITC), and differential interference contrast (DIC) filters on an Olympus IX81 automated inverted microscope with Slidebook software (version 6.0). Cell enumeration was performed using at least 10 different fields of view and at least 200 cells per treatment from replicates in three independent experiments.

sEntV Toxicity Assay

Toxicity of sEntV68 on murine macrophages (RAW 264.7) and human cervical epithelial cells (HeLa) was examined by monitoring cytotoxicity affects of C. albicans on macrophages using the CytoTox96 Non-Radioactive Cytotoxicity assay (Promega). This assay measures release of lactate dehydrogenase (LDH) release (32). RAW264.7 murine macrophages in

RPMI-10 and HeLa cells in Dulbecco’s Modified Eagle Medium with high glucose and 10% fetal bovine serum (DMEM-HG-10) were seeded in a 96-well plate at a concentration of 2.5 x 105 cells/ml and incubated overnight at 37°C with 5% CO2. Media from the seeded plates was

68 replaced with media containing 10 μM sEntV and incubated at 37°C with 5% CO2 for up to 72

54 hours. LDH release was measured every 24 hours and the percentage of maximum LDH was calculated as a ratio of LDH from treated cells to LDH from chemically lysed cells.

Oropharyngeal Candidiasis Model

The efficacy of sEntV68 was tested in the oropharyngeal candidiasis (OPC) model as previously described (317). Mice were immunosuppressed by injecting cortisone acetate subcutaneously in the dorsum of the neck at a concentration of 225mg/kg of body weight one day before inoculation, and subsequently on days 1 and 3 of the infection. Before inoculation, with C. albicans mice were anesthetized with an intraperitoneal injection of 0.1 ml/10g of body weight with 10mg/ml ketamine and 1 mg/ml xylazine and placed on pre-warmed hot water blankets. C. albicans was grown at 30°C in YPD broth overnight and sub-cultured twice before washing with PBS twice and preparing cell suspensions in Hanks’ balanced salt solution

(HBSS) at 1 x 106 cells/ml. Calcium alginate swabs were immersed in the C. albicans suspension with 100 nM sEntV68 or DMSO for 5 minutes and then used to inoculate the mice sublingually for 75 minutes.

The mice were monitored continuously for signs of waking up (movement of extremities and/or blinking of the eyes) and were given additional doses of ketamine, as necessary

(without xylazine, 50 mg ketamine/kg of body weight). After inoculation mice were treated with

100 nM of sEntV68 or DMSO in the drinking water for the remainder of the experiment. Mice were euthanized at 3 and 5 days post-inoculation. The tongues were excised for tissue histology and assessment of the fungal burden.

Tongues were fixed in zinc-buffered formalin followed by paraffin embedding. Thin sections of the tongues stained with periodic acid–Schiff (317). Tissue histology was observed using a light microscope. Images were captured at 40X and histology quantified by measuring the percentage of infected epithelium relative to the entire epithelial area (318). The complete epithelial area and infected epithelium were measured from 40X images taken of the entire

55 tissue section of each sample. For each image the total area of epithelium and infected epithelium were measured using Image J (version 1.5). Measurements were totaled and expressed as a percentage of total infected epithelium relative to the entire totaled epithelial area.

For examination of fungal burden, quantitative PCR (qPCR) was utilized by amplifying a

269 basepair fragment of internal transcribed spacer 2 (ITS2) between the 5.8S and 28S ribosomal RNA genes of C. albicans using 5.8S forward primer and 28S-1 reverse primer

(Table 2.1) (319, 320)38,39]. DNA was extracted using the Yeast DNA Extraction Kit (Thermo

Scientific) according to the protocol with the following modifications. A portion of tongue from each mouse was homogenized in 300μl of Y-PER reagent for 8–10 second increments with incubation on ice until samples were completely homogenized followed by incubation at 65°C for 10 minutes. Samples were centrifuged at 13,000 x g for 10 minutes, supernatant discarded,

400ul of Reagent A and 400ul of Reagent B was added, vortexed, and incubated at 90°C for 3 hours. After treatment protein removal agent DNA was precipitated with isopropyl alcohol and washed with 70% ethanol and resuspended in sterile ultra pure water.

The ITS2 fragment was amplified from the DNA extractions and qPCR was performed with a CFX96TM Real-Time System with a C1000 TouchTM thermal cycler (BioRad). A standard curve was generated using C. albicans genomic DNA ranging from 0.5–5000 pg. C. albicans

DNA was quantified using FastStart Universal SYBR Green master mix with ROX (Roche).

Reactions were set-up according to the manufacturer’s recommendations. To screen for contamination and background fluorescence during qPCR amplification, no template controls were used. Non-infected mouse tongues and extraction-negative controls were processed in parallel with tissue samples to test for possible contamination during homogenization and extraction.

56 Proteolytic Cleavage of rEntV136 by GelE

Since EntV136 is larger than expected based on size exclusion centrifugation of the inhibitory activity and GelE and SprE cleave E. faecalis surface and secreted proteins, proteolytic cleavage of rEntV136 by GelE was examined. Proteolytic activity of GelE was examined as previously described by Pinkston et al. (321). rEntV136 (30 μg/ml) was incubated with increasing concentrations of purified GelE (1.5, 4.5, 15, and 30 μg/ml) in PBS at 37°C for one hour. Samples were boiled at 100°C for five minutes in SDS-PAGE sample buffer with

BME to terminate the reaction. To evaluate cleavage of rEntV136, reactions were subjected to

Tricine-SDS-PAGE and stained with SYPRO Ruby (Lonza) protein gel stain and visualized using a 300 nm UV transilluminator (322).

Homozygous Deletion Mutant Library Screens

To identify possible inhibitory targets of the morphogenesis pathways and stress response pathways a C. albicans mutant library of homozygous deletions in non-essential transcription factors was screened for resistance to E. faecalis supernatant. Biofilms were grown in a 96-well microtitered plates in YNBM as described above. Biofilm biomass was quantified using resazurine (Sigma), as a measure of biofilm biomass and was quantified using a fluorescence plate reader. E. faecalis supernatant was harvested as described above and

50μl added to 96 well plates with 1μl of mutant culture and 50μl YNBAS. Biofilms were grown for 24 hrs at 37°C with shaking (200 rpm) (42). The 50μl of E. faecalis supernatant was replaced for 50μl of sterile bacterial medium as non-treatment controls for each mutant strain.

Resistance to supernatant was determined by comparing treated cells to non-treated controls.

Wildtype (SC5314) and the respective parent strains (SN152 and Day286) were tested in the same manner. Statistically significance differences between treated cells and non-treated controls was determined by Student’s t-test. Mutant strains that had no statistically significance

57 differences between treated cells and non-treated controls were test in a secondary screen using the in vitro biofilm protocol described above.

sEntV68-FITC localization on C. albicans

C. albicans SC5314 expressing constitutive mcherry (ADH1p::mCherry) were grown overnight at 30°C in YPD with aeration. Overnight cultures were sub-cultured in YPD for 5 h at

30°C and washed three times with phosphate-buffered saline (PBS). Washed cells where

7 transferred to chambered polystyrene slides (Ibidi) at a final OD600 of 0.2 (~1 x 10 cell/ml) in

YPD with or without 100nM of sEntV68. Cells were grown for 1 hour at 30°C or 37°C with aeration. To observe sEntV68 localization on hyphal cells, cells were grown at 37°C for 1 hour prior to addition of sEntV68. sEntV68 localization was observed by fluorescence microscopy

(Olympus IX81) using slidebook 6 (3i, Intelligent Imaging Innovations).

58 Table 2.1: Strains and Plasmids

Candida albicans Strains

Source or Strain Designation Relevant genotype/characteristic Parent reference SC5314 Wild-type Prototroph - (323)

DHC271 HWP1-GFP ura3/ura3ENO1/eno1::HWP1p-GFP-URA3 SC5314 (324)

HWP1-GFP ADH1- ura3/ura3ENO1/eno1::HWP1p-GFP-URA3 CEGC1 DHC271 This study mCherry RPS10/rps10::ADH1p-mCherry-SAT1 ura3::imm434/ura3::imm434 his1::hisG/his1::hisG Day286 Parent BWP17 (325) arg4::hisG/arg4::hisG pRS-ARG4-URA3 arg4::dpl200/arg4::dpl200 his1::dpl200/his1::dpl200 SN152 Parent RM1000 (326) leu2::dpl200/leu2::dpl200 URA3/ura3 ura3/ura3 his1/his1 JC50 hog1Δ/Δ (327) hog1∆::LoxP/hog1∆LoxP-HIS1-LoxP RPS10/rps10::CIp20 ura3/ura3 his1/his1 JC52 hog1Δ/HOG1 (327) hog1∆::LoxP/hog1∆LoxP-HIS1-LoxP RPS10/rps10::CIp20-HOG1

Other Fungal Strains

Source or Strain Species Relevant genotype/characteristic reference

MYA3404 Candida tropicalis Prototroph (328)

CLIB298 Candida glabrata Prototroph (328)

J941367 Candida parapsilosis Prototroph (328)

Enterococcus faecalis Strains

Source or Strain Designation Relevant genotype/characteristic Parent reference

OG1RF Wild-type Gel+ Spr+ RfR FaR OG1 (329)

fsrB in-frame deletion mutant (bp 79 to 684); TX5266 ΔfsrB OG1RF (201) Gel- Spr- RifR FaR KanR

R CK111 Conjugative donor upp4::P23repA4, Sp OG1Sp (307)

CEGF1 ΔentV Markerless deletion of entV; RfR FaR OG1RF This study

Reconstituted entV with a silent nucleotide CEGF2 ΔentV/ entV CEGF1 This study change, Δef1097 [entV*], RfR FaR

Escherichia coli Strains and Plasmids

59 Source or Strain Relevant genotype/characteristic reference

EC1000 E. coli cloning host for pCJK47-based plasmids; provides RepA in trans (330)

DH5α E. coli cloning host for routine cloning Invitrogen

BL21 E. coli cloning host for routine cloning, suitable for protein expression Invitrogen (DE3)

CEGE1 BL21 (DE3) with pET28a containing entV136 This study pCJK47 Plasmid for markerless exchange, oriTpCF10 and P-pheS*; EmR (307)

Recombinant expression vector, N-terminal His•Tag/thrombin/T7•Tag configuration pET28a Novagen plus an optional C-terminal His•Tag sequence, IPTG or lactose inducible

60

CHAPTER 3:

The Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm

formation, and virulence of Candida albicans

Portions of this chapter are based on my first author publication. I have received permission to reproduce data or text from this article through license number. I have contributed significantly to this publication. The article is listed for reference: Graham, C. E., M. R. Cruz, D. A. Garsin, and M. C. Lorenz. 2017. Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans. Proc. Natl. Acad. Sci.

U.S.A. 114: 4507–4512 (303).

61 Introduction:

The mucosal surfaces, such as the orogastrointestinal tract of mammals, are ideal niches for microorganisms and it is here where the balance between microbe-microbe interaction and the host play an important role (331-333). Most of the microorganisms in the orogastrointestinal tract are benign or beneficial to the host. However, the status of the host is a major determinant of microbial pathogenesis. As discussed previously, microorganisms typically found as part of orogastrointestinal tract microbiome can cause life-threatening infections. In order for this to occur the microorganisms must cross host protective barriers and/or evade the immune response and must be capable of colonizing other host niches throughout the body (9).

Although E. faecalis and C. albicans are part of the orogastrointestinal microbiome, they are among the most important and problematic, opportunistic nosocomial pathogens (186, 235,

334). When the host defenses are compromised and/or the microbiome is altered, both are capable of crossing anatomical barriers and evading the host immune response (335).

Furthermore, the ability to form biofilms is an important virulence trait of both E. faecalis and C. albicans, as both are capable of forming biofilms on biotic or abiotic surfaces that complicate treatment strategies (39, 336). Specifically, C. albicans forms formidable biofilms that can exceed 200µm in depth and is often found in polymicrobial biofillms with bacteria that have enhanced resistance to anti-microbial drugs and immune surveillance (39, 42, 149, 337).

Since C. albicans and E. faecalis occur in overlapping niches as commensals and are commonly co-isolated during infection, it is plausible that diverse interactions exist between these microbes. Using a Caenorhabditis elegans model infections model, we demonstrated antagonistic interactions between C. albicans and E. faecalis (115). Attenuation of virulence is observed in both microorganisms during co-infection of Caenorhabditis elegans. As illustrated in Figure 3.1, these findings demonstrate a commensal-like interaction within this model host; co-colonization with both microbes does not cause disease unlike individual colonization.

62 Further characterization of the activity by which E. faecalis inhibited C. albicans virulence, identified a secreted factor that exerts its protective effect by inhibiting C. albicans hyphal morphogenesis and biofilm formation (115, 338).

Taking into account the interactions observed in the C. elegans model, environments where these commensal-like interactions might occur in a more relevant mammalian host were considered. C. albicans infections of the oral cavity occur in immunocompetent and immunosuppressed individuals (339). Oropharyngeal candidiasis (OPC), a superficial fungal infection of the oral cavity, is one such infection (339-341). OPC most commonly occurs in immunocompromised individuals, such as those that on immunosuppressant drugs, chemotherapy, or suffering from HIV/AIDS (342-344). In immunocompetent individuals, some common risk factors include use of dentures, corticosteroid inhalers, cigarettes and broad- spectrum antibiotics (345-347).

Formation of thick white plaques (biofilms) of C. albicans on the surface of the tongue, buccal mucosa, soft palate and pharynx are the clinical manifestations of OPC (347). While superficially infecting the oral cavity, C. albicans invades the oral epithelial cells by inducing endocytosis of C. albicans by the epithelial cells and active penetration of epithelial cells (348-

351). Invasion of the oral epithelial by C. albicans causes cell damage that elicits production of the proinflammatory response that in turn stimulates production of AMPs and recruitment professional phagocytes (339, 341). Professional phagocytes, neutrophils and macrophages, are crucial elements of the innate immune response to C. albicans during oral infections (352,

353). Depletion of neutrophils and elimination of macrophages in a murine OPC model increased susceptibility to C. albicans infection (352).

63

Figure 3.1: E. faecalis-C. albicans interactions during infection of Caenorhabditis elegans.

During mono-infection (top, left) C. albicans has pathogenic association with the host, primarily through the production of hyphal cells. As seen in the scanning electron (SEM) image (bottom, left) C. albicans forms hyphal cells that are capable of disseminating throughout the nematode.

In the presence of E. faecalis during co-infection (top, right), C. albicans has commensal like association with the nematode. Apparent in the SEM image (bottom, right) C. albicans and E. faecalis are able to colonize the GI tract of the nematode and hyphal morphogenesis is inhibited. Hyphal morphogenesis is inhibited by the presence of E. faecalis via production of a secreted factor(s) (115).

64 Here I identify the E. faecalis secreted inhibitory factor as EntV, a secreted bacteriocin with reported antibacterial activity. When synthetically produced, this peptide inhibited C. albicans hyphal morphogenesis and biofilm formation at sub-nanomolar concentrations. Furthermore,

EntV was active against mature biofilms, reducing the depth and biofilm biomass. EntV significantly protected murine macrophages against C. albicans infection and was protective in a murine model of OPC. As the rise of drug-resistant fungal infections threatens to undermine the current small arsenal of available treatments, I propose that EntV, or similar compounds, may offer a viable therapeutic alternative, either alone or in combination with existing antifungal agents.

Results:

E. faecalis supernatant inhibits C. albicans hyphal morphogenesis and biofilm formation

E. faecalis secretes an inhibitor of C. albicans virulence and biofilm formation (115). To facilitate identification of this inhibitor, I developed a C. albicans in vitro biofilm model in which cells are grown on a polystyrene substrate in an artificial saliva medium (YNBAS, adapted from

(306)). This media supported biofilm growth over 24 hours (Fig. 3.2A and B), while supplementation with supernatant from E. faecalis cultures decreased both biofilm biomass and hyphal morphogenesis as compared to biofilms grown in medium alone (Fig. 3.2). The E. faecalis-mediated inhibition was observed microscopically in biofilms stained with calcofluor white (Fig. 3.2A and B) and quantitated using the redox reactive dye resazurin to measure biomass (311) (Fig. 3.2C). The reduction in hyphal formation was assayed using a reporter strain expressing a transcriptional fusion of the hyphal-specific HWP1 gene to GFP

(HWP1p::GFP) (Fig. 3.2D-F) (324). A direct correlation between decreases in HWP1-induced fluorescence (Fig. 3.2F) and biofilm biomass (Fig. 3.2C) was

65 A. D.

B. E.

C. F.

e e c

c 1.5 1.5

n n

e e

c c

s s

e e r

r 1.0 1.0

o o

u u

l l

F F

e e v

v 0.5 ****$ 0.5

i i t

t ****$

a a

l l

e e R R 0 0 Control Spent Control Spent Media Media

Figure 3.2: E. faecalis supernatant inhibits C. albicans hyphal morphogenesis and biofilm formation.

C. albicans biofilms grown for 24 hours in YNBAS. Representative images of C. albicans (strain

SC5314) biofilms in the (A) absence and (B) presence of E. faecalis supernatant and stained with calcofluor white. (C) Biofilm density was quantified by measuring resazurin fluorescence.

Representative images of C. albicans hyphal reporter strain (HWP1p::GFP) in the (D) absence or (E) presence of E. faecalis supernatant. (F) Hyphal morphogenesis was quantified by measuring GFP fluorescence as a ratio to the OD600. Experiments were performed three times and analyzed using Student’s t-test (****, p<0.0001).

66 observed, which is in agreement with previous studies demonstrating that hyphal deficient mutants have reduced biofilm biomass (354, 355).

EntV inhibition of hyphal morphogenesis and biofilm formation in C. albicans

Our previous studies in the C. elegans infection model suggested that the secreted inhibitor is regulated by the Fsr two-component quorum sensing system mediated by the

Gelatinase Biosynthesis Activating Pheromone (GBAP) (115, 338). The Fsr system regulates a number of virulence-related traits, including secreted proteases GelE and SprE, as well as a bacteriocin identified systematically from the genome of strain V583 as ef1097 (356, 357). ef1097 encodes a 170 amino acid pre-pro-peptide, and cleavage of the secretion leader sequences results in the export of a 136-residue pro-peptide that has bactericidal activity against Gram-positive bacteria (245, 358). Bacteriocins are typically named after the strain in which they are found, and thus the ef1097 gene product was called enterococcin V583 and enterocin O16 in previous publications (245, 358). However, because the gene is present in all

E. faecalis strains sequenced to date (245), we propose to name the gene product EntV, for enterocin originally found in V583.

I hypothesized that EntV is the secreted inhibitor of C. albicans hyphal morphogenesis and biofilm formation and tested this genetically. I constructed a deletion mutant of entV and tested the supernatant for activity against C. albicans biofilms using the hyphal specific reporter strain (HWP1p::GFP). Indeed, the biofilm inhibitory activity was lost in supernatants derived from the entV mutant strain relative to those from the WT E. faecalis strain (Fig 3.3A), confirming that EntV is necessary for the inhibition of biofilm formation. The mutant strain was complemented by introducing the gene back into the endogenous locus with a silent mutation to distinguish it from the original wild type strain (308). Supernatant from the complemented strain inhibited biofilm formation as well as that of wild type E. faecalis.

67

A.

1.5

e

c n

e ***$ c

s ***$ ***$

e 1.0

r

o

u

l

F

e 0.5

v

i

t

a

l e

R 0 Control WT ΔfsrB ΔentV ΔentV/entV E. faecalis Supernatant

B.

IC50= 0.3 nM IC50= 1000 nM

e 1.5

c

n

e

c s

e 1.0 r 136136N

o rEnrEF1tV097

u 6868

l sEnsEF1tV097 F

0.5

e

v

i

t

a l

e 0 R

Figure 3.3: EntV inhibition of hyphal morphogenesis and biofilm formation in C. albicans.

(A) C. albicans SC5314 biofilms grown for 24 hours in YNBAS with sterile media (M9HY) or spent supernatant from wild-type and mutant E. faecalis strains. Hyphal morphogenesis was quantified by measuring GFP fluorescence as a ratio to the OD600. The experiment was performed three times and analyzed using one-way ANOVA (***, p<0.0008). (B) Biofilms were

136 68 grown with increasing concentrations of rEntV or sEntV and the IC50 calculated at 1000 nM and 0.3 nM, respectively.

68 To test if EntV136 was sufficient for the inhibitory activity, rEntV136 was expressed and purified from E. coli with a hexa-histidine tag (Table 2.1). I detected rEntV136 biofilm inhibitory activity with an IC50 of ~1000nM (Fig. 3.3B). Thus, the genetic and biochemical evidence suggests that EntV is both necessary and sufficient for inhibition of C. albicans biofilm formation. The 136-amino acid EntV is larger than the 3-10 kDa indicated by our preliminary characterization. However, recent work from Dundar et al. suggests that EntV136 is further processed in a GelE-dependent manner to a 7.2 kDa peptide encompassing the 68 carboxy- terminal amino acids (EntV68) (245). This mature form has a predicted disulfide bond that encompasses nearly the entire length of the active protein (from amino acid 4 to 65 of the 68 amino acid peptide) that is necessary for its anti-bacterial activity (245, 358). Attempts to express and purify EntV68 were unsuccessful due to apparent toxicity in E. coli. To surmount this obstacle, the peptide was synthetically produced (sEntV68) in a form that included a disulfide between the cysteine residues. As seen in Fig. 3.3B, sEntV68 inhibited biofilms with an

IC50 of 0.3nM, and therefore was ~3,000-fold more active than the unprocessed form of the peptide.

Characterization of sEntV68 inhibitory activity

To investigate the dynamics of C. albicans biofilms in the presence of sEntV68 I turned to confocal microscopy, with which I could generate a 3-D projection allowing me to measure the overall biofilm depth. When formed in the nutrient-poor artificial saliva medium, control biofilms were 25-30 µm thick after 24 hours, but only 5-10 µm thick in the presence of sEntV68, roughly the length of a C. albicans yeast cell (Fig.3.4A and B). I next examined the effectiveness of sEntV68 against mature biofilms grown for 24 hours. As viewed by confocal microscopy, when biofilms that had developed for 24 hours were treated with sEntV68, a reduction in biofilm depth from ~30μm to ~15μm (Fig. 3.4A) was observed, indicating

69 A.

55 µm 50 µm 45 µm 40 µm

35 µm 30 µm

25 µm 20 µm 15 µm 10 µm

5 µm 0 µm DMSO sEntV DMSO sEntV added at 24hrs sEntV 24 hours 48 hours

B. ****$

1.5 **$ e

c ****$ n

e ****$

c s

e 1.0

r ***$

o

u

l

F

e

v 0.5

i

t

a

l

e R 0 DMSO sEntV DMSO sEntV added sEntV at 24hrs 24 hours 48 hours

Figure 3.4: Characterization of sEntV68 inhibitory activity.

C. albicans SC5314 biofilms were grown with 0.01% DMSO or 100 nM sEntV68 added at different time points during biofilm formation (‘-‘, not added; ‘0hr’, added at the beginning of the experiment. (A) Representative image of 24 hour and 48 hour biofilms observed by confocal microscopy. (B) Hyphal morphogenesis was quantified by measuring GFP fluorescence as a ratio to the OD600 for three separate experiments (**, p=0.0058; ***, p=0.0005; ****, p<0.0001).

70 that this peptide could dismantle mature biofilms. Correspondingly, when the HWP1p::GFP reporter was used to measure hyphal morphogenesis, a decrease in signal was observed at 48 hours following treatment with sEntV68 at 24 hours compared to biofilms grown with the vehicle control after 24 and 48 hours of growth (Fig. 3.4B).

Protection of Murine Macrophages by sEntV68 during C. albicans infection

C. albicans is readily phagocytosed by murine macrophages, whereupon it activates a complex transcriptional and morphogenetic program that promotes fungal survival and results in hyphal-dependent lysis of the macrophage (316, 359, 360). I asked whether sEntV68 could protect phagocytes from fungal-dependent killing. In these experiments, C. albicans was incubated with sEntV68 for 1.5 hours before being added to the macrophages. sEntV68 was also added to the macrophage cell culture medium immediately prior to infecting with C. albicans. A decrease in C. albicans survival was observed when the macrophage infection was carried out with sEntV68 as compared to the control infections (Fig. 3.5A), suggesting that sEntV68 works in synergy with murine macrophages to make C. albicans more sensitive to macrophage attack.

Additionally, I observed a significant decrease in macrophage cytotoxicity in the presence of sEntV68 compared to the controls (Fig. 3.5B), suggesting that sEntV68 protects murine macrophages during C. albicans infection. To assess whether sEntV68 inhibits hyphal morphogenesis within macrophages I used a hyphal specific reporter strain (HWP1p::GFP) constitutively expressing mCherry (ADH1p::mCherry). Cells treated with sEntV68 had a decrease in hyphal growth and GFP fluorescence as compared to control treated cells (Fig.

3.5C and D). No toxicity of the peptide towards macrophages or HeLa cells was observed (Fig.

3.6A and B). Taken together these results suggest that sEntV68 protects murine macrophages by inhibition of C. albicans hyphal morphogenesis.

71 A. HWP1p- ADH1p- Merge B.

GFP mCherry Hyphae Yeast

w/DIC l

o

r

t

O n

o 73% 27%

S

C

M

D O

S 77% 26%

M

D

8

6

8

6

V

t

V n

t 27% 73%

n

E

s E s 0 50 100 C. albicans Inside Macrophages (%) C. D. 100

100

)

x

) a

80 %

m

(

80

l

a

%

(

v

i e

60 v r

s 60 u

a ***$ **$

S

e

l

s

e 40

n R

40

a

c

H

i

b

D

l L

20 a

20

. C 0 0 Control DMSO sEntV68 68 Control DMSO sEntV

Figure 3.5: Protection of Murine Macrophages by sEntV68 during C. albicans infection.

(A) Murine derived macrophages (RAW264.7) were infected with C. albicans (HWP1p::GFP,

68 ADH1p::mCherry) at an MOI of one for one hour with 0.01% DMSO or 100nM sEntV followed by fixation with paraformaldehyde and visualized by fluorescence microscopy. (B)

Filamentation of phagocytosed cells was scored after 2 hours of co-culture. At least 200 cells in at least 10 different fields of view were counted per replicate and the experiment was repeated three times. (C) Macrophage killing was evaluated using the LDH cell toxicity assay and the percentage of killed macrophages calculated. (D) C. albicans survival in murine macrophages was assessed using the XTT cell viability assay and the percent survival was calculated.

Statically significant differences were calculated using one-way ANOVA (* p<0.02).

72 A.

60

) Spontaneous Lysis

DMSO (1%) %

( 68 sEntV (10µM)

H 40

D

L

m

u 20

m

i

x

a M 0 24 hours 48 hours 72 hours

B. 60

Spontaneous Lysis ) DMSO (1%)

% 68

( sEntV (10µM)

H 40

D

L

m

u 20

m

i

x

a M 0 24 hours 48 hours 72 hours

Figure 3.6: Activity of sEntV68 in mammalian cells.

Lactate dehydrogenase (LDH) was measured from (A) Murine macrophages (RAW 264.7) and

(B) Human cervical epithelial cells (HeLa) exposed to 10 μM sEntV68 or 1% DMSO for up to 72 hours. Cell toxicity was evaluated using the LDH release assay and the percentage LDH calculated for replicates of three separate experiments.

73 Efficacy of sEntV68 in the Murine Oropharyngeal Candidiasis Model

Recall, that C. albicans grows on the mucosal surfaces of the oral cavity as part of the normal microflora. However, in patients with defective immune responses it can cause a superficial infection and invade the epithelial layer causing oropharyngeal candidiasis (OPC) that leads to increased morbidity (348-351). To test the efficacy of sEntV68 in host relevant infection model, I used a murine OPC model (317). In this model, mice were given a sublingual inoculation of C. albicans and then given sEntV68 or DMSO in sterile drinking water for up to 5 days. The mice were euthanized and tongues excised at day 3 and day 5 to examine the histology and fungal burden. Control mice showed classical signs of OPC, including extensive invasion of the epithelium by fungal hyphae, disruption of the outer layers of epithelial cells, and infiltration of neutrophils (Fig. 3.7A). In contrast, mice that were treated with 100 nM sEntV68 has significantly reduced invasion; most fungal cells were in the yeast or pseudohyphal form, indicating that the peptide can inhibit morphological differentiation in vivo as well (Fig 3.7A). To provide a quantitative measure of the epithelial invasion, I scored the proportion of the epithelium infected in multiple tongues from control or sEntV68-treated mice, as recently described (318). This data demonstrates a clear difference in the extent of epithelial damage between treated and control animals (Fig. 3.7B). Finally, I assessed fungal burden on the tongues. Because of the significant morphological differences in the two conditions, I considered that colony forming unit plating would not accurately reflect differences in burden between the primarily yeast morphology in the sEntV68-treated animals and the multinucleate and highly adherent hyphae in the controls. Instead, I quantitated fungal DNA using qPCR as described (319, 320), which showed a significant decrease in fungal burden in peptide-treated mice relative to control mice (Fig. 3.7C). These results indicate that sEntV68 is effective in a complex mammalian infection model that simulates clinically relevant infection conditions.

74 68 A. B. Fig. 6. sEntV68Fig.isp6.rotsEntVective inisapmroutreincteivOePinCmaomduerli.ne OPCmodel. ImmunosuppreImssemdumnoicseuwpperreessineodcmulaicteewd esurebliinnogcuualallyted sublingually with C. albicanwisSthC5C31. 4alwbicitahn0s.S0C15%31D4MwSOitho0r.0110%0 nDMMSO or 100 nM 68 68 sEntV68. WatersEcontnVtain.iWngatesErntVco68nta(1in00inngMsE)notVr veh(1ic00le nM) or vehicle (DMSO) alonew(DaMspSOro)viadloendeawdalisbpitroumvi.dAedfteard3liobrit5umd,. After 3 or 5 d, mice were eutmhainceizewderaendeuththeantoizengduesandwethreeextocingseudes were excised for hist ologicafol erxahmistinoalotgioican loef xDaMmSinOatcioontroofl D(AM)oSOr control (A)or sEntV68-treatedsEnt(BV) 68m-ticre.ateRedd(Ba)rromwicheea. Rdsedinadricarotewheads indicate hyphal cells, anhdybplhaaclkcaerlrlso,wanhdeabdlascinkdaircraotweyheeaasdtscienlldsi.cateyeast cells. (C) The percen(tCa)gTehoef ptherecenptitahgeeliaolfstuhrefaecepitsheolwiailnsgurface showing evidence of fungevidaleninvacesiofonfuwangsalcainlcuvalasiteondwaforscocanlcutrolal ted for control and treated tonangduetrs.e(aDte) dDNAtonwguaesse.x(Dtr)aDcteNAd frwoamsethxtreacted from the tongues and thtoenfugunegsaal nbdurtdheenfuwnagsaelsbtiumradteendwfraosmestimated from qPCRamplificaqtiPoCnRoafmthpeli5fi.ca8StiIoTnS2orfethgioen5..8SStaItTiSst2icarellgyion. Statistically significant diffseirgenniceficasnwtedreiffcearlecunlacetesdwebryeucsianlcgulaotened- by usi ng one- way ANOVA (*wa*Py

25 20

e

)

u

s

%

(

s

i

20 t

n 15 wasexpressedwfarosmexppEreTs2s8eadinfroEscherichiam pET28a incoliEscherichiaBL21 and purifcoli BL21ied onandTALONpurified on TALON g

o nearly the enearlyntire ptheroteeinnt.irBeeptwroeteenint.hBeectywseteinneths,ethcyestsetrinuectsu,rtehe structure

i

/

) s

g resin, as describedresin, (48).as describedAttempts(48).to purifyAttemptthes fullyto purifyactivethe68-aafullypeptideactive 68-aa peptide

a o15f the matuorfethfoermaotuf rEenftoVrmisopfreEdnictVtedisinprseildicicotetdo icnonsisliisctootfo consist of

µ

v

( n from E.coli were unsuccessful; thepeptide wassynthesized with thedisulfide

I 10 from E.coli were unsuccessful; the peptide wassynthesized with the disulfide

two helicaltweloemheelnictsa,l seelpeamraetnetds,bsyepaaA rflaetxeidblbeyloaoflperxeibgiloenlo(o2p5,region (25,

l

a N

i bond (Lifeteinbond). The(Lifeteinhyphal-spec). Theifichyphal-specHWP1p-GFPificrepoHWP1prter strain-GFPrepoDHC271rter strain(21) DHC271 (21)

l 10

27). The b27act)e.rTichideabl aacctteivrictyidoafl EacnttivVD ityisonfoEt ndtuVe itsonloystisduanedto lysis and

e

l

h expressing a constitutiveexpressing amCherconstitutivery was generatedmCherry wasviageneratedtransformatviaiontransformatwith ion with

a t i thereisno evidenceof an immunityprot5e in, so themechanism

thereisno evidenceof an ig mmunityprotein, so themechanism p 5

n plasmid pADH1-mChplasmiderrypADH1-mCh(49). erry (49). E remains enigmaticremains (25,enigmatic27). L(25,ikewise,27).u theLikewise,mechanismthe mofechanismthe of the a0n tihyphal aancttiihvyitpyhiasluanctcilveiatry, iasltuhnoculgeF har0i,t adltoheosungohtiltydseoefusnngoatl lyse fungal cellseithDeary. 3I n fact, growDathy 5a nd chemical sensitDivayity 3 of C. al bi -Day In5 Vitro BiofilmInAssays.Vitro BiofilmBiofilmAssays.assaysBiofilin conditionsm assaysmimickiin conditionsng themimickioral cavityng the oral cavity cellseither. In fact, growth and chemical sensitivity ofwCe.realmbiod-ified from published reports(15, 50, 51) by using an artificial saliva cans cells is unaffected, even when exposed to concentrations DMSO (0.0w1%e)re modified from published reports(15, 50, 51) by using an artificial saliva cans cells is unaffected, even when exposed to concenmediatratiosE[0.17%nnstV (100nMYeast) Nitrogen Base (vol/vol), 0.5% casamino acids (vol/vol), four logs higher than the IC .Thus,EntV exerts a true anti- media [0.17% Yeast Nitrogen Base (vol/vol), 0.5% casamino acids (vol/vol), four logs higher th50an the IC50.Thus,EntV exerts a tr0u.e25a%ntmi-ucin 0(v.2o5l/%volm), u1c4inm(Mvolp/vootla),ss1iu4mmchMloproidtaes,s8iumMchsolordiiduem, 8chmloMridsoe,dium chloride, virulence effect68 on thisimportant fungal pathogen. It has been Figure 3.7: sEntVvirulence is protectiveeffect in aon murinethisimportant OPC model.f ungal pathogen. It h100as bμeenM cholin100e chμlMoridche,ol5in0eμMchlosodiuride,m50citrate,μM sodiu5 μMm ascorbate]citrate, 5 μadaptedM ascorbate] adapted speculated that therapeuticsthat target virulencemight beless speculated that therapeuticsthat target virulencemightfrbomelreesf.s19. Tfrhoismmreedf.ia19w.aTshsisupmpeledmiaewntaesdsuwpitphlecmonendtiteiodnwedithbaccotnedriaitilosnue-d bacterial su- Immunosuppressed mice were inoculated sublingually with C. albicans SC5314 with 0.01% likely to selliectkelyfortoresiselectstance.for resiIn sustance.pport Inof suthipps ortidea,ofathismals idlea, pernatanta small or recombinanpernatant ort orrecombinansynthetic peptt or synthide, aseticindicated.peptide,Biofilas indicated.m devel- Biofilm devel- molecule moal solecuexhleibialtinsog exhC. ibialtinbi cgansC.bialofibilcmansanbid ofihyplmhalandophypmenhtalwas allowed to proceed for 24–48 h and then assessed in three DMSO or 100 nM of sEntV68. Water containing sEntV68 (100 nM) or vehicle (DMSO) alone was opment was allowed to proceed for 24–48 h and then assessed in three morphogenmesoirsp-ihnohgibeintiensgis-pinrohpibeirttiinegs dpirdopneorttieinsdduicdenroestisintadnuccee reways:sistanbyceestimatinways:g bybioestimatinmassusingg btheiomassredouxsingdye resazurin,the redox dasyedescribedresazurin,(20),asdescribed (20), providedafte r adre p libitum.eaatefted Afterrexrpe po threeesuarte d or(4 e0 fivex)p.o days,sure ( mice40) . were euthanized and the tonguesby m wereeasu ringbfylumoreeasscuernicneg ffrloumorethsceenHWP1pce from-GFPthehyphal-sHWP1ppecific-GFP hyphal-sreporterpecific reporter In the OPCInmodel,the OPCEntVmodel,reduced,EntVbutreduced,did notbuteliminate,did not eliminate,strain (21), or sthtrraoinug(2h1c),onofrothcarlomugichrocsocnofpoy.cal microscopy. excisedfungal for histologicalcolonization.fungal examinationcolonization.In contrast, of (A)In fungalDMSOcontrast, controlinvasionfungal or (B)of invasion thesEntVepithe-68-treatedof the mice.epithe- Red lium and inflammation,lium and inflammation,which are responsiblewhich are responsiblefor the pathologyfor the pathologyNematode InfectionNematodeModInfectionel. C. elegansModel.glp-4C. (elegansbn2);sek-1glp-4(km4(bn2)n);esek-1mato(dkm4es )nematodes arrows indicate hyphal cells and black arrows indicate yeast cells. (C) The percentage of the of OPC, wereof OPC,almostwereentirelyalmostabsent.entirelyThus,absent.in theThus,presencein theofpresencewere profopagawteedrebpyruospiangastteadndbayrdusteincghnstiaqnudeasrodnteE.chcoliniqustraines onOP50E. colionstrain OP50 on EntV, C. albicansrevertstoabenign commensal interaction with nematode gronwetmhamtoeddeiugmro(NwtGhMm) eadgiaurm(5(N2).GTMh)ealigqaurid(5in2)f.eTchtieonliqaussidayinwfeacstion assay was epithelial surface EntV, showingC. evidencealbicans ofreverts fungal invasiontoabenign was calculatedcommensal for controlinteraction and treatedwith thehost. Wethehavehost.speculatedWe havethatspeculatedthesetwothatspeciesthesetwomightspeciesfind itmightperfindformited asdpesrcfroirbmeded(1a7s,d2e8s);crbirbiedfly(,1y7o, u28n)g; bardiuelftlsy,wyeorueningcaudbualtesdwfeorre4inhcubated for 4 h on solid medium with the wild-type C. albicans SC5314 strain, then the tongues.advantageous (D) DNAadvantageous wasto extractedsuppress fromtovirulence suppressthe tonguesattributesvirulence and the fungalinattributesfavor burdenof incolo-wasfavor estimatedof colo- from on solid medium with the wild-type C. albicans SC5314 strain, then the nization (10); indeed, C. albicanspromotes E. faecaliscoloniza- wormswere cowlolercmtesdw, weraeshcoeldle,catnedd,trwaanshfeerdr,eadntod tsrixa-nwsfeellrrpeldattoesscioxn-wtaeinllipnlgatescontaining nization (10); indeed, C. albicanspromotesE. faecaliscoloniza-20% Brain Heart Infusion (BHI) and 80% M9W, after which viability was qPCRtion amplificationinthegastrointestinaltion of theinthe 5.8Sgastrointestinal ITS2tract region.(41, Statistically42).tractThis(41, significantproposal42). This differencesiscounterproposal wereis calculatedcounter 20% Brain Heart Infusion (BHI) and 80% M9W, after which viability was to studies that suggest these species are commonly isolated to- assayed daily.assayedFilamentatdaily.ionFilamentatof C. albicansion witof C.hinalbithecanswormswithinwasthescoredwormsviawasscored via using one-way ANOVAto studies (**, p<0.01)that suggest these species are commonly isolatedmicroscopito- c analysis. gether in manygetherclinicalin manysamplesclinical(6)samplesbut this,(6)too,butmightthis,representtoo, might represent microscopic analysis. a coordination a coordinationof behaviorofthroughbehaviorsensingthroughhostsensingweaknesseshost weaknessesto to 68 switch cocommensal to copathogen. In support of this notion, Murine MacrophageMurineInfectionMacrophage. To assessInfectionwhether. To assessEntVwhetherprotectsEntVphag68ocytesprotects phagocytes 68 switch cocommensal to copathogen. In support of thisfrnotion,om fungal-induced damage, we used a coculture assay in which C. albicans 68 75 from fungal-induced damage, we used a coculture assay in which C. albicans EntV doesEntVnot affectdoeshyphalnot affectgrowthhyphalstimulatedgrowthbystimulatedmammalianby mammaliancells were incubated with RAW264.7 murine macrophage-like cells as de- serum, the strongest inducing factor (17) and an obvious host cells were incubated with RAW264.7 murine macrophage-like cells as de-

serum, the strongest inducing factor (17) and an obviousscribehostd (32). Fungal viability was assessed by using a modified end-point di- Y Y

scribed (32). Fungal viability was assessed by using a modified end-poG int di-

signal during disseminated infection. A precedent for this ap- G

signal during disseminated infection. A precedent for lthisutionap-assay in which respiratory activity ismeasured using a tetrazolium dye, O L

lution assay in which respiratory activity ismeasured using a tetrazolium dye, O L parent contradiction is seen in the interaction between C. albi- O XTT (53, 54). Lactate dehydrogenase wasassayed by using the Cytotox96 kit I

parent contradiction is seen in the interaction between C. albi- O I

XTT (53, 54). Lactate dehydrogenase wasassayed by using the CytotoB x96 kit B

cansand P. aeruginosa, speciesthat areantagonistic in vitro (13, (Promega). C. albicansmorphology wasassayed microscopically. O O

cansand P. aeruginosa, speciesthat areantagonistic in vitro (13, (Promega). C. albicansmorphology wasassayed microscopically. R

R C

43), but associated with significantly worse clinical outcomes I C

43), but associated with significantly worse clinical outcomes I M

when found together in several clinical settings (44, 45). It is Murine Oropharyngeal Candidiasis Model. Adult BALB/c mice were immuno- M when found together in several clinical settings (44, 45). It is Murine Oropharyngeal Candidiasis Model. Adult BALB/c mice were immuno- possible that some of the protective effectsof EntV in the OPC suppressed with subcutaneous injections of cortisone acetate before su b- possible that some of the protective effectsof EntV in the OPC suppressed with subcutaneous injections of cortisone acetate before su b- model aredue to itsactivityagainst other bacterial speciesinthe lingual inoculation with C. albicans according to So lis and Filler (36). Mice model aredue to itsactivityagainst other bacterial speciesinthe lingual inoculation with C. albicans according to So lis and Filler (36). Mice mouth, although we would expect this to enhance fungal colo- were given EntV or vehicle in drinking water ad libitum. Three and five days mouth, although we would expect this to enhance fungal colo- were given EntV or vehicle in drinking water ad libitum. Three and five days nization rather than suppress it. We are thus only beginning to after inoculation, mice wereeuthanized and thetonguesexcised and halved nization rather than suppress it. We are thus only beginning to after inoculation, mice were euthanized and the tonguesexcised and halved longitudinally. Part of the tongue was processed for histopathological understand the complex interactions among the microbiota and longitudinally. Part of the tongue was processed for histopathological understand the complex interactions among the microbiotaanalyandsis, whereas the rest was homogenized for assessment of fungal bur- their varied effects on the human host, but the knowledge is analysis, whereas the rest was homogenized for assessment of fungal bur- their varied effects on the human host, but the knowledgeden viaisqPCR. DNA was extracted by using the Yeast DNA Extraction Kit likely to have important impacts on the development of den via qPCR. DNA was extracted by using the Yeast DNA Extraction Kit likely to have important impacts on the development(ThermoofScientific), and the Internal Transcribed Spacer (ITS1) of the rDNA novel antimicrobials. (Thermo Scientific), and the Internal Transcribed Spacer (ITS1) of the rDNA novel antimicrobials. genes was amplified as described (38, 39). Experiments were performed in genes was amplified as described (38, 39). Experiments were performed in accordance with protocols approved by the Animal Welfare Committee of Materials and Methods accordance with protocols approved by the Animal Welfare Committee of Materials and Methods the University of TexasHealth Science Center at Houston. Microbiological and Molecular Biological Methods. Candida and Enterococcus the University of Texas Health Science Center at Houston. Microbiological and Molecular Biological Methods. Candida and Enterococcus strainsused in thisstudy are listed in Table S1 . Standard culture media were strainsused in thisstudy are listed in Table S1 . Standard culture meACKNOWdia wereLEDGMENTS. Wethank R. Wheeler, D. Hogan, and P. Sundstromfor used for routine propagation, asdescribed in SI Materialsand Methods.The ACKNOWLEDGMENTS. Wethank R. Wheeler, D. Hogan, and P. Sundstromfor used for routine propagation, asdescribed in SI Materialsand Methodsreage.Tnthse; other members of the M.C.L. and D.A.G. laboratories for helpful entV mutant strain was constructed as described to create an in-frame reagents; other members of the M.C.L. and D.A.G. laboratories for helpful entV mutant strain was constructed as described to create ancoinn-vfrearsmaetions and advice; and Drs. Barrett Harvey, Ambro van Hoof, and markerless deletion by using a P- pheS* counterselection system (46) and conversations and advice; and Drs. Barrett Harvey, Ambro van Hoof, and markerless deletion by using a P- pheS* counterselection systemM(4ic6h)aaenl dGustin for their input. Thiswork wassupported in part by National complemented by using a reported strategy to regenerate the wild-type Institutes of MHeicahlthaelAwaGustrdinsfoRr01thAeI0ir7in5091put.(tTohisMw.Co.Lrk.),waRs01AsupI0p7o6r406ted inanpdart by National complemented by using a reported strategy to regenerate the wild-type locus (47). The recombinant hexahistidine-tagged 136-aa EntV propeptide R01AI110432 (IntostiDtu.Ate.Gs.),oafndHeFa31lthAI1Awa2272rd5s(toR0C1.EA.GI0.7).5091 (to M.C.L.), R01AI076406 and locus (47). The recombinant hexahistidine-tagged 136-aa EntV propeptide R01AI110432 (to D.A.G.), and F31AI122725 (to C.E.G.).

Graham et al. PNAS | April 25, 2017 | vol. 114 | no. 17 | 4511 Graham et al. PNAS | April 25, 2017 | vol. 114 | no. 17 | 4511

Discussion:

I observed a distinct antagonistic interaction between E. faecalis and C. albicans, in which hyphal morphogenesis, an important virulence factor, is inhibited by a secreted bacteriocin, EntV. Hyphal morphogenesis is important for many aspects of C. albicans pathogenicity, including biofilm formation, resistance to phagocytes, and dissemination through tissues (102, 361, 362). The implications of this inhibitory activity are clearly seen in the decreased virulence of C. albicans cells exposed to this peptide in murine macrophages, and in the mouse OPC model.

Biofilm-related infections pose numerous clinical challenges. Biofilms provide a measure of protection to the microbes encased within them from phagocytic attack and antibiotics (20). Protection of persister cells within the biofilm allows C. albicans to disseminate to distant sites, seeding relapses and systemic infections (88). Biofilms of C. albicans are robust and form both on implanted medical devices (catheters, heart valves, dentures) and on epithelial surfaces (OPC) (39). Our in vitro experiments were designed to model the oral cavity, and I observed biofilms exceeding 50 µm in depth; in other media conditions, they can be greater than 200 µm thick. Often, device removal is the only effective treatment option, though this may not be feasible or desirable given a patient’s underlying conditions. Agents that directly disrupt biofilms would be very useful chemotherapeutics in conjunction with existing drugs. Our work suggests that EntV is a candidate anti-biofilm agent.

Currently, nearly all antimicrobials in clinical use work by killing or preventing the growth of the infectious agent. Certainly, this is true for C. albicans. There are presently only three families of commonly used antifungal drugs, polyenes, azoles and echinocandins, all of which target the cell membrane or the cell wall (80, 363). Resistance to this limited arsenal of antifungals is a growing problem (364). It has long been speculated that therapeutics that target virulence might effectively treat infection and not select for resistance as strongly as traditional

76 antimicrobials. In support of this idea, a small molecule also exhibiting C. albicans biofilm and hyphal morphogenesis-inhibiting properties did not induce resistance after repeated exposure

(365).

Many clinical Candida infections are polymicrobial. In one survey of these polymicrobial infections, the most common bacterial co-habitant was E. faecalis (109). In contrast, our work suggests that these two microbes have evolved a relationship in which one inhibits a key virulence trait of the other. A precedent for this apparent contradiction is seen in the interaction between C. albicans and P. aeruginosa, species that are antagonistic in vitro (126, 128), but are associated with significantly worse outcomes when found together in patients, including the lungs of cystic fibrosis patients, during ventilator-associated pneumonia, and in burn trauma

(123, 124).

I previously speculated that the presence of both C. albicans and E. faecalis might promote a commensal interaction with the host, in which virulence attributes are suppressed to facilitate colonization (Fig.1) (338). C. albicans ability to colonize the GI tract in a nonpathogenic state and switch to a pathogenic state upon disruption of the host immune response or microflora has been well documented (4, 8, 11). However, C. albicans is able to shape the bacterial microflora during antibiotic recovery. In a murine colonization model C. albicans reduced spp. and promoted E. faecalis colonization after antibiotic treatment (366, 367). These data thus suggest a model in which C. albicans and E. faecalis promote each other’s commensalism during nonpathogenic colonization of the orogastrointestinal tract, but when the status of the host immune response is disrupted, invasive infection might be stimulated.

We are only beginning to understand the complex interactions amongst the microbiota and their varied effects on the human host, but the knowledge is likely to have important impacts on the development of novel antimicrobials. In conclusion, I report the identification of an anti-fungal, virulence-targeting compound, EntV68, which does not slow the growth of C. albicans, even at high concentrations, but can elicit protection in the sub-nanomolar range by 77 inhibiting hyphal morphogenesis. This study provides impetus for further discovery of novel antimicrobial therapeutics with virulence-targeting activity.

78

CHAPTER 4:

Post-Translational Processing of EntV influences the inhibition of hyphal

morphogenesis and biofilm formation of Candida albicans

Portions of this chapter are based on my first author publication. I have received permission to reproduce data or text from this article through license number. I have contributed significantly to this publication. The article is listed for reference: Graham, C. E., M. R. Cruz, D. A. Garsin, and M. C. Lorenz. 2017. Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans. Proc. Natl. Acad. Sci.

U.S.A. 114: 4507–4512 (303).

79 Introduction:

Bacteria use quorum sensing to communicate via accumulation and detection of extracellular signaling molecules that enable them to respond to these environmental cues based on cell density (368). Gram-positive bacteria QS signaling molecules are peptides that are post-translationally processed from propeptides. These peptides bind the extracellular receptors on specific transmembrane proteins that trigger specific signal transduction pathways

(369).

The Accessory gene regulator (Agr) system in Staphylococcus Spp. is one of the most- studied cyclic peptide-mediated QS systems found in Gram-positive bacteria (Novick &

Geisinger, 2008). The autoinducing peptide (AIP) is produced from the propeptide AgrD that is post-translationally processed and secreted by the transmembrane peptidase AgrB. Upon accumulation of AIP, the canonical two-component system consisting of the histidine kinase

(AgrC) and response regulator (Agr A) transduces the signal that results in phosphorylation of

AgrA (370-374). Phosphorylation activates AgrA, which positively regulated gene expression.

This type of QS system is widespread among Gram-positive bacteria, cognate gene cluster containing these four components have been identified in Enterococcus, , Clostridium,

Lactobacillus, and Bacillus spp., suggesting the retention of this type of QS system is important for the survival of many Gram-positive bacteria (371, 372, 375, 376).

A homolog of the Agr system, the Fsr system in E. faecalis regulates expression of pathogenicity-associated genes and is important for virulence in different infection models (197,

201, 202, 377, 378). The fsr gene cluster contains four genes that encode a histidine kinase

(FsrA), response regulator (FsrC), transmembrane peptidase (FsrB), and the cyclic signal peptide GBAP (FsrD) (376). Phosphorylated FsrA directly regulates the fsr genes, gelE, sprE, and entV and indirectly regulates genes involved in autolysis, adhesion, and biofilm formation

(201, 356, 376, 379). Using microarray transcriptional profiling, Bourgogne et. al. (356) found that fsrB influences expression of numerous genes throughout the growth phases of E.

80 faecalis. In addition to the Fsr regulon, gelE, sprE, and entV all contain consensus sequences necessary for FsrA binding and expression is dependent on cell density where an increase in gene expression is seen in late log phase (356).

Here I demonstrate the post-translational modifications of EntV necessary for inhibition of C. albicans hyphal morphogenesis and biofilm formation. The mature inhibitory activity was

3-10 kDa and a synthetic peptide containing the C-terminal 68 amino acids was functional, indicating that the EntV protein is post-translationally processed via proteolytic cleavage. I show here that recombinant EntV is cleaved in vitro by GelE. Furthermore, EntV is predicted to have a disulfide bond and reduction and alklylation of the supernatant abolished inhibitory activity. Finally, characterization of EntV structure and activity provides useful insight for the future design of novel antifungal and antibiofilm therapeutics based on EntV inhibitory activity against C. albicans.

Results:

Characterization of E. faecalis supernatant inhibitory activity

To characterize the activity of the inhibitor, I used the C. albicans in vitro biofilm model I developed using polystyrene as the growth substrate grown in YNBAS (306). Biofilms grown for

24 hours in the presence of supernatant from E. faecalis had decreased biomass and hyphal growth as compared to biofilms grown in medium alone (Fig 4.1.). This inhibitory activity was retained in the E. faecalis supernatant fraction between 3 to 10 kDa that had been boiled for 20 minutes at 100°C (Fig. 4.1), consistent with what was seen in the C. elegans infection model

(115). In addition, the inhibitory activity was abolished with proteinase K treatment, suggesting a small (3 to 10kDa), heat-stable protein is responsible for the inhibitory activity observed in both the in vitro biofilm model and the in vivo C. elegans infection model.

81

1.5

e

c

n

e

c s

e 1.0

r

o

u

l

F

e v i 0.5 t *$

a **$

l **$

e R 0 Control WT >10kDa <10kDa >3kDa <3kDa ΔfsrB WT ΔentV WT Supernatant Fractions Proteinase K E. faecalis Supernatant

Figure 4.1:. Characterization of inhibitory activity from E. faecalis Supernatants.

C. albicans (HWP1p::GFP) biofilms grown at 37°C for 24 hours in YNBAS. Hyphal morphogenesis was quantified by measuring GFP fluorescence as a ratio to the OD600.

Inhibitory activity from E. faecalis supernatant was retained between 3-10 kDa after size exclusion centrifugation. A reduction of inhibitory activity was observed in supernatants treated with proteinase K and from deletion mutants of fsrB or entV. Experiments were repeated at least three times and statistically significant differences relative to treatment with the WT supernatant were calculated using a one-way ANOVA, and are indicated by asterisks (**, p<0.006; *, p<0.01).

82 Post-translational processing of EntV

As discussed previously (Chapter 1), the inhibitory activity was lost with disruption of the

Fsr system and deletion of entV (Fig. 3.3A). Furthermore, deletion of gelE or sprE resulted in decreased inhibitory activity and a double deletion of gelE and sprE further decreased inhibitory activity, indicating that GelE and SprE are necessary for maximum inhibitory activity. According to the entV gene sequence, EntV is slightly larger than the inhibitory factor characterization based on size exclusion centrifugation (Fig. 4.2). Proteolytic processing of EntV is a plausible explanation for this size discrepancy. Since GelE and SprE cleave E. faecalis surface and secreted proteins, they are candidates for processing EntV (321, 379).

To test the hypothesis that GelE and/or SprE proteolytically process EntV, in silico and in vitro tests were performed. A member of the M4 family of proteases thermolysin, a homolog of GelE from Bacillus thermoproteolyticus, preferentially cleaves sites with position P1' that have bulky and aromatic residues (Ile, Leu, Val, Ala, Met, Phe) and aromatic sites in position

P1 [392]. Glutamyl endopeptidases V8 from Staphylococcus aureus is a homolog of serine protease (SprE) from E. faecalis and preferentially cleaves at Glu in position P1, Pro/Val or Phe in position P2, Ala/Val in position P3, and Asp in position P4 (380). Thus, proteolytic cleavage of EntV by these homologous proteases was examined in silico using PeptideCutter (ExPasy)

[394].

EntV contains two cysteines that form a putative disulfide bond that is necessary for bactericidal activity (358). To examine the role of the disulfide bond for activity against C. albicans, E. faecalis supernatant was reduced followed by alkylation to permanently reduce any cysteine in the supernatant. As illustrated in Figure 4.3A, a reduction in the inhibitory activity of

WT supernatant was observed after reductive-alkylation, suggesting that the disulfide bond is also necessary for the inhibitory activity against C. albicans. Disulfide bond formation in Gram- positive bacteria has been understudied and is not fully understood. On the other hand disulfide bond formation in Gram-negative bacteria has been extensively studied. Since Gram-positive

83

1.5

e

c **" n

e **" c 1.0 **"

s **"

e r

o **" **"

u

l F

0.5

e

v

i

t

a

l e R 0 Control WT ΔentV ΔfsrB ΔgelE ΔsprE ΔgelEΔsprE E. faecalis Supernatant

Figure 4.2: The E. faecalis Fsr system is involved in regulating the inhibitory activity.

The Fsr two component quorum-sensing system from E. faecalis regulates expression of EntV and secreted proteases (GelE and SprE) (356). C. albicans (HWP1p::GFP) biofilms grown at

37°C for 24 hours in YNBAS. Hyphal morphogenesis was quantified by measuring GFP fluorescence as a ratio to the OD600. Disruption of gelE and/or sprE decreased inhibitory activity. Experiments were repeated at least three times and statistically significant differences relative to treatment with the WT supernatant were calculated using a one-way ANOVA, and are indicated by asterisks (**, p<0.009).

84 bacteria lack traditional periplasmic spaces it has been thought that these disulfide bonds are formed spontaneously upon secretion or that many secrete few, if any, proteins that have disulfide bonds (381). However, recent studies have demonstrated the importance of disulfide bond formation by thiol-disulfide (DsbA-like proteins) in Gram- positive actinobacteria (382). Interesting, disruption of dsbA by transposon mutagenesis also resulted in reduced inhibitory activity (Fig. 4.3A). Taken together these results demonstrate that protein oxidation is necessary for maximum inhibitory activity, which suggests that proper formation of the disulfide bond is necessary for EntV efficacy.

Disulfide bridges stabilize the formation of β-strands and α-helices, can control substrate accessibility, and protect proteins from damage. Therefore, only cleavage sites outside of the disulfide bonds are shown (383-385). As illustrated in Figure 4.3B, GelE cleaves at A68 and SprE cleaves at residues further toward the N-terminus, which corresponds with previous MS/MS analysis and N-terminal degradation sequencing of EntV from WT E. faecalis

(245). Thus, these data suggest that GelE is important for cleavage of the EntV propeptide to produce the active bacteriocin. Additionally, GelE also cleaves the C-terminus of EntV in silico resulting in production of a 65 amino acid peptide of 6.9 kDa in size (Fig. 4.3B).

To investigate further the proteolytic processing of EntV by GelE, recombinant EntV136

(136 amino acid propeptide) expressed and purified from E. coli (BL21-DE3) was incubated with increasing concentrations (1.5, 4.5, 15, and 30 μg/ml) of purified GelE from E. faecalis

(321). I observed increased amounts of lower molecular weight EntV (~7KDa) with increased concentrations of GelE (Fig. 4.4), indicating proteolytic processing of EntV by GelE. This supports previous reports that demonstrate the propeptide (136 amino acids, ~15kDa) is further modified by the extracellular gelatinase into the active bacteriocin of 68-amino acid (7kDa) peptide that has been shown to be active against bacteria (245). As discussed in Chapter 3, I

85 A.

1.5

e c

n **" ***"

e ***" c **"

s 1.0

e

r

o

u

l

F

e v

i 0.5

t

a

l

e R

0 Control WT Control WT dsbA::tn Reduced and Alkylated E. faecalis Supernatant

B.

Figure 4.3: Protein oxidation is important for inhibitory activity.

(A) C. albicans (HWP1p::GFP) biofilms grown at 37°C for 24 hours in YNBAS. Hyphal morphogenesis was quantified by measuring GFP fluorescence as a ratio to the OD600.

Reductive-Alkylation decreased inhibitory activity of WT E. faecalis supernatant. Disruption of dsbA (predicted dithiol-disulfide ) by transposon mutagenesis also decreased inhibitory activity. Experiments were repeated at least three times and statistically significant differences relative to treatment with the WT supernatant were calculated using a one-way

ANOVA, and are indicated by asterisks (**, p<0.01, ***p<0.001). (B) In silico digestion of EntV by GelE homolog thermolysin (T) and SprE homolog glutamylpeptidase (G) using

PeptideCutter (ExPASy) (242).

86

Figure 4.4:. EntV is proteolystically processed by E. faecalis gelatinase.

GelE cleavage of rEntV136 was analyzed by SDS-PAGE. Increasing amounts of purified GelE was incubated with 30μg/ml rEntV136 N-terminally tagged with hexa-His for 1hr and the reactions terminated by addition of SDS-PAGE sample buffer and boiling. Proteins were stained with SyproRuby (Lonza) and visualized using a 300 nm UV transilluminator. Increased concentrations of GelE resulted in production of smaller molecular weight products (>10kDa).

87 demonstrated this processed peptide of 68-amino acid inhibited hyphal morphogenesis, biofilm formation, and virulence of C.albicans.

Classification and structure of E. faecalis bacteriocin EntV

EntV belongs to the class IIIb bacteriocins, which are the larger (>10kDa), non-lytic bacteriocins produced by Gram-positive bacteria [400]. To date only five bacteriocin have been identified and characterized in this class, dysgalacticin (DysA, Streptococcus dysgalactiae subsp. equisimilisI), SA-M57 (Streptococcus pyogenes M-type 57), EntV (E. faecalis), and

YpkK ( jeikeium) (243, 358, 386). Each of these proteins have conserved secondary structures: (1) unstructured N-terminus, (2) helix-loop-helix motif at the C-terminus, and (3) two cysteine residues that form a putative disulfide bond (242, 243, 358, 386) (Fig. 4.5A and B). In Addition, these class IIIb bacteriocins have a non-lytic mode of action against similar

Gram-positive bacteria and only EntV has been test against fungi (245, 358).

I hypothesized that these other bacteriocins similar to EntV are also proteolytically cleaved by proteases of the M4 family, homologs of thermolysin, aurolysin and gelatinase

(GelE). As seen in Table 4.1, M4 family proteases have been identified in these other bacterial species and proteolytic cleavage by thermolysin was observed in silico (387). Furthermore, all of the class IIIb bacteriocins gain a greater positive charge that is characteristic of efficacious antimicrobial peptides (Table 4.1, ProtParam) [403]. Using the protein structure of dysgalacticin in consort with the secondary structure consensus (PROMALS3D) of the class IIIb bacteriocins the predicted tertiary structure was assembled (I-Tasser) (242, 388). Collectively, these results suggest that the class IIIb bacteriocins are post-translational processing into small (<10kDa), positively charged, amphipathic bacteriocins.

88

A EntV LGSCVANKIKDEFFAMISISAIVKAAQKKAWKELAVTVLRFAKANGLKTNAIIVAGQLALWAVQC---- SA-M57 LGNCMANKIKDELLAMISVGTIIKYAQKKAWKELAKIVIKYVAKAGVKTNAALIAGQLAIWGLQC---- EntVYpkK L -LGTCSICKGVKANKIKDEASDDIKSFFVFDAMISVNVVIAASAAIIGQVKAAQKKAWKEKDYAKAAKEAVLKYAVTLAKVLRFQGVKAKANRNAAGALLKTNAAGMFAIIYWVGAGQWQCLRGSWALW AVQC---- SADysA-M57 I QRLGCNLCEEAWGYAMANKIKDEIS-LLVTLLAMISVKGIIGTNLFKAGKIIKYAQKKAWKEFEAAAAKLASLAK--IAVTIAGKY---VAKAGVKTNAARIAGMAALFAFVLIAAGQ-TCL----AIW GLQC---- YpkK :L - T*:CI KG K AS D:D I KS:. V:FD V:NV V AA A IGQ : K D Y*AKA AK EA V KY * L AK .Q GVK :*.RNAA :*A L AG. MF *A Y W G WQCRGSW DysAConsensus lgsCvankikdei IQRCLEEAWGYA amisvIS-LVTL aiikaaqkkawkeKGIINLFKAGK AkFEAA vlkyvakaGvktnaaAAKLAS--ATAG ---vAgqRI A GwavqCMAALF A FV A-TC---- : *: : :. : : : * * . :*. :* . * C onsensus lgsCvankikdei amisv aiikaaqkkawke Ak vlkyvakaGvktnaa vAgq A wavqC

B

Conservation: 9 5 5 5 5 9 5 595 5 59 995 9 55 55 DysA 1 I QRCLEEAWGYAISLVTLKGIINLFK AGKFEAAAAKLASA------TAGRIAGMAALFAFVA TC-- 58 YpkK 1 -LTCIKGKASDDIKSVFDVNVVAAAIGQKDYAKAAKEAVKYLAK QGVKRNAAALAGMFAYWGWQCRGSW 68 Conservation:SA-M57 1 LGNCMANKIKDELLAMISVGTIIKYAQKKAWKELAKIVIKYVAK 9 5 5 5 5 9 5 595 5AGVK TNAALIAGQLAIWGL 59 99QC5---- 9 55 6555 DysAEntV 1 1 LGSCVANKIKDEFFAMISISAIVKAAQKKAWKELAVTVLRFAKA I QRCLEEAWGYAISLVTLKGIINLFK AGKFEAAAAKLASANGLK----TNAIIVAGQLALWAVQC-----TAGRIAGMAALFAFVA---- 65 TC -- 58 YpkKConsensus aa: 1 ..p -CLTCIKGKASDDIKSVFDVNVVAAAIGQKDYAKAAKEAVKYLAKh...h...h..hh..shl..h...K.b..hA..h...... [email protected].... QCRGSW 68 SAConsensus-M57 ss: 1 hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhLGNCMANKIKDELLAMISVGTIIKYAQKKAWKELAKIVIKYVAK hhhhhhhhhhhhhhhhhAGVKTNAALIAGQLAIWGL QC---- 65 EntV 1 LGSCVANKIKDEFFAMISISAIVKAAQKKAWKELAVTVLRFAKA NGLKTNAIIVAGQLALWAVQC ---- 65 Consensus aa: ..pCh...h...h..hh..shl..h...K.b..hA..h...... [email protected].... FigureConsensus 4.5: Comparative ss: primary hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh and secondary structures of class IIIb hhhhhhhhhhhhhhhhh bacteriocins after proteolytic processing.

The first amino acid was determined by in silico digestion of each bacteriocin with M4 family protease thermolysin (PeptideCutter, ExPASy). All bacteriocins have conserved cysteines involved in disulfide bond formation. (A) ClustalW alignment of EntV, SA-M57, DysA, and YpkK bacteriocins (242, 358). * indicates conserved amino acids; : indicates amino acids with strongly similar function; and . indicates amino acids with weakly similar function (Network

Protein Sequence Analysis, NPS) (358). (B) Multiple alignment with predicted secondary structure of EntV, SA-M57, DysA, and YpkK bacteriocins (PROMALS3D) (389). The conservation indices for positions with a conservation index above 4 are shown at the top of the alignment block. Sequences are colored according to predicted secondary structures (red: alpha-helix). Just below the alignment sequence is the consensus amino acid symbols: conserved amino acids are in bold and uppercase letters; aliphatic (I, V, L): l; aromatic (Y, H,

W, F): @; hydrophobic (W, F, Y, M, L, I, V, A, C, T, H): h; polar residues (D, E, H, K, N, Q, R, S,

T): p; tiny (A, G, C, S): t; small (A, G, C, S, V, N, D, T, P): s; bulky residues (E, F, I, K, L, M, Q,

R, W, Y): b. Below the consensus amino acid symbols are the consensus predicted secondary structure symbols (alpha-helix: h).

89

Table 4.1. Computation of physical and chemical properties of class IIIb bacteriocins.

Based on the sequence analysis of the propeptide and proteolytically processed active peptide the size and theoretical pI was calculated using ProtPram (ExPASy). Identification of M4 family proteases was preformed using the NCBI gene database.

Species Bacteriocin Gene M4 Family Protease E. faecalis Enterococcin V entV Gelatinase (gelE) S. dysgalactiae Dysgalacticin dysA Oligoendopeptidase F (pepF) S. pyogenes SA-M57 scnM57 Peptidase F (pepF) C. jeikeium Corynicin JK ypkK Zn-dependent metalloprotease (JKRS06030)

Bacteriocin Propeptide Size Theoretical pI Active Peptide Size Theoretical pI (kDa) (kDa) Enterococcin V 14 (136 a.a.) 4.87 6.9 (65 a.a.) 9.84 Dysgalacticin 16.8 (152 a.a.) 4.77 7.0 (65 a.a.) 9.75 SA-M57 14.4 (137 a.a.) 5.40 7.3 (68 a.a.) 9.60 Corynicin JK 21.5 (192 a.a.) 4.46 6.0 (58 a.a.) 9.24

90 Discussion:

Important for competition and commensal growth in the GI tract of the host, post- translational processing of bacteriocins is common for efficient production of antimicrobial peptide against bacteria and fungi (390). Here I demonstrate that EntV is processed in an Fsr- dependent manner into a mature form of 68 amino acids that has activity at nanomolar or lower concentrations in most of our assays, demonstrating that it is highly efficacious.

EntV is a bacteriocin that belongs to the class IIIb bacteriocins, which are the larger

(>10kDA) bacteriocins (391). The structure of the mature form of EntV is predicted in silico to consist of two helical elements, separated by a flexible loop region. The conserved cysteines located immediately before and after these two helices are thought to form a disulfide bond

(392, 393). The immature form contains an additional N-terminal segment predicted to be unstructured (245), which is removed by GelE to form the mature and active form of the peptide

(245). While many bacteriocins encode an immunity protein, Dundar et al. could find no evidence of one, and E. faecalis appears to be intrinsically resistant (358). The bacteriocidal activity of EntV against sensitive species is not due to lysis, and the mechanism remains enigmatic (242).

The class IIIb bacteriocins that have been identified to date have a similar predicted secondary, though are not well conserved at the amino acid level. Considering the structure similarity and conserved cysteines necessary for activity and in silico cleavege, it is plausible that these other class IIIb bacteriocins, like EntV are also post-translational modified resulting in production of a smaller (<10kDA) highly active positively charged antimicrobial peptide. Thus, characterization and classification of these bacteriocins may need revision (242, 243, 358,

394). EntV-like bacteriocins have been identified in multiple host-associated bacteria suggesting these bacteriocins are important for both pathogenic and commensal association with the host (303). Furthermore, given the physiochemical similarities of these bacteriocins it is possible that they have overlapping target spectra. This remains to be further tested and

91 suggests that these bacteriocins may play an important role in bacterial-fungal interactions in other Gram-positive bacteria.

92

CHAPTER 5:

Assessment of EntV mechanism of action against C. albicans

Portions of this chapter are based on my first author publication. I have received permission to reproduce data or text from this article through license number. I have contributed significantly to this publication. The article is listed for reference: Graham, C. E., M. R. Cruz, D. A. Garsin, and M. C. Lorenz. 2017. Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans. Proc. Natl. Acad. Sci.

U.S.A. 114: 4507–4512 (303).

93 Introduction:

C. albicans biofilms are complex, structured communities that form on biotic and abiotic surfaces and are a growing concern in the clinical setting. One of the leading opportunistic fungal pathogens, C. albicans biofilms are often difficult to eradicate and can lead to more severe systemic infection if left untreated. Comprised of both yeast and hyphal cells C. albicans biofilms form via a highly regulated process that begins with adhesion, followed by initiation and formation of hyphal cells, then maturation, and ultimately dispersion (Fig. 1.1) (41).

Adherence is the first essential stage in C. albicans biofilms development and is a highly regulated process. Factors such as cell surface composition, extracellular matrix production, adhesins and other cell surface proteins are all coordinated during attachment to a substrate. Finkel et al. (395), identified 30 transcriptional regulators that are important for adherence during biofilm development, which regulate the expression of 37 surface-associated genes among other genes. Furthermore, a functional relationship between cell wall stress responses and regulation of adherence was identified recently. Transcriptional repression of

ARC18, a member of the Arp2/3 complex important for regulation of endocytosis and the actin , resulted in reduced adhesion and biofilm formation (395). Additionally, cell surface hydrophobicity and exposure of cell wall chitin and β-glucans was increased with depletion of the Arp2/3 complex leading to impaired cell wall integrity and activation of Rho1- mediated cell wall stress responses (50, 396-398).

C. albicans cell wall proteins are essential for maintaining cell wall integrity, adherence, enzymatic functions, biofilm development, and morphological transition. In C. albicans, a MAPK network regulates responses to environmental stresses such as changes in temperature, osmolarity, ionic stress, cell wall stress, and oxidative stress. Three interconnected MAPK pathways, Hog1, Mkc1 and Cek1, regulate this core transcriptional response to environmental stress (50, 398-400). Different membrane sensors sense environmental stress signals; the signals are transduced via the MAPK pathways, resulting in cooperative phosphorylation of

94 Hog1 and Cek1 followed by phosphorylation of Mkc1. Collectively, this results in generation of a core transcriptional stress response (3). While components of the core stress responses have been identified many of the sensors have not been identified or remain uncharacterized in C. albicans.

I hypothesize that EntV is eliciting a cell surface stress that disrupts cell surface proteins, adhesion and thus biofilm formation and hyphal morphogenesis. Here I identify C. albicans genes that when deleted decrease sensitivity to E. faecalis inhibitory activity during biofilm development. Deletion in genes involved in regulation of hyphal morphogenesis, cell wall stress, and cell wall architecture were identified. Furthermore, assessment of sEntV68 localization by fluorescence microscopy in yeast and hyphal cells demonstrated surface association in all cell types. Although sEntV68 inhibits hyphal morphogenesis, biofilm formation and virulence of C. albicans, sEntV68 did not kill C. albicans, nor inhibit its growth, even at high concentrations. sEntV68 inhibits hyphal morphogenesis and biofilm formation in clinical isolates of C. albicans, as well as other pathogenic Candida spp. EntV68, or similar compounds, may offer a viable therapeutic alternative. Understanding the mechanism of action is important for development and use of EntV68 based therapeutics. Identification of genes dysregulated by E. faecalis will provide insight into possible gene targets for development of therapeutic strategies that target hyphal morphogenesis and biofilm development (303).

Results:

Role of hyphal morphogenesis and surface stress response

E. faecalis secretes an inhibitor of C. albicans virulence and biofilm formation that I have identified as the bacteriocin EntV (23, 42, 49). Hyphal morphogenesis was reduced in both in vitro and in vivo assays, suggesting that the E. faecalis mediated inhibition C. albicans is due to dysregulation of hyphal morphogenesis pathways. Thus, I hypothesized that E. faecalis dysregulates hyphal-specific genes in C. albicans that are important for hyphal

95 morphogenesis and thus biofilm development. As mentioned previously, hyphal morphogenesis and biofilm formation of C. albicans are interconnected and regulated by the Ras1-cAMP-PKA signal pathway (401, 402). This is important when considering the mechanism of action of E. faecalis-mediated inhibition of hyphal morphogenesis and biofilm formation.

To understand the mechanism of action I assessed C. albicans mutant libraries of transcription factors and cell wall proteins (303) for decreased sensitivity to E. faecalis supernatant. Biofilms were grown as described above using the resazurin redox assay to quantify biofilm biomass (401). Resistance to WT E. faecalis supernatant was determined by comparing supernatant treated cells to media treated controls. Homozygous deletion mutants in non-essential transcription factors and cell wall proteins that were resistant to E. faecalis supernatant are listed in Table 5.1. Theses homozygous deletion mutants have known functions in hyphal morphogenesis, cell wall biogenesis, mating, cell cycle and unknown function.

24 Transcription factor mutants were identified that were less sensitive to E. faecalis supernatant (Table 5.1) (401, 403, 404). Some of the transcription factor mutants identified are involved in negatively regulating hyphal morphogenesis (NRG1, SSN6, RAP1), deletion of these genes results in a hyperfilamentous phenotype (400, 405). Transcription factor mutants identified involved in cell wall biogenesis genes and that are linked to the cell wall integrity

(CWI) or Hog1 stress response signal cascades (CAS5, SKO1, FCR1, ORF19.2476) were also identified (398, 406, 407). 15 cell wall protein deletion mutants were identified that decreased sensitivity to E. faecalis supernatant (Table 2.2). Moreover, cell wall protein deletion mutants identified are also linked to hyphal morphogenesis (PDE2) and surface stress response

(WSC1, WSC2, WSC4, PGA26, CCW14, ECM4) (107). Taken together these data suggest E. faecalis supernatant is triggering an adaptive stress response in C. albicans that leads to repression of morphogenesis (49).

The Ras1-cAMP-PKA signal pathway regulates hyphal morphogenesis during biofilm development (48). Therefore, inhibition of C. albicans biofilm formation would likely involve 96 Table 5.1: Homozygous deletion mutants in non-essential transcription factors resistant to E. faecalis supernatant.

C. albicans homozygous deletion mutants were screened for resistance to E. faecalis supernatant biofilm inhibition. Biofilms were grown in YNBAS for 24 hours and biofilm biomass was quantified using the resazurin redox assay comparing biofilms grown in the presence or absence of WT E. faecalis supernatant.

S. cerevisiae Gene Systematic Name Function Ortholog HMS1 C5_00670C_A HMS1 Filamentation GLN3 C5_04280C_A GLN3 Nitrogen Stress, Filamentation C5_04410C_A C5_04410C_A AZF1 Unknown (Cell Cycle in S. cerevisiae) GRF10 C5_05080W_A PHO2 Filamentation, Adenine Biosynthesis ZCF21 C4_00760W_A LYS14 Cell Wall Biogensis and Stress, Pathogenesis CAS5 C4_01190W_A MOT3 Cell Wall Biogensis and Stress, Filamentation, Pathogenesis CWT1 CR_05530C_A RDS2 Cell Wall Biogensis and Stress, Nitrogen Stress, Filamentation MBP1 C3_04110C_A MBP1 Cell Cycle TRY4 C3_05050W_A ADR1 Cell Adhesion WOR2 C3_05170W_A UME6 Filamentation, Phenotypic Switching C2_05640W_A C2_05640W_A BAS1 Filamentation STP4 C2_03220C_A STP3 Filamentation SKO1 C1_03770W_A SKO1 Filamentation, Cell Wall Stress, Oxidative Stress, Cell CRZ2 CR_07060C_A CRZ1 Cell Adhesion, pH Response C1_05750C_A C1_05750C_A ECM5 Oxidative Stress MSN4 C1_08940C_A MSN2 Oxidative Stress SSN6 C3_07020W_A CYC8 Filamentation, Pathogenesis FCR1 C3_06850W_A CAT8 Filamentation NRG1 C7_04230W_A NRG2 Filamentation RIM101 C1_14340C_A RIM101 Filamentation, pH Response AAF1 C3_06470W_A - Filamentation, Cell Adhesion CPH2 C6_00280W_A HMS1 Filamentation KAR4 CR_02330C_A KAR4 Mating RAP1 C2_10080W_A RAP1 Filamentation

S. cerevisiae Gene Systematic Name Function Ortholog PGA26 C1_05760C_A - Filamentation, Cell Wall Stress PGA7 C4_00120W_A - Biofilm ECM, Pathogenesis PDE2 C1_02840W_A PDE2 Filamentation, Adhesion, Cell Wall Organization WSC1 C3_04250W_A WSC2 Cell Wall Stress, Cell Wall Organization WSC2 C6_02670C_A WSC2 Cell Wall Stress WSC4 C1_14360C_A WSC4 Cell Wall Stress MNN4-4 C6_02830W_A MNN4 Cell Wall Biogensis and Stress MNN10 C4_00310C_A Mnn10 Cell Wall Biogensis MP65 C2_10030C_A SCW10 Cell Wall Biogensis and Stress, Filamentation, Adhesion, Pathogensis CCW14 C5_01010W_A - Cell Wall Stress VTI1 C3_03400C_A VTI1 Golgi to Vacuole Transport ECM4 CR_02130W_A ECM4 Core Stress Response, Filamentation ECM21 C1_10180C_A ECM21 Endocytosis ECM25 C1_13310W_A ECM25 Filamentation SAP98 C2_03680W_A PEP4 Phenotypic Switching

97 S. cerevisiae Gene Systematic Name Function Ortholog HMS1 C5_00670C_A HMS1 Filamentation GLN3 C5_04280C_A GLN3 Nitrogen Stress, Filamentation C5_04410C_A C5_04410C_A AZF1 Unknown (Cell Cycle in S. cerevisiae) GRF10 C5_05080W_A PHO2 Filamentation, Adenine Biosynthesis ZCF21 C4_00760W_A LYS14 Cell Wall Biogensis and Stress, Pathogenesis CAS5 C4_01190W_A MOT3 Cell Wall Biogensis and Stress, Filamentation, Pathogenesis CWT1 CR_05530C_A RDS2 Cell Wall Biogensis and Stress, Nitrogen Stress, Filamentation TableMBP1 5.2: HomozygousC3_04110C_A deletionMBP1 mutants inCe cellll Cycl walle proteins resistant to E. faecalis TRY4 C3_05050W_A ADR1 Cell Adhesion supernatant.WOR2 C3_05170W_A UME6 Filamentation, Phenotypic Switching C2_05640W_A C2_05640W_A BAS1 Filamentation STP4 C2_03220C_A STP3 Filamentation SKO1 C1_03770W_A SKO1 Filamentation, Cell Wall Stress, Oxidative Stress, Cell C.CRZ2 albicans homozygousCR_07060C_A deletionCRZ1 mutantsC e well Adrehe sio screenedn, pH Respo nse for resistance to E. faecalis C1_05750C_A C1_05750C_A ECM5 Oxidative Stress MSN4 C1_08940C_A MSN2 Oxidative Stress supernatantSSN6 biofilC3_07020W_Am inhibition. BiofilmsCYC8 were grownFilamen tinati oYNBASn, Pathoge nforesis 24 hours and biofilm biomass FCR1 C3_06850W_A CAT8 Filamentation wasNRG1 quantified C7_04230W_Ausing the resazurin NRG2 redox assayFilame ton tacomparetion biofilms grown in the presence or RIM101 C1_14340C_A RIM101 Filamentation, pH Response absenceAAF1 of WTC3_06470W_A E. faecalis supernatant- . Filamentation, Cell Adhesion CPH2 C6_00280W_A HMS1 Filamentation KAR4 CR_02330C_A KAR4 Mating RAP1 C2_10080W_A RAP1 Filamentation

S. cerevisiae Gene Systematic Name Function Ortholog PGA26 C1_05760C_A - Filamentation, Cell Wall Stress PGA7 C4_00120W_A - Biofilm ECM, Pathogenesis PDE2 C1_02840W_A PDE2 Filamentation, Adhesion, Cell Wall Organization WSC1 C3_04250W_A WSC2 Cell Wall Stress, Cell Wall Organization WSC2 C6_02670C_A WSC2 Cell Wall Stress WSC4 C1_14360C_A WSC4 Cell Wall Stress MNN4-4 C6_02830W_A MNN4 Cell Wall Biogensis and Stress MNN10 C4_00310C_A Mnn10 Cell Wall Biogensis MP65 C2_10030C_A SCW10 Cell Wall Biogensis and Stress, Filamentation, Adhesion, Pathogensis CCW14 C5_01010W_A - Cell Wall Stress VTI1 C3_03400C_A VTI1 Golgi to Vacuole Transport ECM4 CR_02130W_A ECM4 Core Stress Response, Filamentation ECM21 C1_10180C_A ECM21 Endocytosis ECM25 C1_13310W_A ECM25 Filamentation SAP98 C2_03680W_A PEP4 Phenotypic Switching

98 perturbation of this signal cascade (408). Interestingly, deletion of negative regulators of the signal pathway, NRG1 and PDE1, conferred resistance to E. faecalis, demonstrating the importance of the Ras1-cAMP-PKA signal pathway regulation of hyphal morphogenesis in response to E. faecalis (Fig 5.1A). This suggests that E. faecalis-mediated inhibition requires repressors of hyphal morphogenesis that are most likely induced during inhibition. Nrg1 is essential for the hyphal to yeast transition during the dispersal stage of biofilm development and negatively regulates the biofilm transcriptional network (155). Pde2 is a high affinity cyclic nucleotide phosphodiesterase that moderates signaling by cAMP in the Ras1-cAMP-PKA signal pathway (50, 397, 400).

The stress response MAPK network consists of three terminal MAPKs, Hog1, Mkc1 and

Cek1, that are phosphorylated as a result of membrane sensors signaling in responses to environmental stress, such as surface stress (48, 399). Deletion of transcription factors regulated by the MAPK network, CAS5, SKO1, and FCR1, conferred resistance to E. faecalis, demonstrating a possible role of the MAPK pathway during inhibition hyphal morphogenesis by

E. faecalis (Fig 5.1B). Furthermore, deletion of the poorly understood cell wall integrity sensors,

WSC1, WSC2, and WSC4, decreased sensitivity to E. faecalis supernatant (Fig. 5.1B). At the center of the MAPK network is Hog1-MAPK, which at basal concentrations represses Cek1 and controls phosphorylation of Mkc1 (5, 409). Phosphorylated Hog1 acts a repressor of hyphal morphogenesis independently of Efg1 and Cph1 by regulating Sko1-Ssn6-Tup1 mediated repression. Thus, I hypothesized that the Hog1-MAPK stress response pathway is activated in

C. albicans due to E. faecalis-mediated perturbation of the cell wall and membrane. Deletion of

HOG1 resulted in decreased sensitivity to E. faecalis supernatant (Fig 5.2). Taken together these results suggest that the stress response MAPK network is activated in response to E. faecalis-mediated stress.

99 No Bacteria A. WT Supernatant No Bacteria

A. 1.5 WT Supernatant

e

c 1.5

n

e

e

c

c s

n 1.0

e

e

r

c

o s

u 1.0 * l

e **" *" r

F *" *

o e u *

l **" *" v

i 0.5

F *" *

t

a

e

l

v e

i 0.5

t

R

a

l e

R 0 Wildtype Parent nrg1Δ/Δ Parent pde2Δ/Δ 0 (SN152) (Day286) Wildtype Parent nrg1Δ/Δ Parent pde2Δ/Δ No Bacteria B. (SN152) (Day2WT86 )S upernatant No Bacteria

B. 1.5 WT Supernatant

e

c 1.5

n

e

e

c

c

s

n e

e 1.0

r

c

o

s

u e l 1.0 **"

r *"

F o

*"

u e

l **" v

i *"

F t

0.5 *"

a

e

l

v

e i

t 0.5

R

a

l e

R 0 Wildtype Parent cas5Δ/Δ sko1Δ/Δ fcr1Δ/Δ Parent wsc1Δ/Δ wsc2Δ/Δ wsc4Δ/Δ 0 (SN152) (Day286) Wildtype Parent cas5Δ/Δ sko1Δ/Δ fcr1Δ/Δ Parent wsc1Δ/Δ wsc2Δ/Δ wsc4Δ/Δ (SN152) (Day286)

Figure 5.1: Deletion of hyphal morphogenesis and cell wall stress genes confers resistance to E. faecalis supernatant.

C. albicans homozygous deletion mutants were screened for resistance to E. faecalis supernatant biofilm inhibition. Biofilm biomass was quantified using the resazurin redox assay comparing no treatment biofilms (blue) to supernatant treated biofilms (orange). (A) Deletion of genes involved in repression of the Ras1-cAMP pathway resulted in decreased sensitivity to E. faecalis supernatant. (B) Deletion of genes involved in cell wall stress resulted in decreased sensitivity to E. faecalis supernatant. Statistically significant differences relative to treatment with the WT supernatant were calculated using a Student’s t-test, and are indicated by asterisks (**, p<0.007; *, p<0.04).

100

1.5

e

c

n e

c 1.0

s

e

r o

u *"

l

F

e **"""""" **""""""

v

i

t a

l 0.5

e R

0 Wildtype Parent hog1Δ/Δ hog1Δ/HOG1 (RM1000)

Figure 5.2: Deletion of the central stress response MAPK, HOG1, decreased C. albicans sensitivity to E. faecalis supernatant.

Treatment with E. faecalis WT supernatant did not decrease biofilm biomass of a HOG1 homozygous deletion mutant and complementation of the WT HOG1 restored sensitivity. was resistance to E. faecalis supernatant biofilm inhibition. Biofilm biomass was quantified using the resazurin redox assay comparing no treatment biofilms (blue) to supernatant treated biofilms

(orange). Statistically significant differences relative to treatment with the WT supernatant were calculated using a Student’s t-test, and are indicated by asterisks (**, p<0.006; *, p<0.03).

101 Host relevant environmental stresses impact on EntV activity

Functional stress response is important for C. albicans survival by initiating adaptation to changes in environmental conditions (401). Induction of MAPK stress response pathways has been observed in C. albicans in response to various external factors that lead to inhibition of hyphal morphogenesis and biofilm formation. Thus, I considered whether sEntV might synergize with host-relevant stresses, including oxidative, nitrosative, cell wall, and cell membrane stresses. The addition of sEntV68 did not affect susceptibility to any of these stresses, reinforcing the idea that it has a novel mechanism of action (Fig 5.3). These results do not correspond with our previous observations in the in vitro biofilm model. There are some plausible explanations: (1) there is no stress response, (2) the stress response is specific to biofilm growth (e.g. adhesion), and/or (3) the stress growth conditions impedes the activity of

EntV68 (e.g. cell surface remodeling). This remains to be fully elucidated and will need to be further examined.

EntV activity on C. albicans in different growth conditions

To better understand the sEntV68 mechanism of action, I considered whether EntV might inhibit biofilm formation as a result of a general inhibition of fungal growth. To address this, I grew C. albicans in the presence of increasing concentrations of sEntV68 and found no reduction in growth as high as 10 µM, 30,000-fold higher than the in vitro biofilm inhibitory concentration (Fig 5.4). Although sEntV68 was very effective at inhibiting biofilm formation in artificial saliva medium (YNBAS), I questioned the efficacy of sEntV68 in stronger hyphal inducing conditions. As illustrated in Figure 5.5, sEntV68 is highly active in a variety of media conditions, including fetal bovine serum (FBS), which is a robust hyphal inducer (Fig. 5.5)

Role of cell wall composition in response to E. faecalis mediated inhibition

While many of the genes identified in the homozygous deletion mutants screen have hyper-filamentous phenotypes, deletion of ZCF21 conferred resistance to E. faecalis 102

B.

A. 1.5 Medium Only 1.5 Medium Only

DMSO

) DMSO

)

0 0

CFW 0

0 SDS 6 6 CFW + DMSO D SDS + DMSO D 68

O 68 O CFW + EntV

( SDS + EntV

(

1.0 68 1.0 68 e

e EntV EntV

c

c n

n

a

a

b

b

r

r o

o

s

s b

b 0.5 0.5 A A

0 0 0 5 10 15 0 5 10 15 Time (Hours) Time (Hours)

1.5 D. 1.5

C. Medium Only Medium Only

) ) DMSO DMSO

0

0

0 0

H O 6 Menadione

6 2 2 D

D H2O2 + DMSO Menadione + DMSO O

O 68 68 (

( H O + EntV Menadione + EntV

1.0 2 2 1.0 e

e EntV68 EntV68

c

c

n

n a

a

b

b

r

r

o o

s

s b

b 0.5 0.5 A A

0 0 0 5 10 15 0 5 10 15 Time (Hours) Time (Hours)

E. 1.5 F. 1.5

Medium Only Medium Only

DMSO ) DMSO

0

)

0 0

NaCl 6 Sorbitol 0

6

NaCl + DMSO D Sorbitol + DMSO D 68 O 68 ( Sorbitol + EntV

O NaCl + EntV

1.0 1.0

( e

EntV68 EntV68

c

e n

c

a

n

b

a

r

b o

r

s

o b

s 0.5 0.5 b A A

0 0 0 5 10 15 0 5 10 15 Time (Hours) Time (Hours)

Figure 5.3: Impact of host relevant stresses on sEntV68 activity.

C. albicans (SC5314) was grown for 15 hours in YPD at 30° C with sEntV68 (100 nM) in different stress conditions: cell wall stress (A) calcofluor white (100 μg/ml) and (B) SDS (10%); cell membrane stress (C) sorbitol (1 M) and (D) Sodium Chloride (1 M); oxidative stress (E) hydrogen peroxide (5 mM) and (F) menadione (100 μM) in a 96 well plate. OD600 was measured every two minutes in a microplate reader.

103

1.5 DMSO

0nM

)

0 0.1nM

0 6

D 1.0 0.5nM

O

(

e 1nM c

n 5nM

a b

r 10nM o

s 0.5

b 100nM A 1000nM 10,000nM 0 0 5 10 15 Time (Hours)

Figure 5.4: sEntV68 activity on planktonic C. albicans yeast cells.

C. albicans was grown at 30°C in YPD for 15 hours with shaking in a 96 well plate with increasing concentrations of sEntV68. Growth of was quantified by measuring absorbance

(OD600) every two minutes in a microplate reader (BioTek).

104

1.5

e

c

n e

c 1.0

s

e

r

o

u

l F

**"""""" ***""""""

e v

i 0.5 t

a ***""""""

l ***""""""

e R

0 DMSO YNBAS RPMI Spider YPD + 10%FBS

Figure 5.5:. sEntV68 inhibitory activity in different media types.

C. albicans (HWP1p::GFP) biofilms grown for 24 hours at 37°C in YNBAS, RPMI, Spider, or

YPD with 10% fetal bovine serum with 0.01% DMSO (blue) or 100 nM sEntV (orange). Hyphal morphogenesis was quantified by measuring GFP fluorescence as a ratio to the OD600 for three separate experiments and statically significant differences were calculated using a Student’s t- test (*p<0.01).

105 supernatant in a hyper-filamentous independent manner (Fig. 5.6, Table 2.1) (9). Zcf21p negatively regulates expression of genes that encode cell surface proteins important for C. albicans virulence (410). Bohm et al. (280), found that ZCF21 regulates cell wall structure and composition, including proteins and carbohydrates in the cell wall. Furthermore, the homozygous ZCF21 deletion mutant had decreased sensitivity to sEnV68, suggesting that deletion of ZCF21 causes changes in the cell wall structure and composition preventing sEntV68 inhibition.

EntV localization in C. albicans

Analysis of the primary and predicted secondary structure (Chapter 4) indicate EntV68 has similar physicochemical properties to human antimicrobial peptides active against C. albicans that elicit a surface stress response that is initiated via interactions with the cell wall and cell wall associated proteins (280, 295, 302). Similar to EntV68, the cationic properties of

LL-37, β-d3, and Hst-5 are thought to promote interactions with the anionic microbial surface and the amphipathic structures allow the peptides to interact with microbial membranes (411).

Thus, I hypothesized that EntV68 may interact with the cell surface of C. albicans in a similar fashion. As demonstrated in Figure 5.7, sEntV68 with a C-terminal FITC tag localized to the cell surface of C. albicans yeast and hyphal cells, suggesting that the EntV68 mechanism of action is cell surface dependent.

EntV68 activity target spectrum

Since sEntV68 is efficacious in both in vitro and in vivo conditions and has therapeutic potential, I tested the efficacy of sEntV68 against clinical isolates of C. albicans and other pathogenic Candida species to gain further insight into the spectrum of fungal targets and mechanism of action. I found that sEntV68 significantly reduced biofilm formation of clinical isolates, including azole resistant strains (Fig. 5.8A) (395, 412). Furthermore, sEntV68 was also active against biofilms formed by other C. glabrata, C. tropicalis, and C. parapsilosis (Fig.. 106

AA..

1.51.5

e

e

c

c

n

n

e

e

c

c

s s

e 1.0 r

e 1.0

r

o

o

u l

u **

l **

F

F **

e **

e

v

i

v

t

i t

a 0.5 l

a 0.5

l

e

e

R R

0 0 WWiillddttypypee PaParerenntt zcfzcf2211ΔΔ//ΔΔ (SN(SN115522)) BB..

1.51.5

e

e

c

c

n

n

e

e

c c

s 1.0

s 1.0

e

r

e

r

o

o

u

l

u

l

F

F

e

e

v

i v

t 0.5 ** ** i ** t 0.5 ** **

a **

l

a

l

e

e

R R

0 0 WWiillddttypypee PaParerenntt zcfzcf2211ΔΔ//ΔΔ (SN(SN115522))

Figure 5.6: Deletion of ZCF21 decreased C. albicans sensitivity to E. faecalis supernatant and sEntV68.

C. albicans biofilms grown for 24 hours in YNBAS at 37°C for 24 hours with (A) sterile medium

(blue) or supernatant (orange) and (B) 0.01% DMSO (blue) or 100 nM sEntV (orange). Biofilm biomass was quantified using the resazurin redox assay. Statistically significant differences relative to treated and control biofilms were calculated using a Student’s t-test, and are indicated by asterisks (**, p<0.007).

107 A. B. C.

IC = 0.5 nM

D. 1.5 50

e

c

n

e c

s 1.0

e

r

o

u

l

F

e

v i

t 0.5

a

l

e R

0

Figure 5.7: Localization patterns of sEntV68 on the surface of C. albicans.

Planktonic cultures of C. albicans expressingsEn constitutivetV68 mCherry were incubated in YNBAS

sEntV68-FITC with 100nM sEntV68-FITC at (A) 30°C for hour, (B) 37°C for 1 hour, or (C) 37°C for 1 hour without sEntV68-FITC to allow for germ-tube formation followed by 1 hour incubation with sEntV68-FITC. Localization of sEntV68-FITC was observed by fluorescence microscopy

(Olympus IX81) using slidebook 6 (3i, Intelligent Imaging Innovations). (D) Biofilms were grown

68 68 with increasing concentrations of sEntV or sEntV -FITC and the IC50 calculated at 0.3 nM and 0.5 nM, respectively.

108 A. A. 1.5

1.5

e

c

n

e

c

e

c

n s

e 1.0

e

c r

s 1.0

o

e

r

u l

o *"*""""" **"""""" *"""""" F

u *""""""

l *"""""" *"""""" *"""""" *"""""" *"""""" * e * *""*"""" **"""""" F **""""""

v *"""""" 0.5 ****"""""" i *"""""" *"""""" *""""""

t *

e *""*"""" **"""""" a

v 0.5 ****""""""

l

i

t

e

a

l

R e

R 0 0 Wildtype Can16 Can17 Can18 Can19 Can92 Can95 Can893 Can897 Can906 Wildtype Can16 Can17 Can18 Can19 Can92 Can95 Can893 Can897 Can906

B. B. 1.5

1.5

e

c

n

e

e

c

c

n s

e 1.0

e c

r *""""""

s 1.0

o

e u r *""""""

l **"""""" o

F *""""""

u

l **"""""" **"""""" e

F *"""""" v

0.5 i

t **""""""

e a

v 0.5

l

i

t

e

a

l

R e

R 0 0 C. albicans C. tropicalis C. parapsilosis C. glabrata C. albicans C. tropicalis C. parapsilosis C. glabrata

Figure 5.8:. sEntV68 target spectrum.

(A) sEntV68 inhibitory activity against clinical isolates of C. albicans. Stain names in red are resistant clinical isolates that resistant to azoles. C. albicans biofilms grown for 24 hours in

YNBAS at 37°C for 24 hours with 0.01% DMSO (blue) or 100 nM sEntV (orange). (B) sEntV68 inhibitory activity against different Candida species. C. albicans, C. tropicalis, C. parapsilosis and C. glabrata biofilms grown for 24 hours in RPMI at 37°C for 24 hours with 0.01% DMSO

(blue) or 100 nM sEntV (orange). Biofilm density was quantified by measuring resazurin fluorescence for three separate experiments and statically significant differences were calculated using a Student’s t-test (*, p<0.05; **, p<0.01).

109

5.8B). Taken together these results suggest that EntV68 is a specific inhibitor of hyphal and biofilm growth with a broad spectrum of activity against C. albicans and Candida spp .

Discussion:

The mechanism by which EntV inhibits C. albicans hyphal growth and biofilm formation is unresolved. However, homozygous deletion mutants resistant to E. faecalis supernatant revealed that hyphal morphogenesis and surface stress response functions are important for the mechanism of action. Localization of the fluorescent peptide suggests a cell surface (wall or membrane) localization, and I have no evidence that it exerts any cytotoxic or lytic stress against C. albicans. Quite the contrary: while sub-nanomolar concentrations of EntV are sufficient to inhibit biofilms, concentrations 10,000-fold higher do not reduce fungal growth, even in the presence of concurrent stresses that damage the cell wall or membrane.

Plausible mechanisms include EntV interfering with a or a hyphal inducing factor or blocking cell-cell or cell-substrate binding mediated by one or more of the C. albicans adhesins. Several of the homozygous deletion mutants, including ZCF21, disrupt the cell wall composition that could lead to masking of a binding target protein or component of the cell wall or membrane. Disruption of the cell wall composition and structure can also impair biofilm development and is often mediated by cell stress response (20). Genetic and biochemical studies will be necessary to elucidate the mechanism of action.

Biofilms provide protection to microbes by production of the ECM that encases the microbes protecting them from the immune response and antibiotics (39). Thus, biofilm related infections present many clinical challenges by complicating treatment strategies (80). With the limited antifungals available fungal infections are difficult to treat. Furthermore, resistance to traditional antifungals is an increasing concern. Traditional antifungals interfere with essential

110 biological processes to kill or inhibit fungal growth and can have off target effects that can be toxic to the patient (91, 413). To overcome these challenges targeting virulence traits rather than essential fungal processes has recently been proposed to alleviate the selection pressure for resistance (410).

Moreover, targeting virulence factors will keep opportunistic pathogens in check rather than disrupting the entire mycoflora with antifungal treatment. One of the most abundant fungal species found in humans, C. albicans does not have any known environmental reservoir (410).

This suggests that C. albicans has a profound association with humans and has coevolved considerably with the human host. Additionally, the majority of healthy individuals are colonized with C. albicans as part of the normal GI microflora (414, 415). That said, the beneficial effects of C. albicans colonization cannot be excluded, and might provide another rationale for why the use of an alternative antifungal could be advantageous. Collectively, our work suggests that

EntV is a promising alternative therapeutic that targets hyphal morphogenesis and biofilm development with a spectrum of activity against clinical isolates and other pathogenic Candida spp.

111

Chapter 6:

Discussion and Perspectives

112 Summary:

E. faecalis and C. albicans occupy overlapping niches as ubiquitous parts of the gastrointestinal and oral microbiome. As opportunistic pathogens these species are also amongst the most important and problematic nosocomial pathogens. I have observed antagonistic interactions between these two species, where virulence of both microbes is attenuated in the C. elegans infection model. Inhibition of C. albicans hyphal morphogenesis was a striking phenotype observed in this infection model. Inhibition of hyphal morphogenesis was also obtained by addition of E. faecalis spent supernatant in the C. elegans infection model and in vitro biofilm model (115).

I have identified the inhibitory factor as an E. faecalis bacteriocin, EntV, which was necessary and sufficient for the reduction of C. albicans virulence and biofilm formation via its inhibition of hyphal morphogenesis. As illustrated in Figure 6.1, I have demonstrated that the

EntV propeptide is post-translational modified for maximum activity. The synthetic version of the mature 68-amino acid peptide inhibited biofilm development in multiple media conditions and disrupted pre-formed biofilms. sEntV68 was protective in different fungal infection models at nanomolar or lower concentrations.

First, nematodes treated with the peptide at 0.1 nM are completely resistant to killing by

C. albicans (data not shown see (303)). sEntV68 was protective in the macrophage infection model and works in synergy with the macrophages increasing their antifungal activity. Finally,

EntV68 reduced epithelial invasion, inflammation, and fungal burden in a murine model of oropharyngeal candidiasis. Hyphal morphogenesis was inhibited in all three models. However,

EntV68 had no effect on C. albicans viability, even in the presence of significant host-mimicking stresses. These findings demonstrate the therapeutic potential of EntV as a novel antifungal agent that targets virulence rather than viability, which is important considering the increase in resistance to traditional antifungals.

113

Figure 6.1: Summary of EntV processing and activity.

The inhibitory activity produced by E. faecalis was first characterized using an in vitro biofilm.

EntV was identified as the inhibitory factor produced by E. faecalis and is proteolytically cleaved produce the active peptide that inhibits morphogenesis in the biofilm assay, murine macrophage infection model, and murine OPC model. Inhibition of hyphal morphogenesis reduced biofilm formation, and attenuated virulence of C. albicans during infection.

114 Furthermore, I demonstrated the physiochemical properties of EntV and the post-translational modifications that are necessary for activity. Upon assessment of the other closely related class IIIb bacteriocins from different bacteria, I observed prominent similarities in possible post- translational modifications and predicted secondary structures. This preliminary characterization of the class IIIb bacteriocins provides useful insight for the future design of

EntV-based therapeutic strategies and demonstrates the importance of the class IIIb bacteriocins for these Gram-positive bacteria and their conceivable role in fungal-bacterial interactions.

To understand how E. faecalis inhibits C. albicans biofilms and hyphal morphogenesis, I identified C. albicans genes that confer decreased sensitivity to E. faecalis inhibitory activity during biofilm development. Screens of homozygous deletion mutants revealed that deletion of genes involved in regulation of hyphal morphogenesis, cell wall stress, and cell wall architecture decreased sensitivity to E. faecalis supernatant. This suggests that EntV may target processes involved in these functions, since perturbation decreases efficacy of E. faecalis inhibitory activity. Furthermore, sEntV68 localized to the cell surface of both yeast and hyphal cells. As illustrated in Figure 6.2, I have demonstrated that modification of C. albicans cell wall components or hyperfilamentation produces decreased sensitivity to E. faecalis mediated inhibition of biofilm development. Thus, I hypothesize that EntV targets the cell surface, which disrupts cell surface function(s) that are necessary for hyphal morphogenesis and biofilm development, most likely a function that is linked between these important virulence determinants. I also examined the target spectrum of sEntV68 and found that clinical isolates of

C. albicans, including azole restraint strains, and other pathogenic Candida spp. are sensitive to sEntV68. This suggests that the mechanism of action targets a common function amongst these different strains and species. In total, these data demonstrate the therapeutic potential of sEntV68 or similar compounds for development of therapeutic strategies that target hyphal morphogenesis and biofilm development.

115

Figure 6.2: Summary of homozygous deletion mutant functions.

Of the 263 mutant screened 39 were less sensitive to E. faecalis supernatant. Collectively, most of these mutants have functions associated with cell wall composition and/or hyphal morphogenesis. Many of the cell wall mutants have functions associated with cell wall stress and are important for maintenance of cell wall integrity and architecture and deletion of these genes leads to cell surface remodeling. Of the 24 transcription factors, many have functions associated with regulating hyphal morphogenesis and many were hyperfilamentous as result of upregulation of hyphal specific genes.

116 C. albicans-E. faecalis interactions during commensalism and pathogenesis

Given the epidemiology of E. faecalis and C. albicans infections, their overlapping niches, and our current findings, it is plausible that these species have synergism during commensal colonization. As I observed previously in the nematode infection model, virulence of both microbes was attenuated, suggesting that their interactions promote non-pathogenic association with the host (115). Previous studies also demonstrate synergism during commensal colonization. Mason et al. (416) demonstrated that E. faecalis becomes more abundant after antibiotic treatment in mice colonized with C. albicans in both the stomach and the cecum. Considering C. albicans does not naturally colonize the GI tract of mice and antibiotic treatment is necessary to establish C. albicans colonization (417); it is plausible that the increase in E. faecalis also promotes commensal colonization of C. albicans (418).

On the other hand, during infection C. albicans and E. faecalis are commonly co- isolated in a variety of different types and severity of infections (109, 157, 159, 182). One common theme among these co-infections is the status of the host changes due to immune dysfunction and/or disruption of anatomical barriers, suggesting that a complex relation exists between E. faecalis and C. albicans that is dependent on the host. Consistent with clinical reports, Carlson et al. (151) found that co-infection with E. faecalis and C. albicans enhanced dissemination of E. faecalis pathogenesis in a murine intraperitoneal infection model.

Considering these results and the clinical indices of E. faecalis and C. albicans co-infections, it is plausible that when immune dysfunction, loss of anatomical barriers, and/or disruption of the microflora occurs E. faecalis is able to hitch a ride, so to speak, with C. albicans during disseminated infection. A similar occurrence has been demonstrated in C. albicans and P. aeruginosa, where in vitro antagonism is observed and clinical co-infections are more severe

(Chapter 1).

In view of our results, current literature, and clinical reports, Figure 6.3 illustrates our predicted model of the complex relationship that exists between E. faecalis and C. albicans. In

117 the presence of a healthy host both microbes are able to colonize as commensal microflora.

However, with a change in the host status both microbes can become pathogenic (8, 366).

During antibiotic treatment, E. faecalis is capable of surviving because of their genomic plasticity and ability to take up resistance genes and virulence traits via conjugation [436].

Thus, E. faecalis is capable of surviving with antibiotic treatment along with C. albicans.

However, with antibiotic treatment the population of E. faecalis is likely reduced, repressing Fsr and in turn EntV production (356). Under these conditions, C. albicans is able to grow, proliferate, and form hyphae. If the host has a compromised immune response, C. albicans is able to disseminate and survive and possibly allow the remaining E. faecalis a mode of dissemination (8). In the case of disrupted anatomical barriers, such as seen in intraperitoneal infections, both E. faecalis and C. albicans are able to survive in the hostile environment of the intraperitoneal cavity allowing further dissemination of both microbes (183, 184). To substantiate this model many more questions must be addressed, which will be discussed subsequently.

Role of E. faecalis bacteriocins in the host environment

Many of the bacteriocins produced by E. faecalis are found on plasmids and vary from strain to strain. Different bacteriocins-producing E. faecalis strains have been isolated from a broad range of sources, from commensal strains found in food and feces to pathogenic strains found in different types of infections. Interestingly, some bacteriocins produced by E. faecium are also produced by E. faecalis commensal and pathogenic strains (419). Although the majority of bacteriocins produced by E. faecalis target closely related Gram-positive bacteria and are thought to be important for competition with other bacteria, cytolysin is the exception because it targets other bacteria and eukaryotic cells. Cytolysin production was first observed in clinical isolates and conferred increased hemolysis (420). Furthermore, many E. faecalis

118 strains produce multiple bacteriocins and many of the same bacteriocins are produced in strains isolated from extremely different environments, indicating that these bacteriocins are not

Figure 6.3: Model of C. albicans-E. Faecalis interactions in the GI tract.

In the healthy host C. albicans and E. faecalis have commensal like association with the host.

There are abundant populations of E. faecalis. Thus, the Fsr system has been stimulated by accumulation of the QS molecule GABP and active EntV is being produced in sufficient concentrations to inhibit hyphal morphogenesis. In the unhealthy host who has compromised immune function, disrupted microflora, and/or loss of anatomical barriers C. albicans is able to penetrate the epithelial layer allowing E. faecalis to escape the intestinal lumen. As the status of the host deteriorates both C. albicans and E. faecalis become opportunistic pathogens.

119 niche specific and rather have a general advantage in any niche [440].

Role of EntV in the host environment

EntV is found in all sequenced E. faecalis strains, indicating that entV function is important for E. faecalis survival and is not niche specific. As mentioned previously, entV is regulated by the Fsr two-component system. The Fsr system is regulated by accumulation of the QS molecule GABP, suggesting that production and function of EntV is important when E. faecalis populations are at higher concentrations. In an environment such as the human GI tract E. faecalis populations can be 107 CFU/μg. In this environment, competition with neighboring bacterial species that are closely related and likely require similar nutrients is important [441]. Production of a bacteriocin such as EntV could be advantageous.

Moreover, production of EntV in the GI tract could also be beneficial during commensal colonization with C. albicans. As demonstrated in our current and previous findings, EntV promotes commensal-like association with the host in C. albicans by inhibition of hyphal morphogenesis (115, 303). Although hyphal cells are considered the invasive and more pathogenic form of C. albicans, there is much debate on this topic (47). Some literature suggests that the yeast form is the commensal, noninvasive morphology and the hyphal form is the invasive pathogenic morphology. This is an over simplification. During invasive infection both morphologies are necessary and the dominance of one morphology over the other depends on the infected organ (421). Moreover, it is unclear what morphology predominates during commensal colonization. Kumamoto et al. (422, 423), reported the expression of hyphal- associated genes during commensal colonization in a murine GI colonization model, despite the fact that C. albicans was found predominately in the yeast form. That said, C. albicans mutant strains locked in either the yeast form or hyphal form have attenuated virulence, suggesting the transition between the two states is dynamic and necessary for maximum virulence (361, 424, 425). Thus, it is plausible that production of EntV during commensal 120 colonization with C. albicans is capable of inhibiting hyphal morphogenesis and promoting commensalism. Furthermore, yeast cells are far less immunogenic and are not recognized by phagocytes, while hyphal cell are much more immunostimulatory (426-428). Thus, controlling hyphal morphogenesis could benefit the microflora including E. faecalis by keeping the immune response restrained.

The class IIIb bacteriocins, including EntV, are unusual compared to other classes of bacteriocins. The majority of propeptides are small where as the class IIIb bacteriocins are large and only about half of the propeptide is necessary for activity (Chapter 4). Most bacteriocins have an immunity protein that is encoded on a neighboring gene or with the same operon. This is the case with the class IIIb bacteriocins, except for EntV where the immunity protein has not been identified. Dundar et al. (245) attempted to identify the immunity protein to no avail. A translationally coupled gene was identified by bioinformatics as a candidate immunity factor (ef1097b). However, deletion of ef1097b had no effect on E. faecalis sensitivity to EntV. I considered whether the N-terminal portion of the propeptide might be the immunity factor. Nevertheless, ΔentV is not sensitive to sEntV68 (data not shown), suggesting that intrinsic immunity to sEntV68 occurs by another mechanism or gene and remains to be elucidated.

Gene expression data demonstrated that entV expression is the same in the pathogenic strain E. faecalis V583 as the commensal strain E. faecalis OG1RF, suggesting that EntV is expressed in all E. faecalis strains in a similar manner (356, 357). Furthermore, Bourgogne et al. (356), identified a FsrA binding consensus sequence that is present in all strains of sequenced E. faecalis. In addition to the Fsr operons, gelE and sprE, and entV, ef1352 that encodes a putative magnesium-translocating P-type ATPase was unregulated in response to

GABP stimulation (Fsr activation) (357). Nonetheless, ef1352 does not have an FsrA binding consensus sequence, suggesting that ef1352 is not directly regulated by the Fsr system and perhaps is induced by EntV causing ion leakage. This remains to be elucidated and would be in stark contrast to the immunity factors of other class IIIb bacteriocins (243, 358, 429). 121 The EntV mechanism of action against bacteria has not been fully elucidated, aside from the non-lytic manner of bactericidal activity. However, other class IIIb bacteriocins have been studied in much more detail. DysA (dysgalacticin) is produced by Streptococcus dysgalactiae subsp. equisimilis and the mechanism of action for DysA has been investigated in some detail. Swe et al. (394, 429) found that DysA has a non-lytic mode of action that is bactericidal, inhibited glucose fermentation by targeting glucose/mannose-PTS, increased membrane permeability that mediated intracellular ion leakage. Given the similarity in secondary structure I hypothesis that EntV has a similar mode of action that is mediated by insertion into the membrane of target bacteria (Fig 6.4). AMPs with similar physiochemical properties as EntV can target both fungi and bacteria (295, 302, 430). Thus, it is conceivable that EntV also interacts with the membrane of C. albicans, albeit to a lesser degree given the complex structure of the cell wall of C. albicans and differences in lipid composition compared to bacteria (13, 431). Human AMPs that are active against C. albicans, Hst-5, LL-37, hBD-3, are also active against bacteria, and inhibit hyphal morphogenesis and biofilm adhesion at sub- lethal concentrations in C. albicans. Considering this it is plausible that only low concentrations of EntV are able to insert into the membrane of C. albicans causing membrane stress that mediates inhibition of hyphal morphogenesis and biofilm development. However, given the lack of synergism of EntV with known membrane and cell wall stressors, this may be an over simplification of a very complex interaction.

Therapeutic potential of bacteriocins and future application

As previously mentioned, application of bacteriocins as therapeutics for human use has been limited to probiotics and application in food as preservatives (Introduction). Reports of in vivo use of bacteriocins have also focused on the use of probiotic bacteria that express and potentially secrete the bacteriocins in the gastrointestinal tract (432). Although the use of bacteriocin-producing probiotics may be as beneficial as prophylactics, application of purified

122

Figure 6.4: Predicted membrane topology of active EntV.

The two-amphipathic helices of EntV are predicated to insert into the cytoplasmic membrane of susceptible targets. The disulfide bridge is predicted to remain on the outer leaflet of the membrane. Membrane topology was predicted using PHDhtm transmembrane helices predictions (NPS), where the EntV amino acid sequence is compared and aligned to similar sequences to obtain a more accurate predicted membrane topology (310).

123 bacteriocins would be superior at countering established infections. Application of purified nisin has been successful, but has been limited to application in food (244). One of the greatest impediments in the therapeutic application of purified bacteriocins is the stability of the bacteriocin in the host environment (433). Engineering bacteriocins to increase stability and potency will overcome this problem. To design bacteriocins with increased stability and potency an understanding of bacteriocins that are effective in vivo is necessary. The stability and target spectrum of EntV have not been extensively studied, thus in the event that an enhanced version would be ideal strategies for modification of class II bacteriocins can be used for EntV.

Another, important factor to consider is that the size of EntV is twice as large as the class II bacteriocins, which have been extensively used and tested and are more cost effective treatment strategies.

The class IIa bacteriocins structure-function relationship has been extensively studied and the in vivo efficacy has been examined. Dabour et al. (434) found that intra-gastric application of pediocin PA-1, produced by Pediococcus acidilactici, was efficacious in mice with

GI infections of . Infected mice where given repeat doses (250 μg/ day) of purified pediocin PA-1 for three days, which resulted in up to 2-log reductions in L. monocytogenes in feces and translocation into the liver and spleen was slowed. Furthermore,

L. monocytogenes completely disappeared in the liver and spleen within six days of treatment.

Another class IIa bacteriocins that has been studied in vivo is piscicolin 126 from

Carnobacterium piscicola. Ingham et al. (435) found that after tail vein injection into mice piscicolin 126 retain activity against L. monocytogenes. Mice were infected intravenously with

L. monocytogenes followed by intravenous treatment with purified piscicolin 126, which significantly reduced L. monocytogenes in the liver and spleen reducing clinical signs of disease. Likewise, Rihakova et al. (436) demonstrated that the divercin RV41 bacteriocin produced by Carnobacterium divergens significantly reduced L. monocytogenes during invasive infection. The efficacy of structural variants with mutation in different amino acids was

124 also tested and while all retained activity against L. monocytogenes the level of potency varied, demonstrating that certain amino acids are required for activity.

Unlike traditional antibiotics bacteriocins are confronted with challenges related to their structure as peptides. The structure-function relationships and in vivo efficacy observations can provide insight to determine engineering methods to enhance stability and potency of bacteriocins, such as EntV. Some commonly used methods to increase stability are: (1) introduction of additional disulfide bridges (437, 438), (2) amino acid substitutions including incorporation of D-amino acids (439, 440) (3) encapsulation of bacteriocins in liposomes (441-

443). The class IIa bacteriocins have one or more disulfide bridges that are important for activity and target specificity (242). Fimland et al. (437) found that addition of a C-terminal disulfide bridge into sakacin P produced by , which only has an N-terminal disulfide bridge, broadened the target spectrum, increased potency and decreased temperature sensitivity. To reduce proteolytic cleavage of bacteriocins by proteases found in the gastrointestinal tract, O’Shea et al. (439) altered trypsin recognition sites in salivaricin P, a class IIb bacteriocin produced by Lactobacillus salivarius. Antimicrobial activity was retained with these alterations and salivaricin P variants were resistant to trypsin digestion. In the event that modification of the bacteriocin disrupts activity, encapsulation of the bacteriocin can be preserving. Were et al. (443) observed that encapsulation of nisin and lysozyme increased efficacy of nisin against L. monocytogenes by stabilizing the bacteriocin activity and structure.

To enhance the potency of bacteriocins the physiochemical properties that contribute to the mode of action must be considered. As demonstrated in Chapter 4, active EntV is amphipathic and has a net positive charge (~9.84). Proteolytic cleavage of the propeptide increased activity against C. albicans (Chapter 3). This corresponds with enhancement of the positive net charge that promotes the initial electrostatic interaction with the target cell.

Likewise, Kazazic et al. (444) demonstrated that addition of positively charged amino acids at specific residues enhanced activity of sakacin P by increasing cell binding.

125 There are different methods that have been used previously with success to develop engineered bacteriocins (433). Rational substitution of amino acids is one such method that depends on the structure-function relationship of the bacteriocins. Use of error-prone PCR to randomly generate mutants is another approach. However, greater modifications are usually required to improve bacteriocin activity, as bacteriocins have evolved to be as effective as possible (242, 445). An alternative approach that allows for major modification is creating chimeras of with different bacteriocins from the same class to create a DNA-shuffling library to create hybrids with increased activity and broadened spectrum targets (445).

Traditional antimicrobial therapies often have broader-spectrum of activity than bacteriocins, thus having a greater impact on the commensal microflora. Often keeping opportunistic pathogens at bay, the commensal microflora acts a barrier to infection and disruption of the commensal microflora with antimicrobial treatment is a growing concern. An advantage over traditional antimicrobials, bacteriocins often have specific microbial targets, and due to their composition are less likely to be toxic because they are easily broken down into nontoxic amino acids. Correspondingly, our cytotoxicity assays (Chapter 3) demonstrated that sEntV68 is not toxic to murine macrophages or Human cervical epithelial cells. Likewise,

Jasniewski et al. (446) found that two class IIa bacteriocins, Cbn BM1 and Cbn B2 produced by

Carnobacterium maltaromaticum, in combination or alone had no cytotoxic effects on human gastrointestinal cells (Caco-2) at concentration 100-folder than what was required for antimicrobial activity.

Future perspectives and directions

As mentioned previously, C. albicans biofilms are a growing concern in the clinical setting. Due to their complex structure, encasement in the ECM, and production of persister cells traditional antifungals are ineffective at eradicating C. albicans biofilms. In addition there are currently no biofilm-specific therapeutics used clinically further complicating treatment

126 strategies (39). Several chemicals have been identified that specifically target biofilm formation, while others target hyphal morphogenesis (92, 111, 447). These studies are insightful and show promise for the development of biofilm specific treatment strategies. Likewise, our findings demonstrate the possible use of a natural-based peptide for control and treatment of

C. albicans biofilms that has many advantages over the use of other treatment.

Here, I have described very complex and ecologically important interactions between E. faecalis and C. albicans. As described in Chapter 3, I have identified a bacteriocin, EntV that has therapeutic potential in different in vitro and in vivo models. Furthermore, I have characterized EntV activity with respect to post-translational processing that results in production of a highly active version of EntV. Additionally, I have identified genes that when deleted decrease C. albicans sensitivity to E. faecalis supernatant and sEntV68. Lastly, I examined the spectrum of target activity for sEntV68. Although, I have addressed many of the questions I had when starting this project, there are still many more intriguing questions left to answer.

Is sEntV68 efficacious in other mammalian infection models with C. albicans?

C. albicans can cause a variety of different infectious, from superficial infections (OPC, vaginal candidiasis) to systemic infections (endocarditis, candidemia, intraperitoneal infections).

Recall that during invasive infection the yeast-hyphal transition is necessary for maximum virulence. However, the dominance of one morphology over the other depends on the infected organ (421). Thus, it would be insightful to assess the efficacy of EntV an invasive infection model, such as the intravenous tail vein injection model. Considering that other bacteriocins are effective in invasive bacterial infections, EntV would be expected to decrease dissemination of

C. albicans. Nevertheless, EntV does not have fungicidal activity during planktonic growth so the effects of EntV may be moderate.

127 Given our observation in the C. elegans infection model, where commensal-like association with C. elegans was observed, I speculate that these species have synergism during commensal colonization. This could be confirmed using the murine colonization model

(422). Perez et al. (9) screened homozygous transcription factor deletion mutants in both the murine intestinal colonization and systemic infection models. Eight transcriptional regulators were identified that have roles in at least one of the models examined. 800 target genes where identified by genome-wide chromatin immunoprecipitation that are regulated by the eight transcriptional regulators. Interestingly, many pathogenesis associated genes were upregulated during commensal colonization suggesting that commensalism and pathogenicity are genetically intertwined

Addition of EntV to either of these models could change the gene expression profiles or the specific genes identified.

Is sEntV68 efficacious against other fungal pathogenes not closely related to C. albicans?

sEntV68 was active against biofilms formed by other C. glabrata, C. tropicalis, and C. parapsilosis. Although all of these species belong to the Candida genus, only C. tropicalis and

C. parapsilosisi are closely related to C. albicans and are part of the CUG clade that translate the CUG codon as serine instead of leucine (448). C. glabrata is more closely related to S. cerevisiae. The Candida genus originally included yeast species that are able to form hyphae or psuedohyphae and have no observed sexual spores. Although C. albicans remains the leading of cause of candidiasis, isolation of non-albicans Candida (NAC) species is increasing in patients. C. glabrata followed by C. parapsilosis and C. tropicalis were the most common

NAC species isolated worldwide in a 10 year retrospective analysis of NAC species infections

(449). Interestingly, EntV has inhibitory activity against all of these species, suggesting the mechanism of action requires a common feature found in these different species.

128 C. albicans, Cryptococcus neoformans and Aspergillus fumigatus are among the most prevalent fungal pathogens. Resistance to traditional antifungals is also an increasing problem in C. neoformans and A. fumigatus infections (450). Thus, development of novel treatment strategies that do not promote resistance is also critical to treating these infections. Since C. neoformans and A. fumigatus are important pathogens and not closely related to C. albicans, examining the efficacy of EntV would provide useful insight into the mechanism of action and could be used as a novel treatment strategy.

What is the role of the disulfide bridge and how is it formed?

Recall that EntV contains two cysteines that form a putative disulfide bond that is necessary for bactericidal activity and inhibitory activity against C. albicans (Chapter 4) (358).

Until recently disulfide bond formation in Gram-positive bacteria was not well understood and was not thought be important (381). However, recently thiol-disulfide oxidoreductases (DsbA- like proteins) have been characterized in Gram-positive actinobacteria (382). Furthermore, a homolog of dsbA has been identified in E. faecalis and disruption of dsbA by transposon mutagenesis resulted in reduced inhibitory activity suggesting that dsbA is required for producing active EntV68. However, the role of the disulfide bond formation by DsbA remains to be fully elucidated. I are currently examining the role of dsbA in producing active EntV68 and other possible roles in E. faecalis (Armand Brown, Ph.D.). Currently, there are only limited reports on the dsbA homolog (EF1088) in E. faecalis thus this work will not only add to our understand of EntV, but the physiology of E. faecalis.

What role if any does SprE play in post-translational processing of sEntV68?

I hypothesized that GelE and/or SprE proteolytically process EntV. However, I did not determine the role of SprE for EntV68 activity. To address this, we are currently working on examining the in vitro processing of EntV in deletion mutant of E. faecalis (ΔgelE, ΔsprE,

129 ΔgelEΔsprE) compared to WT. EntV will be purified from the supernatant and cell lysates of the

E. faecalis strains and subjected to SDS-PAGE and visualized by Istern blotting polyclonal antibodies. Our previous attempts to observe in vivo cleavage of EntV did not work because we was expressing EntV with a C-terminal Hexa-HIS tag expressed from the endogenous promoter in the mutant strains and WT E. faecalis. However, I was not able to visualize the

EntV-6XHIS by western blot with HIS antibodies. The EntV-6XHIS construct fully complemented the ΔentV mutant, suggesting EntV is being produced. This along with our in silico analysis of proteolytic cleavage by the GelE homolog, thermolysin, suggests that the

Hexa-HIS tag is being cleaved in vivo by GelE.

What is the sEntV68 mode of action against C. albicans?

As previously mentioned, I speculate that EntV interacts with the membrane of C. albicans at low concentration causing minimal perturbation of the membrane that in turn disrupts biofilm formation and hyphal morphogenesis. This hypothesis also corresponds with our mutant library screens, where deletion of genes that disrupt the cell wall components or increase filamentation are less sensitive to E. faecalis supernatant. Thus, it is plausible that disruption of the cell wall components masks or interferes with EntV targeting the membrane. If indeed EntV68 is causing membrane stress by leakage of ions then there should be a change in membrane potential and intracellular ATP, which can be measured and quantified using fluorescence assays and bioluminescence assays, respectively (382).

Furthermore, treatment of C. albicans with human AMPs LL-37, β-defensin-3 hβD-3, or

Hst-5 causes vacuolar expansion, which is thought to occur as a result of osmotic stress from membrane permeabilization and cytosolic ion leakage (280, 451). The vacuole can be visualized by staining with FM4-64 (Invitrogen), a red fluorescent lipophilic stain that intercalates into the membrane and then is transported to the vacuole, allowing assessment of

130 the physical characteristics of the vacuole (302, 452). This could determine if E. faecalis causes membrane perturbation resulting in an increase in vacuole size.

Are the other class IIIb bacteriocins active against C. albicans?

Examining the efficacy of the other class IIIb bacteriocins against C. albicans could provide further insight into the mechanism of action and would also suggest that the interactions observed between E. faecalis and C. albicans are more widespread, which has ecological implications. Furthermore, understanding the structure differences and possible activity differences could contribute to engineering possibilities of EntV68 (51, 453). As mentioned previously, the potency and stability can be improved by enhancing the physiochemical properties that contribute to the mode of action. If these bacteriocins are active against C. albicans the construction of chimeric bacteriocins is possible using different portion of the class IIIb bacteriocins to create hybrids that may have increased activity and broadened target spectrums (433). This could also provide insight into what portions of the peptides are necessary for activity increasing our ability to develop EntV-based treatment strategies.

How does C. albicans gene expression change in the presence of E. faecalis?

The EntV mechanism of action is one of the most important questions left to answer.

Changes in C. albicans gene expression in the presence of E. faecalis, E. faecalis supernatant, and EntV68 can be used to elucidate the mechanism of action. The most comprehensive way to address this question would be by RNA sequencing, which would tell us the global changes in gene expression. This assay can be done in C. albicans grown in the in vitro biofilm assay grown in YNBAS for 24 hours. Previous studies have observed gene expression changes in C. albicans grown as monoculture and polymicrobial biofilms with different bacteria and growth conditions (445). While the transcription factor mutant library screen gives some insight into gene regulation in the presence E. faecalis supernatant only a select number of genes can be 131 assessed, which only include non-essential genes and only a small portion of the genes expressed by C. albicans. RNA sequencing can also provide insight into gene expression in different infection models that have already been tested (nematode, macrophage, OPC) or new models to understand the effects of EntV during colonization and systemic infections. Not only will RNA sequencing provide insight into gene expression of C. albicans, but also gene expression of E. faecalis.

I also observed attenuation of E. faecalis virulence in the C. elegans infection model, where survival of the nematode was increased during co-infection with C. albicans and E. faecalis compared to nematodes infected with E. faecalis only. The E. faecalis mode of virulence is less striking than that of C. albicans, where hyphal cells invade through the body of the nematode. Nematodes infected with E. faecalis become impacted with the bacteria and are unable to expel them and eventually die (LD~10). Preliminary observation demonstrate that spent media from C. albicans-E. faecalis biofilms inhibits biofilm formation of E. faecalis

(Melissa Cruz). Furthermore, alteration of bacterial gene expression by C. albicans has been previously reported, such as what is seen in S. aureus and C. albicans biofilms (42, 454, 455).

This project has overall provided valuable insight into the growing knowledge of polymicrobial interactions. Moreover, I have demonstrated that a bacteriocin produced by E. faecalis has therapeutic potential against C. albicans in different in vitro and in vivo assays. The future directions of this study can lead to advancements in treatment of antifungal resistant C. albicans by exploiting bacterial mediated mechanisms to treat C. albicans infections.

132 Bibliography:

1. Sardi, J. C. O., L. Scorzoni, T. Bernardi, A. M. Fusco-Almeida, and M. J. S. Mendes Giannini. 2013. Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options. Journal of Medical Microbiology 62: 10–24.

2. Kojic, E. M., and R. O. Darouiche. 2004. Candida infections of medical devices. Clinical Microbiology Reviews 17: 255–267.

3. Jacobsen, I. D., D. Wilson, B. Wächtler, S. Brunke, J. R. Naglik, and B. Hube. 2012. Candida albicans dimorphism as a therapeutic target. Expert Review of Anti-infective Therapy 10: 85– 93.

4. Hube, B. 2004. From commensal to pathogen: stage- and tissue-specific gene expression of Candida albicans. Current Opinion in Microbiology 7: 336–341.

5. Sorgo, A. G., C. J. Heilmann, S. Brul, C. G. de Koster, and F. M. Klis. 2012. Beyond the wall: Candida albicanssecret(e)s to survive. FEMS Microbiol Lett 338: 10–17.

6. Mayer, F. L., D. Wilson, and B. Hube. 2014. Candida albicans pathogenicity mechanisms. Virulence 4: 119–128.

7. Duggan, S., I. Leonhardt, K. Hünniger, and O. Kurzai. 2015. Host response to Candida albicans bloodstream infection and sepsis. Virulence 6(4): 316–326.

8. Gow, N. A. R., F. L. van de Veerdonk, A. J. P. Brown, and M. G. Netea. 2011. Candida albicans morphogenesis and host defence: discriminating invasion from colonization. Nature Review Microbiology 10: 112–122.

9. Pérez, J. C., C. A. Kumamoto, and A. D. Johnson. 2013. Candida albicans Commensalism and Pathogenicity Are Intertwined Traits Directed by a Tightly Knit Transcriptional Regulatory Circuit. 11: e1001510.

10. Lionakis, M. S. 2014. New insights into innate immune control of systemic candidiasis. Med Mycol 52: 555–564.

11. Naglik, J. R., S. J. Challacombe, and B. Hube. 2003. Candida albicans Secreted Aspartyl Proteinases in Virulence and Pathogenesis. Microbiology and Molecular Biology Reviews 67: 400–428.

12. Gow, N. A. R., A. J. P. Brown, and F. C. Odds. 2002. Fungal morphogenesis and host invasion. Current Opinion in Microbiology 5: 366–371.

13. Gow, N. A., and B. Hube. 2012. Importance of the Candida albicans cell wall during commensalism and infection. Current Opinion in Microbiology 15: 406–412.

14. Calderone, R. A., and W. A. Fonzi. 2001. Virulence factors of Candida albicans. Trends in Microbiology 9: 327–335.

15. Pappas, P. G., J. H. Rex, J. D. Sobel, S. G. Filler, W. E. Dismukes, T. J. Walsh, J. E. Edwards, Infectious Diseases Society of America. 2004. Guidelines for treatment of candidiasis. Clin. Infect. Dis. 38: 161–189.

133 16. Pappas, P. G., C. A. Kauffman, D. R. Andes, K. A. Marr, L. Ostrosky-Zeichner, A. C. Reboli, M. G. Schuster, J. A. Vazquez, T. J. Walsh, T. E. Zaoutis, and J. D. Sobel. 2016. Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America. Clin. Infect. Dis. 62: e1–50.

17. Wenzel, R. P. 1995. Nosocomial candidemia: risk factors and attributable mortality. Clinical Infectious Diseases 20: 1531–1534.

18. Kullberg, B. J., and A. M. L. Oude Lashof. 2002. Epidemiology of opportunistic invasive mycoses. Eur J Med Res 7: 183–191.

19. Weig, M. 1998. Clinical aspects and pathogenesis of Candida infection. Trends in Microbiology 6: 468–470.

20. Nett, J. E., H. Sanchez, M. T. Cain, K. M. Ross, and D. R. Andes. 2011. Interface of Candida albicans biofilm matrix-associated drug resistance and cell wall integrity regulation. Eukaryotic Cell 10: 1660–1669.

21. Kumamoto, C. A. 2005. A contact-activated kinase signals Candida albicans invasive growth and biofilm development. Proc Natl Acad Sci USA 102: 5576–5581.

22. Chandra, J., D. M. Kuhn, P. K. Mukherjee, L. L. Hoyer, T. McCormick, and M. A. Ghannoum. 2001. Biofilm Formation by the Fungal Pathogen Candida albicans: Development, Architecture, and Drug Resistance. Journal of Bacteriology 183: 5385–5394.

23. Fox, E. P., and C. J. Nobile. 2012. A sticky situation: untangling the transcriptional network controlling biofilm development in Candida albicans. Transcription 3: 315–322.

24. Ramage, G., E. Mowat, B. Jones, C. Williams, and J. Lopez-Ribot. 2009. Our Current Understanding of Fungal Biofilms. Critical Reviews in Microbiology 35: 340–355.

25. Ramage, G., S. P. Saville, D. P. Thomas, and J. L. Lopez-Ribot. 2005. Candida biofilms: an update. Eukaryotic Cell 4: 633–638.

26. Dominic, R. M., S. Shenoy, and S. Baliga. 2007. Candida biofilms in medical devices: evolving trends. Kathmandu Univ Med J (KUMJ) 5: 431–436.

27. Pfaller, M. A., and D. J. Diekema. 2007. Epidemiology of invasive candidiasis: a persistent public health problem. Clinical Microbiology Reviews. 20(1): 133-163.

28. Wisplinghoff, H., T. Bischoff, S. M. Tallent, H. Seifert, R. P. Wenzel, and M. B. Edmond. 2004. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin. Infect. Dis. 39: 309–317.

29. Cauda, R. 2009. Candidaemia in Patients with an Inserted Medical Device. Drugs 69: 33– 38.

30. Donlan, R. M., and J. W. Costerton. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clinical Microbiology Reviews 15: 167–193.

31. Seddiki, S. M. L., Z. Boucherit-Otmani, K. Boucherit, S. Badsi-Amir, M. Taleb, and D. Kunkel. 2013. Assessment of the types of catheter infectivity caused by Candida species and their biofilm formation. First study in an intensive care unit in Algeria. International Journal of

134 General Medicine 6: 1–7.

32. National Nosocomial Infections Surveillance (NNIS). 2001. National Nosocomial Infections Surveillance (NNIS) System Report, Data Summary from January 1992-June 2001, issued August 2001. American Journal of Infection Control 29: 404–421.

33. Andes, D. R., N. Safdar, J. W. Baddley, G. Playford, A. C. Reboli, J. H. Rex, J. D. Sobel, P. G. Pappas, and B. J. Kullberg. 2012. Impact of Treatment Strategy on Outcomes in Patients with Candidemia and Other Forms of Invasive Candidiasis: A Patient-Level Quantitative Review of Randomized Trials. Clinical Infectious Diseases 54: 1110–1122.

34. Cornely, O. A., M. Bassetti, T. Calandra, J. Garbino, B. J. Kullberg, O. Lortholary, W. Meersseman, M. Akova, M. C. Arendrup, S. Arikan Akdagli, J. Bille, E. Castagnola, M. Cuenca Estrella, J. P. Donnelly, A. H. Groll, R. Herbrecht, W. W. Hope, H. E. Jensen, C. Lass Flörl, G. Petrikkos, M. D. Richardson, E. Roilides, P. E. Verweij, C. Viscoli, and A. J. Ullmann. 2012. ESCMID guideline for the diagnosis and management of Candida diseases 2012: non‐ neutropenic adult patients. Clinical Microbiology and Infection 18: 19–37.

35. Lepak, A., and D. Andes. 2011. Fungal sepsis: optimizing antifungal therapy in the critical care setting. Crit Care Clin 27: 123–147.

36. Lortholary, O., G. Petrikkos, M. Akova, M. C. Arendrup, S. Arikan Akdagli, M. Bassetti, J. Bille, T. Calandra, E. Castagnola, O. A. Cornely, M. Cuenca Estrella, J. P. Donnelly, J. Garbino, A. H. Groll, R. Herbrecht, W. W. Hope, H. E. Jensen, B. J. Kullberg, C. Lass Flörl, W. Meersseman, M. D. Richardson, E. Roilides, P. E. Verweij, C. Viscoli, and A. J. Ullmann. 2012. ESCMID guideline for the diagnosis and management of Candida diseases 2012: patients with HIV infection or AIDS. Clinical Microbiology and Infection 18: 68–77.

37. Mermel, L. A., M. Allon, E. Bouza, D. E. Craven, P. Flynn, N. P. O’Grady, I. I. Raad, B. J. A. Rijnders, R. J. Sherertz, and D. K. Warren. 2009. Clinical practice guidelines for the diagnosis and management of intravascular catheter-related infection: 2009 Update by the Infectious Diseases Society of America. Clin. Infect. Dis. 49: 1–45.

38. Viudes, A., J. Pem x000E1 n, E. Cant x000F3 n, P. x000DA beda, J. L x000F3 pez-Ribot, and M. Gobernado. 2002. Candidemia at a Tertiary-Care Hospital: Epidemiology, Treatment, Clinical Outcome and Risk Factors for Death. European Journal of Clinical Microbiology & Infectious Diseases 21: 767–774.

39. Nobile, C. J., and A. D. Johnson. 2015. Candida albicans Biofilms and Human Disease. Annu. Rev. Microbiol. 69: 71–92.

40. Finkel, J. S., and A. P. Mitchell. 2010. Genetic control of Candida albicans biofilm development. Nature review Microbiology 9: 109–118.

41. Finkel, J. S., W. Xu, D. Huang, E. M. Hill, J. V. Desai, C. A. Woolford, J. E. Nett, H. Taff, C. T. Norice, D. R. Andes, F. Lanni, and A. P. Mitchell. 2012. Portrait of Candida albicans adherence regulators. PLoS Pathog 8: e1002525.

42. Nobile, C. J., E. P. Fox, J. E. Nett, T. R. Sorrells, Q. M. Mitrovich, A. D. Hernday, B. B. Tuch, D. R. Andes, and A. D. Johnson. 2012. A Recently Evolved Transcriptional Network Controls Biofilm Development in Candida albicans. Cell 148: 126–138.

43. Gow, N. A. R., F. L. van de Veerdonk, A. J. P. Brown, and M. G. Netea. 2012. Candida

135 albicans morphogenesis and host defence: discriminating invasion from colonization. Nat Rev Micro 10: 112–122.

44. Nobile, C. J., and A. P. Mitchell. 2005. Regulation of Cell-Surface Genes and Biofilm Formation by the C. albicans Transcription Factor Bcr1p. Current Biology 15: 1150–1155.

45. Richard, M. L., C. J. Nobile, V. M. Bruno, and A. P. Mitchell. 2005. Candida albicans Biofilm-Defective Mutants. Eukaryotic Cell 4: 1493–1502.

46. Brown, A. J., and N. A. Gow. 1999. Regulatory networks controlling Candida albicans morphogenesis. Trends in Microbiology 7: 333–338.

47. Berman, J., and P. E. Sudbery. 2002. Candida albicans: A molecular revolution built on lessons from budding yeast. Nat Rev Genet 3: 918–932.

48. Biswas, S., P. Van Dijck, and A. Datta. 2007. Environmental Sensing and Signal Transduction Pathways Regulating Morphopathogenic Determinants of Candida albicans. Microbiology and Molecular Biology Reviews 71: 348–376.

49. Song, Y., N. Sahni, K. J. Daniels, K. L. Lu, T. Srikantha, G. Huang, A. M. Garnaas, and D. R. Soll. 2011. Alternative mating type configurations (a/α versus a/a or α/α) of Candida albicans result in alternative biofilms regulated by different pathways. PLoS Biology 9: e1001117.

50. Dunkel, N., K. Biswas, E. Hiller, K. Fellenberg, S. V. Satheesh, S. Rupp, and J. Morschhauser. 2014. Control of morphogenesis, protease secretion and gene expression in Candida albicans by the preferred nitrogen source ammonium. Microbiology 160: 1599–1608.

51. Maidan, M. M., J. M. Thevelein, and P. Van Dijck. 2005. Carbon source induced yeast-to- hypha transition in Candida albicans is dependent on the presence of amino acids and on the G-protein-coupled receptor Gpr1. Biochem. Soc. Trans. 33: 291–293.

52. Bockmühl, D. P., and J. F. Ernst. 2001. A potential phosphorylation site for an A-type kinase in the Efg1 regulator protein contributes to hyphal morphogenesis of Candida albicans. Genetics 157: 1523–1530.

53. Noffz, C. S., V. Liedschulte, K. Lengeler, and J. F. Ernst. 2008. Functional Mapping of the Candida albicans Efg1 Regulator. Eukaryotic Cell 7: 881–893.

54. Banerjee, M., P. Uppuluri, X. R. Zhao, P. L. Carlisle, G. Vipulanandan, C. C. Villar, J. L. Lopez-Ribot, and D. Kadosh. 2013. Expression of UME6, a key regulator of Candida albicans hyphal development, enhances biofilm formation via Hgc1- and Sun41-dependent mechanisms. Eukaryotic Cell 12: 224–232.

55. Fox, E. P., C. K. Bui, J. E. Nett, N. Hartooni, M. C. Mui, D. R. Andes, C. J. Nobile, and A. D. Johnson. 2015. An expanded regulatory network temporally controls Candida albicans biofilm formation. Molecular Microbiology 96: 1226–1239.

56. Mitchell, A. P., and K. Lagree. 2017. Fungal Biofilms: Inside Out. Microbiology Spectrum 5(2): 1–14.

57. Kadosh, D., and A. D. Johnson. 2005. Induction of the Candida albicans filamentous growth program by relief of transcriptional repression: a genome-wide analysis. Mol. Biol. Cell 16: 2903–2912.

136 58. Banerjee, M., D. S. Thompson, A. Lazzell, P. L. Carlisle, C. Pierce, C. Monteagudo, J. L. Lopez-Ribot, and D. Kadosh. 2008. UME6, a novel filament-specific regulator of Candida albicans hyphal extension and virulence. Mol. Biol. Cell 19: 1354–1365.

59. Zeidler, U., T. Lettner, C. Lassnig, M. Müller, R. Lajko, H. Hintner, M. Breitenbach, and A. Bito. 2009. UME6is a crucial downstream target of other transcriptional regulators of true hyphal development in Candida albicans. FEMS Yeast Research 9: 126–142.

60. Chaffin, W. L. 2008. Candida albicans Cell Wall Proteins. Microbiology and Molecular Biology Reviews 72: 495–544.

61. Nobile, C. J., J. E. Nett, D. R. Andes, and A. P. Mitchell. 2006. Function of Candida albicans Adhesin Hwp1 in Biofilm Formation. Eukaryotic Cell 5: 1604–1610.

62. Nobile, C. J., H. A. Schneider, J. E. Nett, D. C. Sheppard, S. G. Filler, D. R. Andes, and A. P. Mitchell. 2008. Complementary adhesin function in C. albicans biofilm formation. Current Biology 18: 1017–1024.

63. Nobile, C. J., D. R. Andes, J. E. Nett, F. J. Smith, F. Yue, Q.-T. Phan, J. E. Edwards, S. G. Filler, and A. P. Mitchell. 2006. Critical Role of Bcr1-Dependent Adhesins in C. albicans Biofilm Formation In Vitro and In Vivo. PLoS Pathog 2: e63.

64. Li, F., M. J. Svarovsky, A. J. Karlsson, and J. P. Wagner. 2007. Eap1p, an adhesin that mediates Candida albicans biofilm formation in vitro and in vivo. Eukaryotic Cell 6(6): 931-939.

65. Theberge, S., A. Semlali, A. Alamri, K. P. Leung, and M. Rouabhia. 2013. C. albicans growth, transition, biofilm formation, and gene expression modulation by antimicrobial decapeptide KSL-W. BMC Microbiology 13: 1–1.

66. Monniot, C., A. Boisramé, G. Da Costa, and M. Chauvel. 2013. Rbt1 protein domains analysis in Candida albicans brings insights into hyphal surface modifications and Rbt1 potential role during adhesion and biofilm formation. PLoS One 8(12): e82395.

67. Ene, I. V., and R. J. Bennett. 2009. Hwp1 and related adhesins contribute to both mating and biofilm formation in Candida albicans. Eukaryotic Cell 8: 1909–1913.

68. Granger, B. L., M. L. Flenniken, D. A. Davis, A. P. Mitchell, and J. E. Cutler. 2005. Yeast wall protein 1 of Candida albicans. Microbiology 151: 1631–1644.

69. Granger, B. L. 2012. Insight into the antiadhesive effect of yeast wall protein 1 of Candida albicans. Eukaryotic Cell 11: 795–805.

70. Cabral, V., S. Znaidi, L. A. Walker, H. Martin-Yken, E. Dague, M. Legrand, K. Lee, M. Chauvel, A. Firon, T. Rossignol, M. L. Richard, C. A. Munro, S. Bachellier-Bassi, and C. d’Enfert. 2014. Targeted Changes of the Cell Wall Proteome Influence Candida albicans Ability to Form Single- and Multi-strain Biofilms. PLoS Pathog 10: e1004542–21.

71. Ramage, G., B. Coco, L. Sherry, J. Bagg, and D. F. Lappin. 2012. In Vitro Candida albicans Biofilm Induced Proteinase Activity and SAP8 Expression Correlates with In Vivo Denture Stomatitis Severity. Mycopathologia 174: 11–19.

72. Rajendran, R., D. P. Robertson, P. J. Hodge, D. F. Lappin, and G. Ramage. 2010. Hydrolytic Production is Associated with Candida Albicans Biofilm Formation from

137 Patients with Type 1 Diabetes. Mycopathologia 170: 229–235.

73. Joo, M. Y., J. H. Shin, H.-C. Jang, E. S. Song, S. J. Kee, M. G. Shin, S. P. Suh, and D. W. Ryang. 2013. Expression of SAP5 and SAP9 in Candida albicans biofilms: comparison of bloodstream isolates with isolates from other sources. Med Mycol 51: 892–896.

74. Winter, M. B., E. C. Salcedo, M. B. Lohse, N. Hartooni, M. Gulati, H. Sanchez, J. Takagi, B. Hube, D. R. Andes, A. D. Johnson, C. S. Craik, and C. J. Nobile. 2016. Global Identification of Biofilm-Specific Proteolysis in Candida albicans. mBio 7(5): e01514-16.

75. Staib, P., M. Kretschmar, T. Nichterlein, H. Hof, and J. Morschhauser. 2000. Differential activation of a Candida albicans virulence gene family during infection. Proc Natl Acad Sci USA 97: 6102–6107.

76. Nailis, H., S. Kucharíková, M. Ricicová, P. Van Dijck, D. Deforce, H. Nelis, and T. Coenye. 2010. Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression. BMC Microbiology 10: 114.

77. Denning, D. W., and W. W. Hope. 2010. Therapy for fungal diseases: opportunities and priorities. Trends in Microbiology 18: 195–204.

78. Brown, G. D., D. W. Denning, N. A. Gow, S. M. Levitz, M. G. Netea, and T. C. White. 2012. Hidden killers: human fungal infections. Sci Transl Med 4(165): 165rv13.

79. Moudgal, V., and J. Sobel. 2010. Antifungals to treat Candida albicans. Expert Opin Pharmacother 11: 2037–2048.

80. Shapiro, R. S., N. Robbins, and L. E. Cowen. 2011. Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease. Microbiology and Molecular Biology Reviews 75: 213–267.

81. Gómez-López, A., O. Zaragoza, J. L. Rodríguez-Tudela, and M. Cuenca-Estrella. 2008. Pharmacotherapy of yeast infections. Expert Opin Pharmacother 9: 2801–2816.

82. Ghannoum, M. A., and L. B. Rice. 1999. Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clinical Microbiology Reviews 12: 501–517.

83. Canuto, M. M., and F. G. Rodero. 2002. Antifungal drug resistance to azoles and polyenes. The Lancet Infectious Diseases 2: 550–563.

84. Deray, G. 2002. Amphotericin B nephrotoxicity. Journal of Antimicrobial Chemotherapy. 49 (1); 37-41.

85. Sheehan, D. J., C. A. Hitchcock, and C. M. Sibley. 1999. Current and emerging azole antifungal agents. Clinical Microbiology Reviews 12: 40–79.

86. Denning, D. W. 2003. Echinocandin antifungal drugs. The Lancet 362: 1142–1151.

87. Holt, S. L., and R. H. Drew. 2011. Echinocandins: Addressing outstanding questions surrounding treatment of invasive fungal infections. American Journal of Health-System Pharmacy 68(13): 1207-1220.

138 88. LaFleur, M. D., C. A. Kumamoto, and K. Lewis. 2006. Candida albicans biofilms produce antifungal-tolerant persister cells. Antimicrobial Agents and Chemotherapy 50: 3839–3846.

89. Tobudic, S., C. Kratzer, A. Lassnigg, and E. Presterl. 2011. Antifungal susceptibility of Candida albicans in biofilms. Mycoses 55: 199–204.

90. Paramonova, E., B. P. Krom, H. C. van der Mei, H. J. Busscher, and P. K. Sharma. 2009. Hyphal content determines the compression strength of Candida albicans biofilms. Microbiology 155: 1997–2003.

91. Gauwerky, K., C. Borelli, and H. C. Korting. 2009. Targeting virulence: A new paradigm for antifungals. 14: 214–222.

92. Pierce, C. G., and J. L. Lopez-Ribot. 2013. Candidiasis drug discovery and development: new approaches targeting virulence for discovering and identifying new drugs. Expert Opinion on Drug Discovery 8: 1117–1126.

93. BAILLIE, G. S., and L. J. DOUGLAS. 2000. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. Journal of Antimicrobial Chemotherapy 46: 397–403.

94. Al-Fattani, M. A. 2006. Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance. Journal of Medical Microbiology 55: 999–1008.

95. Nobile, C. J., J. E. Nett, A. D. Hernday, O. R. Homann, J.-S. Deneault, A. Nantel, D. R. Andes, A. D. Johnson, and A. P. Mitchell. 2009. Biofilm Matrix Regulation by Candida albicans Zap1. 7: e1000133–15.

96. Lattif, A. A., P. K. Mukherjee, J. Chandra, M. R. Roth, R. Welti, M. Rouabhia, and M. A. Ghannoum. 2011. Lipidomics of Candida albicans biofilms reveals phase-dependent production of phospholipid molecular classes and role for lipid rafts in biofilm formation. Microbiology 157: 3232–3242.

97. Mitchell, K. F., R. Zarnowski, and D. R. Andes. 2016. Fungal Super Glue: The Biofilm Matrix and Its Composition, Assembly, and Functions. PLoS Pathog 12: e1005828–6.

98. Zarnowski, R., W. M. Westler, G. A. Lacmbouh, J. M. Marita, J. R. Bothe, J. Bernhardt, A. Lounes-Hadj Sahraoui, J. Fontaine, H. Sanchez, R. D. Hatfield, J. M. Ntambi, J. E. Nett, A. P. Mitchell, and D. R. Andes. 2014. Novel Entries in a Fungal Biofilm Matrix Encyclopedia. mBio 5: e01333–14–e01333–14.

99. Nett, J. E., H. Sanchez, M. T. Cain, and D. R. Andes. 2010. Genetic Basis of Candida Biofilm Resistance Due to Drug‐Sequestering Matrix Glucan. J Infect Dis 202: 171–175.

100. Martins, M., M. Henriques, J. L. Lopez-Ribot, and R. Oliveira. 2011. Addition of DNase improves the in vitro activity of antifungal drugs against Candida albicans biofilms. Mycoses 55: 80–85.

101. Martins, M., P. Uppuluri, D. P. Thomas, I. A. Cleary, M. Henriques, J. L. Lopez-Ribot, and R. Oliveira. 2009. Presence of Extracellular DNA in the Candida albicans Biofilm Matrix and its Contribution to Biofilms. Mycopathologia 169: 323–331.

102. Ramage, G. 2002. Investigation of multidrug efflux pumps in relation to fluconazole

139 resistance in Candida albicans biofilms. Journal of Antimicrobial Chemotherapy 49: 973–980.

103. Cowen, L. E. 2008. The evolution of fungal drug resistance: modulating the trajectory from genotype to phenotype. Nat Rev Micro 6: 187–198.

104. Mateus, C., S. A. Crow, and D. G. Ahearn. 2004. Adherence of Candida albicans to silicone induces immediate enhanced tolerance to fluconazole. Antimicrobial Agents and Chemotherapy 48: 3358–3366.

105. Nett, J. E., A. J. Lepak, K. Marchillo, and D. R. Andes. 2009. Time Course Global Gene Expression Analysis of an In Vivo CandidaBiofilm. J Infect Dis. 200: 307–313.

106. Khot, P. D., P. A. Suci, R. L. Miller, R. D. Nelson, and B. J. Tyler. 2006. A small subpopulation of blastospores in candida albicans biofilms exhibit resistance to amphotericin B associated with differential regulation of ergosterol and beta-1,6-glucan pathway genes. Antimicrobial Agents and Chemotherapy 50: 3708–3716.

107. Brown, A. J. P., S. Budge, D. Kaloriti, A. Tillmann, M. D. Jacobsen, Z. Yin, I. V. Ene, I. Bohovych, D. Sandai, S. Kastora, J. Potrykus, E. R. Ballou, D. S. Childers, S. Shahana, and M. D. Leach. 2013. Stress adaptation in a pathogenic fungus. Journal of Experimental Biology 217: 144–155.

108. Peleg, A. Y., D. A. Hogan, and E. Mylonakis. 2010. Medically important bacterial–fungal interactions. Nature Review Microbiology 8: 340–349.

109. Hermann, C., J. Hermann, U. Munzel, and R. Ruchel. 1999. Bacterial flora accompanying Candida in clinical specimens. Mycoses 42: 619–627.

110. Harriott, M. M., and M. C. Noverr. 2011. Importance of Candida-bacterial polymicrobial biofilms in disease. Trends in Microbiology 19: 557–563.

111. Shareck, J., and P. Belhumeur. 2011. Modulation of Morphogenesis in Candida albicans by Various Small Molecules. Eukaryotic Cell 10: 1004–1012.

112. Liu, Y., and S. G. Filler. 2011. Candida albicans Als3, a Multifunctional Adhesin and Invasin. Eukaryotic Cell 10: 168–173.

113. Peters, B. M., E. S. Ovchinnikova, B. P. Krom, L. M. Schlecht, H. Zhou, L. L. Hoyer, H. J. Busscher, H. C. van der Mei, M. A. Jabra-Rizk, and M. E. Shirtliff. 2012. Staphylococcus aureus adherence to Candida albicans hyphae is mediated by the hyphal adhesin Als3p. Microbiology 158: 2975–2986.

114. Mallick, E. M., and R. J. Bennett. 2013. Sensing of the microbial neighborhood by Candida albicans. PLoS Pathog 9: e1003661.

115. Cruz, M. R., C. E. Graham, B. C. Gagliano, M. C. Lorenz, and D. A. Garsin. 2013. Enterococcus faecalis inhibits hyphal morphogenesis and virulence of Candida albicans. Infection and Immunity 81: 189–200.

116. Xu, H., T. Sobue, A. Thompson, Z. Xie, K. Poon, A. Ricker, J. Cervantes, P. I. Diaz, and A. Dongari-Bagtzoglou. 2013. Streptococcal co-infection augments Candidapathogenicity by amplifying the mucosal inflammatory response. Cell Microbiol 16: 214–231.

140 117. Azizi, El, M. A., S. E. Starks, and N. Khardori. 2004. Interactions of Candida albicans with other Candida spp. and bacteria in the biofilms. Journal of Applied Microbiology 96: 1067– 1073.

118. McAlester, G., F. O'Gara, and J. P. Morrissey. 2008. Signal-mediated interactions between Pseudomonas aeruginosa and Candida albicans. Journal of Medical Microbiology 57: 563–569.

119. Leclair, L. W., and D. A. Hogan. 2010. Mixed bacterial-fungal infections in the CF respiratory tract. Med Mycol 48 Suppl 1: S125–32.

120. Fourie, R., R. Ells, C. W. Swart, O. M. Sebolai, J. Albertyn, and C. H. Pohl. 2016. Candida albicans and Pseudomonas aeruginosa Interaction, with Focus on the Role of Eicosanoids. Front Physiol 7: 64.

121. Williamson, D. R., M. Albert, M. M. Perreault, M.-S. Delisle, J. Muscedere, C. Rotstein, X. Jiang, and D. K. Heyland. 2011. The relationship between Candida species cultured from the respiratory tract and systemic inflammation in critically ill patients with ventilator-associated pneumonia. Can J Anaesth 58: 275–284.

122. Roux, D., S. Gaudry, D. Dreyfuss, J. El-Benna, N. de Prost, E. Denamur, G. Saumon, and J.-D. Ricard. 2009. Candida albicans impairs macrophage function and facilitates Pseudomonas aeruginosa pneumonia in rat. Crit. Care Med. 37: 1062–1067.

123. Neely, A. N., E. J. Law, and I. A. Holder. 1986. Increased susceptibility to lethal Candida infections in burned mice preinfected with Pseudomonas aeruginosa or pretreated with proteolytic . Infection and Immunity 52: 200–204.

124. Azoulay, E., J.-F. Timsit, M. Tafflet, A. de Lassence, M. Darmon, J.-R. Zahar, C. Adrie, M. Garrouste-Orgeas, Y. Cohen, B. Mourvillier, B. Schlemmer, Outcomerea Study Group. 2006. Candida colonization of the respiratory tract and subsequent pseudomonas ventilator- associated pneumonia. Chest 129: 110–117.

125. Gibson, J., A. Sood, and D. A. Hogan. 2009. Pseudomonas aeruginosa-Candida albicans interactions: localization and fungal toxicity of a phenazine derivative. Applied and Environmental Microbiology 75: 504–513.

126. Hogan, D. A., and R. Kolter. 2002. Pseudomonas-Candida Interactions: An Ecological Role for Virulence Factors. Science 296: 2229–2232.

127. Morales, D. K., and D. A. Hogan. 2010. Candida albicans interactions with bacteria in the context of human health and disease. PLoS Pathog 6: e1000886.

128. Hogan, D. A., Å. Vik, and R. Kolter. 2004. A Pseudomonas aeruginosa quorum-sensing molecule influences Candida albicans morphology. Molecular Microbiology 54: 1212–1223.

129. Davis-Hanna, A., A. E. Piispanen, L. I. Stateva, and D. A. Hogan. 2007. Farnesol and dodecanol effects on the Candida albicans Ras1-cAMP signalling pathway and the regulation of morphogenesis. Molecular Microbiology 67: 47–62.

130. Kebaara, B. W., M. L. Langford, D. H. M. L. P. Navarathna, R. Dumitru, K. W. Nickerson, and A. L. Atkin. 2008. Candida albicans Tup1 is involved in farnesol-mediated inhibition of filamentous-growth induction. Eukaryotic Cell 7: 980–987.

141 131. Hall, R. A., K. J. Turner, J. Chaloupka, F. Cottier, L. De Sordi, D. Sanglard, L. R. Levin, J. Buck, and F. A. Muhlschlegel. 2011. The Quorum-Sensing Molecules Farnesol/Homoserine Lactone and Dodecanol Operate via Distinct Modes of Action in Candida albicans. Eukaryotic Cell 10: 1034–1042.

132. Lindsay, A. K., A. Deveau, A. E. Piispanen, and D. A. Hogan. 2012. Farnesol and cyclic AMP signaling effects on the hypha-to-yeast transition in Candida albicans. Eukaryotic Cell 11: 1219–1225.

133. Piispanen, A. E., N. Grahl, J. M. Hollomon, and D. A. Hogan. 2013. Regulated proteolysis of Candida albicans Ras1 is involved in morphogenesis and quorum sensing regulation. Molecular Microbiology 89: 166–178.

134. Boon, C., Y. Deng, L. H. Wang, Y. He, and J. L. Xu. 2008. A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition. The ISME. 2(1): 27-36.

135. Wang, L. H., Y. He, Y. Gao, J. E. Wu, Y. H. Dong, C. He, S. X. Wang, L. X. Weng, J. L. Xu, L. Tay, R. X. Fang, and L. H. Zhang. 2004. A bacterial cell–cell communication signal with cross‐kingdom structural analogues. Molecular Microbiology 51: 903–912.

136. Cugini, C., D. K. Morales, and D. A. Hogan. 2010. Candida albicans-produced farnesol stimulates Pseudomonas quinolone signal production in LasR-defective Pseudomonas aeruginosa strains. Microbiology. 156: 3096–3107.

137. Lemos, J. A., R. G. Quivey Jr, and H. Koo. 2013. Streptococcus mutans: a new Gram- positive paradigm? Microbiology. 159(3): 436-445.

138. Falsetta, M. L., M. I. Klein, J. A. Lemos, B. B. Silva, S. Agidi, K. K. Scott-Anne, and H. Koo. 2012. Novel antibiofilm chemotherapy targets exopolysaccharide synthesis and stress tolerance in Streptococcus mutans to modulate virulence expression in vivo. Antimicrobial Agents and Chemotherapy 56: 6201–6211.

139. Raja, M., A. Hannan, and K. Ali. 2010. Association of oral candidal carriage with dental caries in children. Caries Res. 44: 272–276.

140. Ribeiro, D. G., A. C. Pavarina, L. N. Dovigo, A. L. Machado, E. T. Giampaolo, and C. E. Vergani. 2012. Prevalence of Candida spp. associated with bacteria species on complete dentures. Gerodontology 29: 203–208.

141. Jarosz, L. M., D. M. Deng, H. C. van der Mei, W. Crielaard, and B. P. Krom. 2009. Streptococcus mutans Competence-Stimulating Peptide Inhibits Candida albicans Hypha Formation. Eukaryotic Cell 8: 1658–1664.

142. Vílchez, R., A. Lemme, B. Ballhausen, V. Thiel, S. Schulz, R. Jansen, H. Sztajer, and I. Wagner-Döbler. 2010. Streptococcus mutans Inhibits Candida albicans Hyphal Formation by the Fatty Acid Signaling Molecule trans-2-Decenoic Acid (SDSF). Chem. Eur. J. of Chem. Bio. 11: 1552–1562.

143. Desai, K., L. Mashburn-Warren, M. J. Federle, and D. A. Morrison. 2012. Development of Competence for Genetic Transformation of Streptococcus mutans in a Chemically Defined Medium. Journal of Bacteriology 194: 3774–3780.

142 144. Sztajer, H., S. P. Szafranski, J. U. R. Tomasch, M. Reck, M. Nimtz, M. Rohde, and I. Wagner-Dobler. 2014. Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans. The ISME Journal. 8(11): 2256–2271.

145. Archer, N. K., M. J. Mazaitis, J. W. Costerton, J. G. Leid, M. E. Powers, and M. E. Shirtliff. 2014. Staphylococcus aureusbiofilms. Virulence 2: 445–459.

146. Hoyer, L. L., C. B. Green, S.-H. Oh, and X. Zhao. 2008. Discovering the secrets of the Candida albicans agglutinin-like sequence (ALS) gene family – a sticky pursuit. Med Mycol 46: 1–15.

147. Silverman, R. J., A. H. Nobbs, M. M. Vickerman, M. E. Barbour, and H. F. Jenkinson. 2010. Interaction of Candida albicans Cell Wall Als3 Protein with Streptococcus gordonii SspB Adhesin Promotes Development of Mixed-Species Communities. Infection and Immunity 78: 4644–4652.

148. Holmes, A. R., R. McNab, and H. F. Jenkinson. 1996. Candida albicans binding to the oral bacterium Streptococcus gordonii involves multiple adhesin-receptor interactions. Infection and Immunity 64: 4680–4685.

149. Bamford, C. V., A. d'Mello, A. H. Nobbs, L. C. Dutton, M. M. Vickerman, and H. F. Jenkinson. 2009. Streptococcus gordonii Modulates Candida albicans Biofilm Formation through Intergeneric Communication. Infection and Immunity 77: 3696–3704.

150. Tang, Y.-W., and C. W. Stratton. 2010. Staphylococcus aureus: An old pathogen with new weapons. Clin. Lab. Med. 30: 179–208.

151. 1983. Enhancement by Candida albicans of Staphylococcus aureus, Serratia marcescens, and Streptococcus faecalis in the establishment of infection in mice. 39: 193–197.

152. Carlson, E. 1983. Effect of strain of Staphylococcus aureus on synergism with Candida albicans resulting in mouse mortality and morbidity. Infection and Immunity 42: 285–292.

153. Carlson, E., and G. Johnson. 1985. Protection by Candida albicans of Staphylococcus aureus in the establishment of dual infection in mice. Infection and Immunity 50: 655–659.

154. Peters, B. M., and M. C. Noverr. 2013. Candida albicans-Staphylococcus aureus polymicrobial peritonitis modulates host innate immunity. Infection and Immunity 81: 2178– 2189.

155. Inglis, D. O., and G. Sherlock. 2013. Ras Signaling Gets Fine-Tuned: Regulation of Multiple Pathogenic Traits of Candida albicans. Eukaryotic Cell 12: 1316–1325.

156. Gullberg, R. M., S. R. Homann, and J. P. Phair. 1989. Enterococcal Bacteremia: Analysis of 75 Episodes. Rev Infect Dis 11: 74–85.

157. Klotz, S. A., B. S. Chasin, B. Powell, N. K. Gaur, and P. N. Lipke. 2007. Polymicrobial bloodstream infections involving Candida species: analysis of patients and review of the literature. Diagnostic Microbiology and Infectious Disease 59: 401–406.

158. Sutter, D., D. Stagliano, L. Braun, F. Williams, J. Arnold, M. Ottolini, and J. Epstein. 2008. Polymicrobial bloodstream infection in pediatric patients: risk factors, microbiology, and antimicrobial management. Pediatr. Infect. Dis. J. 27: 400–405.

143 159. Gomes, B. P. F. A., F. Montagner, V. B. Berber, A. A. Zaia, C. C. R. Ferraz, J. F. A. de Almeida, and F. J. Souza-Filho. 2009. Antimicrobial action of intracanal medicaments on the external root surface. Journal of Dentistry 37: 76–81.

160. Peciuliene, V., and A. H. Reynaud. 2001. Isolation of yeasts and enteric bacteria in root‐ filled teeth with chronic apical periodontitis. International Endodontic Journal. 34(6); 429-434.

161. Vidana, R., Å. Sullivan, H. Billström, M. Ahlquist, and B. Lund. 2011. Enterococcus faecalis infection in root canals – host‐derived or exogenous source? Letters in Applied Microbiology 52: 109–115.

162. Kovac, J., and D. Kovac. 2011. Effect of irrigating solutions in endodontic therapy. Bratisl Lek Listy 112: 410–415.

163. da Silva, F. P., and M. C. C. Machado. 2012. Antimicrobial peptides: Clinical relevance and therapeutic implications. Peptides 36: 308–314.

164. Miranda, T. T., C. R. Vianna, L. Rodrigues, A. S. Monteiro, C. A. Rosa, and A. Corrêa. 2009. Diversity and frequency of yeasts from the dorsum of the tongue and necrotic root canals associated with primary apical periodontitis. Int Endod J 42: 839–844.

165. Richards, D., J. K. Davies, and D. Figdor. 2010. Starvation survival and recovery in serum of Candida albicans compared with Enterococcus faecalis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 110: 125–130.

166. Egan, M. W., D. A. Spratt, Y. L. Ng, J. M. Lam, D. R. Moles, and K. Gulabivala. 2002. Prevalence of yeasts in saliva and root canals of teeth associated with apical periodontitis. Int Endod J 35: 321–329.

167. Zehnder, M., and B. Guggenheim. 2009. The mysterious appearance of enterococci in filled root canals. Int Endod J 42: 277–287.

168. Ferrari, P. H. P., S. Cai, and A. C. Bombana. 2005. Effect of endodontic procedures on enterococci, enteric bacteria and yeasts in primary endodontic infections. Int Endod J 38: 372– 380.

169. Kiani, D., E. L. Quinn, K. H. Burch, T. Madhavan, L. D. Saravolatz, and T. R. Neblett. 1979. The increasing importance of polymicrobial bacteremia. JAMA 242: 1044–1047.

170. Cooper, G. S., D. S. Havlir, D. M. Shlaes, and R. A. Salata. 1990. Polymicrobial Bacteremia in the Late 1980s: Predictors of Outcome and Review of the Literature. Medicine 69: 114.

171. Krupova, I., E. Kaiserova, A. Foltinova, G. Kovacicova, M. Kiskova, A. Krchnakova, A. Kunova, J. Trupl, D. West, and V. Krcmery. 1998. Bacteremia and fungemia in pediatric versus adult cancer patients after chemotherapy: comparison of etiology, risk factors and outcome. J Chemother 10: 236–242.

172. González-Barca, E., A. Fernández-Sevilla, J. Carratalá, A. Salar, J. Peris, A. Grañena, and F. Gudiol. 1999. Prognostic Factors Influencing Mortality in Cancer Patients with Neutropenia and Bacteremia. European Journal of Clinical Microbiology & Infectious Diseases 18: 539–544.

144 173. Vallés, J., C. León, and F. Alvarez-Lerma. 1997. Nosocomial bacteremia in critically ill patients: a multicenter study evaluating epidemiology and prognosis. Spanish Collaborative Group for Infections in Intensive Care Units of Sociedad Espanola de Medicina Intensiva y Unidades Coronarias (SEMIUC). Clinical Infectious Diseases 24: 387–395.

174. Reuben, A. G., D. M. Musher, R. J. Hamill, and I. Broucke. 1989. Polymicrobial bacteremia: clinical and microbiologic patterns. Rev Infect Dis 11: 161–183.

175. Vallés, J., J. Rello, A. Ochagavía, J. Garnacho, and M. A. Alcala. 2003. Community- acquired bloodstream infection in critically ill adult patients: impact of shock and inappropriate antibiotic therapy on survival. Chest. 123(5): 1615-1624.

176. Sartelli, M. 2010. A focus on intra-abdominal infections. World J Emerg Surg 5: 9–20.

177. Menichetti, F., and G. Sganga. 2013. Definition and Classification of Intra-abdominal Infections. Journal of Chemotherapy 21: 3–4.

178. Wacha, H., T. Hau, R. Dittmer, and C. Ohmann. 1999. Risk factors associated with intraabdominal infections: a prospective multicenter study. Peritonitis Study Group. Langenbecks Arch Surg 384: 24–32.

179. Schoeffel, U., E. Jacobs, G. Ruf, F. Mierswa, B. S. von, and E. H. Farthmann. 1995. Intraperitoneal micro-organisms and the severity of peritonitis. Eur J Surg 161: 501–508.

180. Mulier, S., F. Penninckx, C. Verwaest, L. Filez, R. Aerts, S. Fieuws, and P. Lauwers. 2003. Factors affecting mortality in generalized postoperative peritonitis: multivariate analysis in 96 patients. World J Surg 27: 379–384.

181. Pieracci, F. M., and P. S. Barie. 2007. Management of severe sepsis of abdominal origin. Scand J Surg 96: 184–196.

182. Vergidis, P., R. K. Shields, S. Y. Park, B. N. Wildfeuer, R. L. Simmons, and M. H. Nguyen. 2016. Intra-Abdominal Candidiasis: The Importance of Early Source Control and Antifungal Treatment. PLoS ONE 11: e0153247.

183. Bassetti, M., E. Righi, F. Ansaldi, M. Merelli, C. Scarparo, M. Antonelli, J. Garnacho- Montero, A. Diaz-Martin, I. Palacios-Garcia, R. Luzzati, C. Rosin, L. Lagunes, J. Rello, B. Almirante, P. G. Scotton, G. Baldin, G. Dimopoulos, M. Nucci, P. Munoz, A. Vena, E. Bouza, V. de Egea, A. L. Colombo, C. Tascini, F. Menichetti, E. Tagliaferri, P. Brugnaro, M. Sanguinetti, A. Mesini, G. Sganga, C. Viscoli, and M. Tumbarello. 2015. A multicenter multinational study of abdominal candidiasis: epidemiology, outcomes and predictors of mortality. Intensive Care Med 41: 1601–1610.

184. Dupont, H., C. Paugam-Burtz, and C. Muller-Serieys. 2002. Predictive factors of mortality due to polymicrobial peritonitis with Candida isolation in peritoneal fluid in critically ill patients. Archives of Surgery. 137(12): 13411346.

185. Rex, J. H. 2006. Antifungal prophylaxis in the intensive care unit: who should get it? Crit. Care Med. 34: 1286–1287.

186. Agudelo Higuita, N. I., and M. M. Huycke. 2014. Enterococcal Disease, Epidemiology, and Implications for Treatment In: Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Boston: Massachusetts Eye and Ear Infirmary. 65-99.

145 187. Arias, C. A., and B. E. Murray. 2012. The rise of the Enterococcus: beyond vancomycin resistance. Nature Publishing Group 10: 266–278.

188. Krogstad, D. J., and A. R. Pargwette. 1980. Defective killing of enterococci: a common property of antimicrobial agents acting on the cell wall. Antimicrobial Agents and Chemotherapy 17: 965–968.

189. Arias, C. A., and B. E. Murray. 2014. Emergence and management of drug-resistant enterococcal infections. Expert Review of Anti-infective Therapy 6: 637–655.

190. Boyce, J. M., S. M. Opal, J. W. Chow, M. J. Zervos, G. Potter-Bynoe, C. B. Sherman, R. L. Romulo, S. Fortna, and A. A. Medeiros. 1994. Outbreak of multidrug-resistant Enterococcus faecium with transferable vanB class vancomycin resistance. J. Clin. Microbiol. 32: 1148–1153.

191. Huycke, M. M., C. A. Spiegel, and M. S. Gilmore. 1991. Bacteremia caused by hemolytic, high-level gentamicin-resistant Enterococcus faecalis. Antimicrobial Agents and Chemotherapy 35: 1626–1634.

192. Kramer, A., I. Schwebke, and G. Kampf. 2006. How long do nosocomial pathogens persist on inanimate surfaces? A systematic review. BMC Infect Dis 6: 318–8.

193. Garsin, D. A., K. L. Frank, J. Silanpää, F. M. Ausubel, and A. Hartke. 2014. Pathogenesis and models of enterococcal infection. In: Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Boston: Massachusetts Eye and Ear Infirmary. 185-257.

194. Clewell, D. B., K. E. Weaver, G. M. Dunny, and T. M. Coque. 2014. Extrachromosomal and mobile elements in enterococci: transmission, maintenance, and epidemiology. In: Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Boston: Massachusetts Eye and Ear Infirmary. 309-420.

195. Palmer, K. L., V. N. Kos, and M. S. Gilmore. 2010. Horizontal gene transfer and the genomics of enterococcal antibiotic resistance. Current Opinion in Microbiology 13: 632–639.

196. de Regt, M. J. A., W. van Schaik, M. van Luit-Asbroek, H. A. T. Dekker, E. van Duijkeren, C. J. M. Koning, M. J. M. Bonten, and R. J. L. Willems. 2012. Hospital and Community Ampicillin-Resistant Enterococcus faecium Are Evolutionarily Closely Linked but Have Diversified through Niche Adaptation. PLoS ONE 7: e30319.

197. Qin, X., K. V. Singh, G. M. Weinstock, and B. E. Murray. 2001. Characterization of fsr, a Regulator Controlling Expression of Gelatinase and Serine Protease in Enterococcus faecalis OG1RF. Journal of Bacteriology 183: 3372–3382.

198. Burns, E. H., A. M. Marciel, and J. M. Musser. 1996. Activation of a 66-kilodalton human endothelial cell matrix metalloprotease by Streptococcus pyogenes extracellular . Infection and Immunity 64: 4744–4750.

199. Lantz, M. S., R. D. Allen, L. W. Duck, J. L. Blume, L. M. Switalski, and M. Hook. 1991. Identification of Porphyromonas gingivalis components that mediate its interactions with fibronectin. Journal of Bacteriology 173: 4263–4270.

200. Novick, R. P., and E. Geisinger. 2008. Quorum Sensing in Staphylococci. Annu. Rev. Genet. 42: 541–564.

146 201. Qin, X., K. V. Singh, G. M. Weinstock, and B. E. Murray. 2000. Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence. Infection and Immunity 68: 2579–2586.

202. Nakayama, J., Y. Cao, T. Horii, S. Sakuda, A. D. L. Akkermans, W. M. de Vos, and H. Nagasawa. 2001. Gelatinase biosynthesis-activating pheromone: a peptide lactone that mediates a quorum sensing in Enterococcus faecalis. Molecular Microbiology 41: 145–154.

203. Del Papa, M. F., L. E. Hancock, V. C. Thomas, and M. Perego. 2007. Full Activation of Enterococcus faecalis Gelatinase by a C-Terminal Proteolytic Cleavage. Journal of Bacteriology 189: 8835–8843.

204. Su, Y. A., M. C. Sulavik, P. He, K. K. Makinen, P. L. Makinen, S. Fiedler, R. Wirth, and D. B. Clewell. 1991. Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp. liquefaciens. Infection and Immunity 59: 415–420.

205. O'Donohue, M. J., B. P. Roques, and A. Beaumont. 1994. Cloning and expression in Bacillus subtilis of the npr gene from Bacillus thermoproteolyticus Rokko coding for the thermostable metalloprotease thermolysin. Biochem. J. 300(2): 599-603.

206. Sabat, A., K. Kosowska, K. Poulsen, A. Kasprowicz, A. Sekowska, B. van Den Burg, J. Travis, and J. Potempa. 2000. Two allelic forms of the aureolysin gene (aur) within Staphylococcus aureus. Infection and Immunity 68: 973–976.

207. Maccallum, W. G., and T. W. Hastings. 1899. A Case of Acute Endocarditis caused by Micrococcus Zymogenes (nov. spec.), with a description of the microorgansim. J. Exp. Med. 4: 521–534.

208. Makinen, P. L., D. B. Clewell, F. An, and K. K. Makinen. 1989. Purification and substrate specificity of a strongly hydrophobic extracellular (“gelatinase”) from Streptococcus faecalis (strain 0G1-10). Journal of Biological Chemistry 264: 3325–3334.

209. Schmidtchen, A., I.-M. Frick, E. Andersson, H. Tapper, and L. Björck. 2002. Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL-37. Molecular Microbiology 46: 157–168.

210. Schmidtchen, A., I.-M. Frick, and L. Björck. 2001. Dermatan sulphate is released by proteinases of common pathogenic bacteria and inactivates antibacterial α‐defensin. Molecular Microbiology 39: 708–713.

211. Zeng, J., F. Teng, and B. E. Murray. 2005. Gelatinase is important for translocation of Enterococcus faecalis across polarized human enterocyte-like T84 cells. Infection and Immunity 73: 1606–1612.

212. Ohara-Nemoto, Y., Y. Ikeda, M. Kobayashi, and M. Sasaki. 2002. Characterization and molecular cloning of a glutamyl endopeptidase from Staphylococcus epidermidis. Microbial ….

213. Yoshikawa, K., H. Tsuzuki, T. Fujiwara, E. Nakamura, H. Iwamoto, K. Matsumoto, M. Shin, N. Yoshida, and H. Teraoka. 1992. Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600. Biochim. Biophys. Acta 1121: 221–228.

214. Garsin, D. A., C. D. Sifri, E. Mylonakis, X. Qin, K. V. Singh, B. E. Murray, S. B.

147 Calderwood, and F. M. Ausubel. 2001. A simple model host for identifying Gram-positive virulence factors. Proc Natl Acad Sci USA 98: 10892–10897.

215. Engelbert, M., E. Mylonakis, F. M. Ausubel, S. B. Calderwood, and M. S. Gilmore. 2004. Contribution of Gelatinase, Serine Protease, and fsr to the Pathogenesis of Enterococcus faecalis Endophthalmitis. Infection and Immunity 72: 3628–3633.

216. Sifri, C. D., E. Mylonakis, K. V. Singh, X. Qin, D. A. Garsin, B. E. Murray, F. M. Ausubel, and S. B. Calderwood. 2002. Virulence Effect of Enterococcus faecalis Protease Genes and the Quorum-Sensing Locus fsr in Caenorhabditis elegans and Mice. Infection and Immunity 70: 5647–5650.

217. Qin, X., K. V. Singh, Y. Xu, G. M. Weinstock, and B. E. Murray. 1998. Effect of disruption of a gene encoding an of Enterococcus faecalis OG1RF. Antimicrobial Agents and Chemotherapy 42: 2883–2888.

218. Kawalec, M., J. Potempa, J. L. Moon, J. Travis, and B. E. Murray. 2004. Molecular Diversity of a Putative Virulence Factor: Purification and Characterization of Isoforms of an Extracellular Serine Glutamyl Endopeptidase of Enterococcus faecalis with Different Enzymatic Activities. Journal of Bacteriology 187: 266–275.

219. Rice, K., R. Peralta, D. Bast, J. de Azavedo, and M. J. McGavin. 2001. Description of staphylococcus serine protease (ssp) operon in Staphylococcus aureus and nonpolar inactivation of sspA-encoded serine protease. Infection and Immunity 69: 159–169.

220. Rouf, S. M. A., Y. Ohara-Nemoto, Y. Shimoyama, S. Kimura, T. Ono, and T. K. Nemoto. 2012. Propeptide processing and proteolytic activity of proenzymes of the staphylococcal and enterococcal GluV8-family protease. Indian J. Biochem. Biophys. 49: 421–427.

221. Thomas, V. C., L. R. Thurlow, D. Boyle, and L. E. Hancock. 2008. Regulation of Autolysis- Dependent Extracellular DNA Release by Enterococcus faecalis Extracellular Proteases Influences Biofilm Development. Journal of Bacteriology 190: 5690–5698.

222. Mundt, J. O. 1963. Occurrence of enterococci in animals in a wild environment. Appl Microbiol 11: 136–140.

223. Martin, J. D., and J. O. Mundt. 1972. Enterococci in insects. Appl Microbiol 24: 575–580.

224. Gilmore, M. S., F. Lebreton, and W. van Schaik. 2013. Genomic transition of enterococci from gut commensals to leading causes of multidrug-resistant hospital infection in the antibiotic era. Current Opinion in Microbiology 16: 10–16.

225. Hayashi, H. 2005. Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism. Journal of Medical Microbiology 54: 1093–1101.

226. Carvalho, M. D. G. S. 2006. Enterococcus caccae sp. nov., isolated from human stools. International Journal Of Systematic And Evolutionary Microbiology 56: 1505–1508.

227. Layton, B. A., S. P. Walters, L. H. Lam, and A. B. Boehm. 2010. Enterococcus species distribution among human and animal hosts using multiplex PCR. Journal of Applied Microbiology 60: 55–9.

148 228. Christoffersen, T. E., H. Jensen, C. R. Kleiveland, G. Dørum, M. Jacobsen, and T. Lea. 2012. In vitro comparison of commensal, probiotic and pathogenic strains of Enterococcus faecalis. Br J Nutr 108: 2043–2053.

229. Franz, C. M. A. P., M. Huch, H. Abriouel, W. Holzapfel, and A. Gálvez. 2011. Enterococci as probiotics and their implications in food safety. International Journal of 151: 125–140.

230. Berg, R. D. 1996. The indigenous gastrointestinal microflora. Trends in Microbiology 4: 430–435.

231. Donskey, C. J. 2004. The role of the intestinal tract as a reservoir and source for transmission of nosocomial pathogens. Clin. Infect. Dis. 39: 219–226.

232. Gomes, B. C., F. deMelo, and E. DeMartinis. 2010. Dualistic aspects of Enterococcus spp. in foods. Appl Microbial Biotechnol. 25(5): 668-675.

233. Foulquié Moreno, M. R., P. Sarantinopoulos, E. Tsakalidou, and L. De Vuyst. 2006. The role and application of enterococci in food and health. International Journal of Food Microbiology 106: 1–24.

234. Khan, H., S. Flint, and P.-L. Yu. 2010. International Journal of Food Microbiology. International Journal of Food Microbiology 141: 1–10.

235. Lebreton, F., R. J. L. Willems, and M. S. Gilmore. 2014. Enterococcus Diversity, Origins in Nature, and Gut Colonization. In: Enterococci: From Commensals to Leading Causes of Drug Resistant Infection. Boston: Massachusetts Eye and Ear Infirmary. 5-63.

236. Franz, C. M., W. H. Holzapfel, and M. E. Stiles. 1999. Enterococci at the crossroads of food safety? International Journal of Food Microbiology 47: 1–24.

237. Franz, C. 2003. Enterococci in foods—a conundrum for food safety. International Journal of Food Microbiology 88: 105–122.

238. Wunderlich, P. F., L. Braun, I. Fumagalli, V. D'Apuzzo, F. Heim, M. Karly, R. Lodi, G. Politta, F. Vonbank, and L. Zeltner. 1989. Double-blind report on the efficacy of lactic acid- producing Enterococcus SF68 in the prevention of antibiotic-associated diarrhoea and in the treatment of acute diarrhoea. J. Int. Med. Res. 17: 333–338.

239. Canganella, F., M. Gasbarri, and S. Massa. 1996. A microbiological investigation on probiotic preparations used for animal feeding. Microbiological research.

240. Fan, Y.-J., S.-J. Chen, Y.-C. Yu, J.-M. Si, and B. Liu. 2006. A probiotic treatment containing Lactobacillus, Bifidobacterium and Enterococcus improves IBS symptoms in an open label trial. J. Zhejiang Univ. - Sci. B 7: 987–991.

241. Agerholm-Larsen, L., A. Raben, N. Haulrik, A. S. Hansen, M. Manders, and A. Astrup. 2000. Effect of 8 week intake of probiotic milk products on risk factors for cardiovascular diseases. Eur J Clin Nutr 54: 288–297.

242. Heng, N., P. A. Wescombe, J. P. Burton, R. W. Jack, and J. R. Tagg. 2007. The diversity of bacteriocins in Gram-positive bacteria, (M. A. Riley, ed). Springer, Heidelberg, Germany; :45–83.

149 243. Heng, N. C. K., G. A. Burtenshaw, R. W. Jack, and J. R. Tagg. 2004. Sequence analysis of pDN571, a plasmid encoding novel bacteriocin production in M-type 57 Streptococcus pyogenes. Plasmid 52: 225–229.

244. Cotter, P. D., C. Hill, and R. P. Ross. 2005. Bacteriocins: developing innate immunity for food. Nat Rev Micro 3: 777–788.

245. Dundar, H., D. A. Brede, S. L. La Rosa, A. O. El-Gendy, D. B. Diep, and I. F. Nes. 2015. The fsr Quorum-Sensing System and Cognate Gelatinase Orchestrate the Expression and Processing of Proprotein EF_1097 into the Mature Antimicrobial Peptide Enterocin O16. Journal of Bacteriology 197: 2112–2121.

246. Schnell, N., K. D. Entian, U. Schneider, F. Götz, and H. Zähner. 1988. Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings. Nature. 333(6170): 276-278.

247. Chatterjee, C., M. Paul, L. Xie, and W. A. van der Donk. 2005. Biosynthesis and Mode of Action of Lantibiotics. Chem. Rev. 105: 633–684.

248. Eijsink, V., L. Axelsson, and D. B. Diep. 2002. Production of class II bacteriocins by lactic acid bacteria; an example of biological warfare and communication. Antonie Van Leeuwenhoek. 81: 639-654.

249. Miescher, S., M. P. Stierli, M. Teuber, and L. Meile. 2000. Propionicin SM1, a Bacteriocin from Propionibacterium jensenii DF1: Isolation and Characterization of the Protein and its Gene. Systematic and Applied Microbiology 23: 174–184.

250. Maqueda, M., A. Gálvez, M. M. Bueno, M. J. Sanchez-Barrena, C. Gonzalez, A. Albert, M. Rico, and E. Valdivia. 2004. Peptide AS-48: prototype of a new class of cyclic bacteriocins. Current Protein and Peptide Science. 5(5): 399-416.

251. Opsata, M., I. F. Nes, and H. Holo. 2010. Class IIa bacteriocin resistance in Enterococcus faecalis V583: The mannose PTS operon mediates global transcriptional responses. BMC Microbiology 10: 224.

252. Haas, W., B. D. Shepard, and M. S. Gilmore. 2002. Two-component regulator of Enterococcus faecalis cytolysin responds to quorum-sensing autoinduction. Nature 415: 84–87.

253. Van Tyne, D., M. Martin, and M. Gilmore. 2013. Structure, Function, and Biology of the Enterococcus faecalis Cytolysin. 5: 895–911.

254. Huycke, M. M., and M. S. Gilmore. 1995. Frequency of aggregation substance and cytolysin genes among enterococcal endocarditis isolates. Plasmid 34: 152–156.

255. Coque, T. M., J. E. Patterson, J. M. Steckelberg, and B. E. Murray. 1995. Incidence of hemolysin, gelatinase, and aggregation substance among enterococci isolated from patients with endocarditis and other infections and from feces of hospitalized and community-based persons. J INFECT DIS 171: 1223–1229.

256. Cebrián, R., A. Baños, E. Valdivia, R. Pérez-Pulido, M. Martínez-Bueno, and M. Maqueda. 2012. Characterization of functional, safety, and probiotic properties of Enterococcus faecalis UGRA10, a new AS-48-producer strain. Food Microbiology 30: 59–67.

150 257. Martínez-Bueno, M., M. Maqueda, A. Gálvez, B. Samyn, J. Van Beeumen, J. Coyette, and E. Valdivia. 1994. Determination of the gene sequence and the molecular structure of the enterococcal peptide antibiotic AS-48. Journal of Bacteriology 176: 6334–6339.

258. Grande Burgos, M. J., R. P. Pulido, M. del Carmen López Aguayo, A. Gálvez, and R. Lucas. 2014. The Cyclic Antibacterial Peptide Enterocin AS-48: Isolation, Mode of Action, and Possible Food Applications. IJMS 15: 22706–22727.

259. Riley, M. A., and J. E. Wertz. 2002. Bacteriocins: evolution, ecology, and application. Annu. Rev. Microbiol. 56: 117–137.

260. Birri, D. J., D. A. Brede, T. Forberg, H. Holo, and I. F. Nes. 2010. Molecular and genetic characterization of a novel bacteriocin locus in Enterococcus avium isolates from infants. Applied and Environmental Microbiology 76: 483–492.

261. López-Cuellar, M. R., A. I. Rodriguez-Hernandez, and N. Chavarria-Hernandez. 2016. LAB bacteriocin applications in the last decade. Biotechnology Biotechnological Equipment 30: 1039–1050.

262. De Kwaadsteniet, M., K. T. Doeschate, and L. M. T. Dicks. 2009. Nisin F in the treatment of respiratory tract infections caused by Staphylococcus aureus. Letters in Applied Microbiology 48: 65–70.

263. Kaur, B., P. P. Balgir, B. Mittu, B. Kumar, and N. Garg. 2013. Biomedical applications of fermenticin HV6b isolated from Lactobacillus fermentum HV6b MTCC10770. BioMed Research International 2013: 168438–8.

264. Lubelski, J., R. Rink, R. Khusainov, G. N. Moll, and O. P. Kuipers. 2007. Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin. Cell. Mol. Life Sci. 65: 455–476.

265. de Arauz, L. J., A. F. Jozala, P. G. Mazzola, and T. C. V. Penna. 2009. Nisin biotechnological production and application: a review. Trends in Food Science & Technology 20: 146–154.

266. Smith, L., and J. D. Hillman. 2008. Therapeutic potential of type A (I) lantibiotics, a group of cationic peptide antibiotics. Current Opinion in Microbiology 11: 401–408.

267. Breukink, E., I. Wiedemann, C. van Kraaij, O. P. Kuipers, H. G. Sahl, and B. de Kruijff. 1999. Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic. Science 286: 2361–2364.

268. Wiedemann, I., E. Breukink, C. van Kraaij, O. P. Kuipers, G. Bierbaum, B. de Kruijff, and H.-G. Sahl. 2001. Specific Binding of Nisin to the Precursor Lipid II Combines Pore Formation and Inhibition of Cell Wall Biosynthesis for Potent Antibiotic Activity. Journal of Biological Chemistry 276: 1772–1779.

269. Cleveland, J., T. J. Montville, I. F. Nes, and M. L. Chikindas. 2001. Bacteriocins: safe, natural antimicrobials for food preservation. International Journal of Food Microbiology 71: 1– 20.

270. Shin, J. M., J. W. Gwak, P. Kamarajan, J. C. Fenno, A. H. Rickard, and Y. L. Kapila. 2016. Biomedical applications of nisin. Journal of Applied Microbiology 120: 1449–1465.

151 271. Mulders, J. W., I. J. Boerrigter, H. S. Rollema, R. J. Siezen, and W. M. de Vos. 1991. Identification and characterization of the lantibiotic nisin Z, a natural nisin variant. Eur. J. Biochem. 201: 581–584.

272. de Vos, W. M., J. W. Mulders, R. J. Siezen, J. Hugenholtz, and O. P. Kuipers. 1993. Properties of Nisin Z and Distribution of. Applied and Environmental Microbiology 59: 213–218.

273. Yuan, J., Z.-Z. Zhang, X.-Z. Chen, W. Yang, and L.-D. Huan. 2004. Site-directed mutagenesis of the hinge region of nisinZ and properties of nisinZ mutants. Appl. Microbiol. Biotechnol. 64: 806–815.

274. Le Lay, C., B. Akerey, I. Fliss, M. Subirade, and M. Rouabhia. 2008. Nisin Z inhibits the growth of Candida albicans and its transition from blastospore to hyphal form. Journal of Applied Microbiology 105: 1630–1639.

275. Akerey, B., C. Le Lay, I. Fliss, M. Subirade, and M. Rouabhia. 2009. In vitro efficacy of nisin Z against Candida albicans adhesion and transition following contact with normal human gingival cells. Journal of Applied Microbiology 107: 1298–1307.

276. Diep, D. B., L. S. Håvarstein, and I. F. Nes. 1996. Characterization of the locus responsible for the bacteriocin production in Lactobacillus plantarum C11. Journal of Bacteriology 178: 4472–4483.

277. Sharma, A., and S. Srivastava. 2014. Anti-Candida activity of two-peptide bacteriocins, plantaricins (Pln E/F and J/K) and their mode of action. Fungal Biology 118: 264–275.

278. Diep, D. B., R. Myhre, O. Johnsborg, A. Aakra, and I. F. Nes. 2003. Inducible bacteriocin production in Lactobacillus is regulated by differential expression of the pln operons and by two antagonizing response regulators, the activity of which is enhanced upon phosphorylation. Molecular Microbiology 47: 483–494.

279. Anderssen, E. L., D. B. Diep, I. F. Nes, V. G. Eijsink, and J. Nissen-Meyer. 1998. Antagonistic activity of Lactobacillus plantarum C11: two new two-peptide bacteriocins, plantaricins EF and JK, and the induction factor plantaricin A. Applied and Environmental Microbiology 64: 2269–2272.

280. De Smet, K., and R. Contreras. 2005. Human Antimicrobial Peptides: Defensins, Cathelicidins and Histatins. Biotechnol Lett 27: 1337–1347.

281. Edgerton, M., S. E. Koshlukova, T. E. Lo, B. G. Chrzan, R. M. Straubinger, and P. A. Raj. 1998. Candidacidal activity of salivary histatins. Identification of a histatin 5-binding protein on Candida albicans. Journal of Biological Chemistry 273: 20438–20447.

282. Helmerhorst, E. J., W. van't Hof, P. Breeuwer, E. C. I. Veerman, T. Abee, R. F. Troxler, A. V. N. Amerongen, and F. G. Oppenheim. 2001. Characterization of Histatin 5 with Respect to Amphipathicity, Hydrophobicity, and Effects on Cell and Mitochondrial Membrane Integrity Excludes a Candidacidal Mechanism of Pore Formation. Journal of Biological Chemistry 276: 5643–5649.

283. Puri, S., and M. Edgerton. 2014. How Does It Kill?: Understanding the Candidacidal Mechanism of Salivary Histatin 5. Eukaryotic Cell 13: 958–964.

284. Jang, W. S., J. S. Bajwa, J. N. Sun, and M. Edgerton. 2010. Salivary histatin 5

152 internalization by translocation, but not endocytosis, is required for fungicidal activity in Candida albicans. Molecular Microbiology 77: 354–370.

285. Li, X. S., M. S. Reddy, D. Baev, and M. Edgerton. 2003. Candida albicansSsa1/2p Is the Cell Envelope Binding Protein for Human Salivary Histatin 5. Journal of Biological Chemistry 278: 28553–28561.

286. Sun, J. N., W. Li, W. S. Jang, N. Nayyar, M. D. Sutton, and M. Edgerton. 2008. Uptake of the antifungal cationic peptide Histatin 5 by Candida albicansSsa2p requires binding to non- conventional sites within the ATPase domain. Molecular Microbiology 70: 1246–1260.

287. Kumar, R., S. Chadha, D. Saraswat, J. S. Bajwa, R. A. Li, H. R. Conti, and M. Edgerton. 2011. Histatin 5 uptake by Candida albicans utilizes polyamine transporters Dur3 and Dur31 proteins. J. Biol. Chem. 286: 43748–43758.

288. Baev, D., X. S. Li, J. Dong, P. Keng, and M. Edgerton. 2002. Human Salivary Histatin 5 Causes Disordered Volume Regulation and Cell Cycle Arrest in Candida albicans. Infection and Immunity 70: 4777–4784.

289. Gyurko, C., U. Lendenmann, R. F. Troxler, and F. G. Oppenheim. 2000. Candida albicans Mutants Deficient in Respiration Are Resistant to the Small Cationic Salivary Antimicrobial Peptide Histatin 5. Antimicrobial Agents and Chemotherapy 44: 348–354.

290. Gudmundsson, G. H., B. Agerberth, J. Odeberg, T. Bergman, B. Olsson, and R. Salcedo. 1996. The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. Eur. J. Biochem. 238: 325–332.

291. Agerberth, B., J. Charo, J. Werr, B. Olsson, F. Idali, L. Lindbom, R. Kiessling, H. Jörnvall, H. Wigzell, and G. H. Gudmundsson. 2000. The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations. Blood 96: 3086–3093.

292. Nijnik, A., and R. Hancock. 2009. The roles of cathelicidin LL-37 in immune defences and novel clinical applications. Current opinion in hematology. 16: 4147.

293. Tsai, P. W., C.-Y. Yang, H. T. Chang, and C. Y. Lan. 2011. Human Antimicrobial Peptide LL-37 Inhibits Adhesion of Candida albicans by Interacting with Yeast Cell-Wall Carbohydrates. PLoS ONE 6: e17755.

294. Hertog, den, A. L., J. van Marle, H. A. van Veen, W. van't Hof, J. G. M. Bolscher, E. C. I. Veerman, and A. V. Nieuw Amerongen. 2005. Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane. Biochem. J. 388: 689–695.

295. Chang, H. T., P. W. Tsai, H. H. Huang, Y. S. Liu, T. S. Chien, and C. Y. Lan. 2012. LL37 and hBD-3 elevate the β-1,3-exoglucanase activity of Candida albicansXog1p, resulting in reduced fungal adhesion to plastic. Biochem. J. 441: 963–970.

296. Hancock, R. E. W., and H.-G. Sahl. 2006. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24: 1551–1557.

297. Lehrer, R. I. 2004. Primate defensins. Nat Rev Micro 2: 727–738.

153 298. Edgerton, M., S. E. Koshlukova, M. W. Araujo, R. C. Patel, J. Dong, and J. A. Bruenn. 2000. Salivary histatin 5 and human neutrophil defensin 1 kill Candida albicans via shared pathways. Antimicrobial Agents and Chemotherapy 44: 3310–3316.

299. Brogden, K. A. 2005. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Micro 3: 238–250.

300. Sang, Y., and F. Blecha. 2008. Antimicrobial peptides and bacteriocins: alternatives to traditional antibiotics. Anim. Health. Res. Rev. 9: 227–235.

301. Tsai, P. W., Y.-L. Cheng, W.-P. Hsieh, and C. Y. Lan. 2014. Responses of Candida albicans to the human antimicrobial peptide LL-37. J Microbiol. 52: 581–589.

302. Ordonez, S. R., I. H. Amarullah, R. W. Wubbolts, E. J. A. Veldhuizen, and H. P. Haagsman. 2014. Fungicidal Mechanisms of Cathelicidins LL-37 and CATH-2 Revealed by Live-Cell Imaging. Antimicrobial Agents and Chemotherapy 58: 2240–2248.

303. Graham, C. E., M. R. Cruz, D. A. Garsin, and M. C. Lorenz. 2017. Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans. Proc. Natl. Acad. Sci. U.S.A. 114: 4507–4512.

304. Del Papa, M. F., and M. Perego. 2008. Ethanolamine activates a sensor histidine kinase regulating its utilization in Enterococcus faecalis. Journal of Bacteriology 190: 7147–7156.

305. Sherman, F. 1991. Getting started with yeast. Methods Enzymol. 194: 3–21.

306. Wong, L., C. Sissons, and C. H. Sissions. 2001. A comparison of human dental plaque microcosm biofilms grown in an undefined medium and a chemically defined artificial saliva. Arch. Oral Biol. 46: 477–486.

307. Kristich, C. J., J. R. Chandler, and G. M. Dunny. 2007. Development of a host-genotype- independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis. Plasmid 57: 131–144.

308. Roh, J. H., K. V. Singh, S. L. La Rosa, A. L. V. Cohen, and B. E. Murray. 2015. The two- component system GrvRS (EtaRS) regulates ace expression in Enterococcus faecalis OG1RF. Infection and Immunity 83: 389–395.

309. Brothers, K. M., Z. R. Newman, and R. T. Wheeler. 2011. Live imaging of disseminated candidiasis in zebrafish reveals role of phagocyte oxidase in limiting filamentous growth. Eukaryotic Cell 10: 932–944.

310. Peters, B. M., M. A. Jabra-Rizk, M. A. Scheper, J. G. Leid, J. W. Costerton, and M. E. Shirtliff. 2010. Microbial interactions and differential protein expression in Staphylococcus aureus-Candida albicans dual-species biofilms. FEMS Immunology & Medical Microbiology. 59: 493-503.

311. Van den Driessche, F., P. Rigole, G. Brackman, and T. Coenye. 2014. Optimization of resazurin-based viability staining for quantification of microbial biofilms. Journal of Microbiological Methods 98: 31–34.

312. Fox, K. A., A. Ramesh, J. E. Stearns, A. Bourgogne, A. Reyes-Jara, W. C. Winkler, and D. A. Garsin. 2009. Multiple posttranscriptional regulatory mechanisms partner to control

154 ethanolamine utilization in Enterococcus faecalis. Proc. Natl. Acad. Sci. U.S.A. 106: 4435– 4440.

313. Miramón, P., and M. C. Lorenz. 2016. The SPS amino acid sensor mediates nutrient acquisition and immune evasion in Candida albicans. Cell Microbiol 18: 1611–1624.

314. Rocha, C. R., K. Schröppel, D. Harcus, A. Marcil, D. Dignard, B. N. Taylor, D. Y. Thomas, M. Whiteway, and E. Leberer. 2001. Signaling through adenylyl cyclase is essential for hyphal growth and virulence in the pathogenic fungus Candida albicans. Mol. Biol. Cell 12: 3631– 3643.

315. Miramon, P., C. Dunker, L. Kasper, I. D. Jacobsen, D. Barz, O. Kurzai, and B. Hube. 2014. A family of glutathione peroxidases contributes to oxidative stress resistance in Candida albicans. Med Mycol 52: 223–239.

316. Vylkova, S., and M. C. Lorenz. 2014. Modulation of phagosomal pH by Candida albicans promotes hyphal morphogenesis and requires Stp2p, a regulator of amino acid transport. PLoS Pathog 10: e1003995.

317. Solis, N. V., and S. G. Filler. 2012. Mouse model of oropharyngeal candidiasis. Nat Protoc 7: 637–642.

318. Moyes, D. L., D. Wilson, J. P. Richardson, S. Mogavero, S. X. Tang, J. Wernecke, S. Höfs, R. L. Gratacap, J. Robbins, M. Runglall, C. Murciano, M. Blagojevic, S. Thavaraj, T. M. Förster, B. Hebecker, L. Kasper, G. Vizcay, S. I. Iancu, N. Kichik, A. Häder, O. Kurzai, T. Luo, T. Krüger, O. Kniemeyer, E. Cota, O. Bader, R. T. Wheeler, T. Gutsmann, B. Hube, and J. R. Naglik. 2016. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 532: 64–68.

319. Khot, P. D., D. L. Ko, and D. N. Fredricks. 2009. Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays. Applied and Environmental Microbiology 75: 1559–1565.

320. Rickerts, V., P. D. Khot, D. L. Ko, and D. N. Fredricks. 2012. Enhanced fungal DNA- extraction from formalin-fixed, paraffin-embedded tissue specimens by application of thermal energy. Med Mycol 50: 667–672.

321. Pinkston, K. L., P. Gao, D. Diaz-Garcia, J. Sillanpaa, S. R. Nallapareddy, B. E. Murray, and B. R. Harvey. 2011. The Fsr Quorum-Sensing System of Enterococcus faecalis Modulates Surface Display of the Collagen-Binding MSCRAMM Ace through Regulation of gelE. Journal of Bacteriology 193: 4317–4325.

322. Schagger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochemistry 166: 368–379.

323. Fonzi, W. A., and M. Y. Irwin. 1993. Isogenic strain construction and gene mapping in Candida albicans. Genetics 134: 717–728.

324. Staab, J. F., Y.-S. Bahn, and P. Sundstrom. 2003. Integrative, multifunctional plasmids for hypha-specific or constitutive expression of green fluorescent protein in Candida albicans. Microbiology 149: 2977–2986.

155 325. Davis, D. A., V. M. Bruno, L. Loza, S. G. Filler, and A. P. Mitchell. 2002. Candida albicans Mds3p, a conserved regulator of pH responses and virulence identified through insertional mutagenesis. Genetics 162: 1573–1581.

326. Noble, S. M., and A. D. Johnson. 2007. Genetics of Candida albicans, a Diploid Human Fungal Pathogen. Annu. Rev. Genet. 41: 193–211.

327. Smith, D. A., S. Nicholls, B. A. Morgan, A. J. P. Brown, and J. Quinn. 2004. A conserved stress-activated protein kinase regulates a core stress response in the human pathogen Candida albicans. Mol. Biol. Cell 15: 4179–4190.

328. Butler, G., M. D. Rasmussen, M. F. Lin, M. A. S. Santos, S. Sakthikumar, C. A. Munro, E. Rheinbay, M. Grabherr, A. Forche, J. L. Reedy, I. Agrafioti, M. B. Arnaud, S. Bates, A. J. P. Brown, S. Brunke, M. C. Costanzo, D. A. Fitzpatrick, P. W. J. de Groot, D. Harris, L. L. Hoyer, B. Hube, F. M. Klis, C. Kodira, N. Lennard, M. E. Logue, R. Martin, A. M. Neiman, E. Nikolaou, M. A. Quail, J. Quinn, M. C. Santos, F. F. Schmitzberger, G. Sherlock, P. Shah, K. A. T. Silverstein, M. S. Skrzypek, D. Soll, R. Staggs, I. Stansfield, M. P. H. Stumpf, P. E. Sudbery, T. Srikantha, Q. Zeng, J. Berman, M. Berriman, J. Heitman, N. A. R. Gow, M. C. Lorenz, B. W. Birren, M. Kellis, and C. A. Cuomo. 2009. Evolution of pathogenicity and sexual reproduction in eight Candida genomes. Nature 459: 657–662.

329. Murray, B. E., K. V. Singh, R. P. Ross, J. D. Heath, G. M. Dunny, and G. M. Weinstock. 1993. Generation of restriction map of Enterococcus faecalis OG1 and investigation of growth requirements and regions encoding biosynthetic function. Journal of Bacteriology 175: 5216– 5223.

330. Leenhouts, K., G. Buist, A. Bolhuis, A. ten Berge, J. Kiel, I. Mierau, M. Dabrowska, G. Venema, and J. Kok. 1996. A general system for generating unlabelled gene replacements in bacterial . Mol. Gen. Genet. 253: 217–224.

331. Walter, J., and R. Ley. 2011. The Human Gut Microbiome: Ecology and Recent Evolutionary Changes. Annu. Rev. Microbiol. 65: 411–429.

332. Iliev, I. D., V. A. Funari, K. D. Taylor, Q. Nguyen, C. N. Reyes, S. P. Strom, J. Brown, C. A. Becker, P. R. Fleshner, M. Dubinsky, J. I. Rotter, H. L. Wang, D. P. B. McGovern, G. D. Brown, and D. M. Underhill. 2012. Interactions Between Commensal Fungi and the C-Type Lectin Receptor Dectin-1 Influence Colitis. Science 336: 1314–1317.

333. Ghannoum, M. A., R. J. Jurevic, P. K. Mukherjee, F. Cui, M. Sikaroodi, A. Naqvi, and P. M. Gillevet. 2010. Characterization of the Oral Fungal Microbiome (Mycobiome) in Healthy Individuals. PLoS Pathog 6: e1000713–8.

334. Oever, J. T., and M. G. Netea. 2014. The bacteriome-mycobiome interaction and antifungal host defense. Eur. J. Immunol. 44: 3182–3191.

335. Mayr, F. B., S. Yende, and D. C. Angus. 2013. Epidemiology of severe sepsis. Virulence 5: 4–11.

336. Dunny, G. M., L. E. Hancock, and N. Shankar. 2014. Enterococcal Biofilm Structure and Role in Colonization and Disease.

337. Desai, J. V., V. M. Bruno, S. Ganguly, R. J. Stamper, K. F. Mitchell, N. Solis, E. M. Hill, W. Xu, S. G. Filler, D. R. Andes, S. Fanning, F. Lanni, and A. P. Mitchell. 2013. Regulatory Role of

156 Glycerol in Candida albicans Biofilm Formation. mBio 4: e00637–12–e00637–12.

338. Garsin, D. A., and M. C. Lorenz. 2014. Candida albicans and Enterococcus faecalis in the gut. Gut Microbes 4: 409–415.

339. Salvatori, O., S. Puri, S. Tati, and M. Edgerton. 2016. Innate Immunity and Saliva in Candida albicans–mediated Oral Diseases. J Dent Res 95: 365–371.

340. Villar, C. C., and A. Dongari-Bagtzoglou. 2008. Immune defence mechanisms and immunoenhancement strategies in oropharyngeal candidiasis. Expert Rev. Mol. Med. 10: 445– 20.

341. Swidergall, M., and S. G. Filler. 2017. Oropharyngeal Candidiasis: Fungal Invasion and Epithelial Cell Responses. PLoS Pathog 13: e1006056–7.

342. Sangeorzan, J. A., S. F. Bradley, X. He, L. T. Zarins, G. L. Ridenour, R. N. Tiballi, and C. A. Kauffman. 1994. Epidemiology of oral candidiasis in HIV-infected patients: colonization, infection, treatment, and emergence of fluconazole resistance. Am. J. Med. 97: 339–346.

343. Rhodus, N. L., C. Bloomquist, W. Liljemark, and J. Bereuter. 1997. Prevalence, density, and manifestations of oral Candida albicans in patients with Sjogren's syndrome. J Otolaryngol 26: 300–305.

344. Willis, A. M., W. A. Coulter, C. R. Fulton, J. R. Hayes, P. M. Bell, and P. J. Lamey. 1999. Oral candidal carriage and infection in insulin-treated diabetic patients. Diabet. Med. 16: 675– 679.

345. Epstein, J. B., and B. Polsky. 1998. Oropharyngeal candidiasis: a review of its clinical spectrum and current therapies. Clin Ther 20: 40–57.

346. Lynch, D. P. 1994. Oral candidiasis: history, classification, and clinical presentation. Oral surgery 78: 189–193.

347. Pankhurst, C. L. 2013. Candidiasis (oropharyngeal). BMJ Clin Evid 2013: 1304.

348. Phan, Q. T., C. L. Myers, Y. Fu, D. C. Sheppard, M. R. Yeaman, W. H. Welch, A. S. Ibrahim, J. E. Edwards, and S. G. Filler. 2007. Als3 Is a Candida albicans Invasin That Binds to Cadherins and Induces Endocytosis by Host Cells. 5: e64–15.

349. Wächtler, B., F. Citiulo, N. Jablonowski, S. Förster, F. Dalle, M. Schaller, D. Wilson, and B. Hube. 2012. Candida albicans-Epithelial Interactions: Dissecting the Roles of Active Penetration, Induced Endocytosis and Host Factors on the Infection Process. PLoS ONE 7: e36952–9.

350. Zhu, W., Q. T. Phan, P. Boontheung, N. V. Solis, J. A. Loo, and S. G. Filler. 2012. EGFR and HER2 receptor kinase signaling mediate epithelial cell invasion by Candida albicans during oropharyngeal infection. Proc. Natl. Acad. Sci. U.S.A. 109: 14194–14199.

351. Sun, J. N., N. V. Solis, Q. T. Phan, J. S. Bajwa, H. Kashleva, A. Thompson, Y. Liu, A. Dongari-Bagtzoglou, M. Edgerton, and S. G. Filler. 2010. Host Cell Invasion and Virulence Mediated by Candida albicans Ssa1. PLoS Pathog 6: e1001181–14.

352. Farah, C. S., S. Elahi, G. Pang, T. Gotjamanos, G. J. Seymour, R. L. Clancy, and R. B.

157 Ashman. 2001. T Cells Augment Monocyte and Neutrophil Function in Host Resistance against Oropharyngeal Candidiasis. Infection and Immunity 69: 6110–6118.

353. McNulty, K. M., J. Plianrungsi, J. E. Leigh, D. Mercante, and P. L. Fidel. 2005. Characterization of CD8+ T Cells and Microenvironment in Oral Lesions of Human Immunodeficiency Virus-Infected Persons with Oropharyngeal Candidiasis. Infection and Immunity 73: 3659–3667.

354. Baillie, G. S., and L. J. Douglas. 1999. Role of dimorphism in the development of Candida albicans biofilms. Journal of Medical Microbiology 48: 671–679.

355. Ramage, G., K. VandeWalle, J. L. Lopez-Ribot, and B. L. Wickes. 2002. The filamentation pathway controlled by the Efg1 regulator protein is required for normal biofilm formation and development in Candida albicans. FEMS Microbiol Lett 214: 95–100.

356. Bourgogne, A., S. G. Hilsenbeck, G. M. Dunny, and B. E. Murray. 2006. Comparison of OG1RF and an Isogenic fsrB Deletion Mutant by Transcriptional Analysis: the Fsr System of Enterococcus faecalis Is More than the Activator of Gelatinase and Serine Protease. Journal of Bacteriology 188: 2875–2884.

357. Teixeira, N., S. Varahan, M. J. Gorman, K. L. Palmer, A. Zaidman-Remy, R. Yokohata, J. Nakayama, L. E. Hancock, A. Jacinto, M. S. Gilmore, and M. de Fátima Silva Lopes. 2013. Drosophila Host Model Reveals New Enterococcus faecalis Quorum-Sensing Associated Virulence Factors. PLoS ONE 8: e64740.

358. Swe, P. M., N. C. K. Heng, Y. T. Ting, H. J. Baird, A. Carne, A. Tauch, J. R. Tagg, and R. W. Jack. 2007. ef1097 and ypkK encode enterococcin V583 and corynicin JK, members of a new family of antimicrobial proteins (bacteriocins) with modular structure from Gram-positive bacteria. Microbiology 153: 3218–3227.

359. Lorenz, M. C., and G. R. Fink. 2001. The glyoxylate cycle is required for fungal virulence. Letters to Nature. 412: 1–4.

360. Lorenz, M. C., J. A. Bender, and G. R. Fink. 2004. Transcriptional response of Candida albicans upon internalization by macrophages. Eukaryotic Cell 3: 1076–1087.

361. Lo, H.-J., J. R. Kohler, B. DiDomenico, D. Loebenberg, A. Cacciapuoti, and G. R. Fink. 1997. Nonfilamentous C. albicans mutants are avirulent. Cell 90: 939–949.

362. Saville, S. P., A. L. Lazzell, C. Monteagudo, and J. L. Lopez-Ribot. 2003. Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection. Eukaryotic Cell 2: 1053–1060.

363. Nett, J. E., and D. R. Andes. 2012. Antifungals: drug class, mechanisms of action, pharmacokinetics/pharmacodynamics, drug-drug interactions, toxicity, and clinical use. Candida and Candidiasis 345–371.

364. Sanguinetti, M., B. Posteraro, and C. Lass-Flörl. 2015. Antifungal drug resistance among Candidaspecies: mechanisms and clinical impact. Mycoses 58: 2–13.

365. Pierce, C. G., A. K. Chaturvedi, A. L. Lazzell, A. T. Powell, S. P. Saville, S. F. McHardy, and J. L. Lopez-Ribot. 2015. A Novel Small Molecule Inhibitor of Candida albicans Biofilm Formation, Filamentation and Virulence with Low Potential for the Development of Resistance.

158 NPJ Biofilms Microbiomes 1: 1–8.

366. Mason, K. L., T. A. Stepien, J. E. Blum, J. F. Holt, N. H. Labbe, J. S. Rush, K. F. Raffa, and J. Handelsman. 2011. From commensal to pathogen: translocation of Enterococcus faecalis from the midgut to the hemocoel of Manduca sexta. mBio 2: e00065–11.

367. Erb Downward, J. R., N. R. Falkowski, K. L. Mason, R. Muraglia, and G. B. Huffnagle. 2013. Modulation of Post-Antibiotic Bacterial Community Reassembly and Host Response by Candida albicans. Sci. Rep. 3.

368. Ng, W.-L., and B. L. Bassler. 2009. Bacterial Quorum-Sensing Network Architectures. Annu. Rev. Genet. 43: 197–222.

369. DebRoy, S., P. Gao, D. A. Garsin, B. R. Harvey, V. Kos, I. F. Nes, and M. Solheim. 2014. Transcriptional and Post Transcriptional Control of Enterococcal Gene Regulation.

370. Lyon, G. J., and R. P. Novick. 2004. Peptide signaling in Staphylococcus aureus and other Gram-positive bacteria. Peptides 25: 1389–1403.

371. Novick, R. P. 2003. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Molecular Microbiology 48: 1429–1449.

372. Qiu, R., W. Pei, L. Zhang, J. Lin, and G. Ji. 2005. Identification of the Putative Staphylococcal AgrB Catalytic Residues Involving the Proteolytic Cleavage of AgrD to Generate Autoinducing Peptide. Journal of Biological Chemistry 280: 16695–16704.

373. Zhang, L., L. Gray, R. P. Novick, and G. Ji. 2002. Transmembrane Topology of AgrB, the Protein Involved in the Post-translational Modification of AgrD in Staphylococcus aureus. Journal of Biological Chemistry 277: 34736–34742.

374. Zhang, L., J. Lin, and G. Ji. 2004. Membrane Anchoring of the AgrD N-terminal Amphipathic Region Is Required for Its Processing to Produce a Quorum-sensing Pheromone in Staphylococcus aureus. Journal of Biological Chemistry 279: 19448–19456.

375. Sturme, M. H. J., J. Nakayama, D. Molenaar, Y. Murakami, R. Kunugi, T. Fujii, E. E. Vaughan, M. Kleerebezem, and W. M. de Vos. 2005. An agr-Like Two-Component Regulatory System in Lactobacillus plantarum Is Involved in Production of a Novel Cyclic Peptide and Regulation of Adherence. Journal of Bacteriology 187: 5224–5235.

376. Nakayama, J., S. Chen, N. Oyama, K. Nishiguchi, E. A. Azab, E. Tanaka, R. Kariyama, and K. Sonomoto. 2006. Revised Model for Enterococcus faecalis fsr Quorum-Sensing System: the Small Open Reading Frame fsrD Encodes the Gelatinase Biosynthesis-Activating Pheromone Propeptide Corresponding to Staphylococcal AgrD. Journal of Bacteriology 188: 8321–8326.

377. Hancock, L. E., and M. Perego. 2004. The Enterococcus faecalis fsr two-component system controls biofilm development through production of gelatinase. Journal of Bacteriology 186: 5629–5639.

378. Pillai, S. K., G. Sakoulas, G. M. Eliopoulos, R. C. Moellering, B. E. Murray, and R. T. Inouye. 2004. Effects of glucose on fsr-mediated biofilm formation in Enterococcus faecalis. J INFECT DIS 190: 967–970.

159 379. Teixeira, N., S. Santos, P. Marujo, R. Yokohata, V. S. Iyer, J. Nakayama, L. E. Hancock, P. Serror, and M. D. F. Silva Lopes. 2012. The incongruent gelatinase genotype and phenotype in Enterococcus faecalis are due to shutting off the ability to respond to the gelatinase biosynthesis-activating pheromone (GBAP) quorum-sensing signal. Microbiology 158: 519– 528.

380. Houmard, J., and G. R. Drapeau. 1972. Staphylococcal Protease: A Proteolytic Enzyme Specific for Glutamyl Bonds. Proc Natl Acad Sci USA 69: 3506–3509.

381. Daniels, R., P. Mellroth, A. Bernsel, F. Neiers, S. Normark, G. von Heijne, and B. Henriques-Normark. 2010. Disulfide Bond Formation and Cysteine Exclusion in Gram-positive Bacteria. Journal of Biological Chemistry 285: 3300–3309.

382. Reardon-Robinson, M. E., and H. Ton-That. 2016. Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria. Journal of Bacteriology 198: 746–754.

383. Bulaj, G. 2005. Formation of disulfide bonds in proteins and peptides. Biotechnology Advances 23: 87–92.

384. Sevier, C. S., and C. A. Kaiser. 2002. Formation and transfer of disulphide bonds in living cells. Nat Rev Mol Cell Biol 3: 836–847.

385. Goldberg, M. E., and Y. Guillou. 1994. Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme. Protein Science 3: 883–887.

386. Heng, N. C. K. 2006. Dysgalacticin: a novel, plasmid-encoded antimicrobial protein (bacteriocin) produced by Streptococcus dysgalactiae subsp. equisimilis. Microbiology 152: 1991–2001.

387. NCBI Resource Coordinators. 2016. Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 44: D7–19.

388. Guaní-Guerra, E., T. Santos-Mendoza, S. O. Lugo-Reyes, and L. M. Terán. 2010. Antimicrobial peptides: General overview and clinical implications in human health and disease. Clinical Immunology 135: 1–11.

389. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice

. Nucleic Acids Res 22: 4673–4680.

390. Combet, C., C. Blanchet, C. Geourjon, and G. Deléage. 2000. NPS@: Network Protein Sequence Analysis. Trends in Biochemical Sciences 25: 147–150.

391. Pei, J., B.-H. Kim, and N. V. Grishin. 2008. PROMALS3D: a tool for multiple protein sequence and structure alignments. Nucleic Acids Res 36: 2295–2300.

392. Yang, J., R. Yan, A. Roy, D. Xu, J. Poisson, and Y. Zhang. 2015. CORRESPONDENCE The I-TASSER Suite: protein structure and function prediction. Nature Publishing Group 12: 7– 8.

393. Arnold, K., F. Kiefer, J. Kopp, J. N. D. Battey, M. Podvinec, J. D. Westbrook, H. M.

160 Berman, L. Bordoli, and T. Schwede. 2008. The Protein Model Portal. J Struct Funct Genomics 10: 1–8.

394. Swe, P. M., G. M. Cook, J. R. Tagg, and R. W. Jack. 2009. Mode of action of dysgalacticin: a large heat-labile bacteriocin. Journal of Antimicrobial Chemotherapy 63: 679– 686.

395. Lee, J. A., N. Robbins, J. L. Xie, T. Ketela, and L. E. Cowen. 2016. Functional Genomic Analysis of Candida albicans Adherence Reveals a Key Role for the Arp2/3 Complex in Cell Wall Remodelling and Biofilm Formation. PLoS Genet 12: e1006452–24.

396. Cannon, R. D., E. Lamping, A. R. Holmes, K. Niimi, K. Tanabe, M. Niimi, and B. C. Monk. 2007. Candida albicans drug resistance another way to cope with stress. Microbiology 153: 3211–3217.

397. Munro, C. A., S. Selvaggini, I. de Bruijn, L. Walker, M. D. Lenardon, B. Gerssen, S. Milne, A. J. P. Brown, and N. A. R. Gow. 2007. The PKC, HOG and Ca 2+signalling pathways co- ordinately regulate chitin synthesis in Candida albicans. Molecular Microbiology 63: 1399– 1413.

398. Enjalbert, B., D. A. Smith, M. J. Cornell, I. Alam, S. Nicholls, A. J. P. Brown, and J. Quinn. 2006. Role of the Hog1 stress-activated protein kinase in the global transcriptional response to stress in the fungal pathogen Candida albicans. Mol. Biol. Cell 17: 1018–1032.

399. Alonso-Monge, R., S. Carvaihlo, C. Nombela, E. Rial, and J. Pla. 2009. The Hog1 MAP kinase controls respiratory metabolism in the fungal pathogen Candida albicans. Microbiology 155: 413–423.

400. Herrero-de-Dios, C., R. Alonso-Monge, and J. Pla. 2014. The lack of upstream elements of the Cek1 and Hog1 mediated pathways leads to a synthetic lethal phenotype upon osmotic stress in Candida albicans. Fungal Genetics and Biology 69: 31–42.

401. 2009. A Phenotypic Profile of the Candida albicans Regulatory Network. 5: e1000783.

402. Blankenship, J. R., and A. P. Mitchell. 2006. How to build a biofilm: a fungal perspective. Current Opinion in Microbiology 9: 588–594.

403. Cleary, I. A., and S. P. Saville. 2010. An analysis of the impact of NRG1 overexpression on the Candida albicans response to specific environmental stimuli. Mycopathologia 170: 1–10.

404. Lu, Y., C. Su, A. Wang, and H. Liu. 2011. Hyphal Development in Candida albicans Requires Two Temporally Linked Changes in Promoter Chromatin for Initiation and Maintenance. 9: e1001105.

405. Alonso-Monge, R., E. Román, D. M. Arana, D. Prieto, V. Urrialde, C. Nombela, and J. Pla. 2010. The Sko1 protein represses the yeast-to-hypha transition and regulates the oxidative stress response in Candida albicans. Fungal Genetics and Biology 47: 587–601.

406. Nikolaou, E., I. Agriafioti, M. Stumpf, J. Quinn, I. Stansfield, and A. J. Brown. 2009. Phylogenetic diversity of stress signaling pathways in fungi. BMC Evol Biol 9: 44.

407. Birkaya, B., A. Maddi, J. Joshi, S. J. Free, and P. J. Cullen. 2009. Role of the Cell Wall Integrity and Filamentous Growth Mitogen-Activated Protein Kinase Pathways in Cell Wall

161 Remodeling during Filamentous Growth. Eukaryotic Cell 8: 1118–1133.

408. 2010. The Transcriptional Regulator Nrg1p Controls Candida albicans Biofilm Formation and Dispersion. 9: 1531–1537.

409. Heilmann, C. J., A. G. Sorgo, S. Mohammadi, G. J. Sosinska, C. G. de Koster, S. Brul, L. J. de Koning, and F. M. Klis. 2013. Surface Stress Induces a Conserved Cell Wall Stress Response in the Pathogenic Fungus Candida albicans. Eukaryotic Cell 12: 254–264.

410. Böhm, L., P. Muralidhara, and J. C. Pérez. 2016. A Candida albicans regulator of disseminated infection operates primarily as a repressor and governs cell surface remodeling. Molecular Microbiology 100: 328–344.

411. Hirakawa, M. P., D. A. Martinez, S. Sakthikumar, M. Z. Anderson, A. Berlin, S. Gujja, Q. Zeng, E. Zisson, J. M. Wang, J. M. Greenberg, J. Berman, R. J. Bennett, and C. A. Cuomo. 2015. Genetic and phenotypic intra-species variation in Candida albicans. Genome Res. 25: 413–425.

412. Su, C., Y. Lu, and H. Liu. 2013. Reduced TOR signaling sustains hyphal development in Candida albicans by lowering Hog1 basal activity. Mol. Biol. Cell 24: 385–397.

413. Jacobsen, I. D., D. Wilson, B. Wächtler, S. Brunke, J. R. Naglik, and B. Hube. 2014. Candida albicans dimorphism as a therapeutic target. Expert Review of Anti-infective Therapy 10: 85–93.

414. Odds, F. C. 1987. Candida infections: an overview. CRC Critical Reviews in Microbiology.

415. Spellberg, B. J., A. S. Ibrahim, V. Avenissian, S. G. Filler, C. L. Myers, Y. Fu, and J. E. Edwards. 2005. The anti-Candida albicans vaccine composed of the recombinant N terminus of Als1p reduces fungal burden and improves survival in both immunocompetent and immunocompromised mice. Infection and Immunity 73: 6191–6193.

416. Mason, K. L., J. R. Erb Downward, N. R. Falkowski, V. B. Young, J. Y. Kao, and G. B. Huffnagle. 2011. Interplay between the Gastric Bacterial Microbiota and Candida albicans during Postantibiotic Recolonization and Gastritis. Infection and Immunity 80: 150–158.

417. Naglik, J. R., P. L. Fidel Jr, and F. C. Odds. 2008. Animal models of mucosal Candida infection. FEMS Microbiol Lett 283: 129–139.

418. Mason, K. L., J. R. Erb Downward, K. D. Mason, N. R. Falkowski, K. A. Eaton, J. Y. Kao, V. B. Young, and G. B. Huffnagle. 2012. Candida albicans and Bacterial Microbiota Interactions in the Cecum during Recolonization following Broad-Spectrum Antibiotic Therapy. Infection and Immunity 80: 3371–3380.

419. Inoue, T., H. Tomita, and Y. Ike. 2006. Bac 32, a novel bacteriocin widely disseminated among clinical isolates of Enterococcus faecium. Antimicrobial Agents and Chemotherapy 50: 1202–1212.

420. Ike, Y., H. Hashimoto, and D. B. Clewell. 1987. High incidence of hemolysin production by Enterococcus (Streptococcus) faecalis strains associated with human parenteral infections. J. Clin. Microbiol. 25: 1524–1528.

421. Lionakis, M. S., J. K. Lim, C.-C. R. Lee, and P. M. Murphy. 2011. Organ-Specific Innate

162 Immune Responses in a Mouse Model of Invasive Candidiasis. J Innate Immun 3: 180–199.

422. White, S. J., A. Rosenbach, P. Lephart, D. Nguyen, A. Benjamin, S. Tzipori, M. Whiteway, J. Mecsas, and C. A. Kumamoto. 2007. Self-Regulation of Candida albicans Population Size during GI Colonization. PLoS Pathog 3: e184–13.

423. Rosenbach, A., D. Dignard, J. V. Pierce, M. Whiteway, and C. A. Kumamoto. 2010. Adaptations of Candida albicans for Growth in the Mammalian Intestinal Tract. Eukaryotic Cell 9: 1075–1086.

424. Zheng, X., Y. Wang, and Y. Wang. 2004. Hgc1, a novel hypha-specific G1 cyclin-related protein regulates Candida albicans hyphal morphogenesis. EMBO J. 23: 1845–1856.

425. 2001. NRG1 represses yeast-hypha morphogenesis and hypha-specific gene expression in Candida albicans. 20: 4742–4752.

426. Wozniok, I., A. Hornbach, C. Schmitt, M. Frosch, H. Einsele, B. Hube, J. Löffler, and O. Kurzai. 2008. Induction of ERK-kinase signalling triggers morphotype-specific killing of Candida albicans filaments by human neutrophils. Cell Microbiol 10: 807–820.

427. Urban, C. F., U. Reichard, V. Brinkmann, and A. Zychlinsky. 2006. Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms. Cell Microbiol 8: 668–676.

428. Marcil, A., C. Gadoury, J. Ash, J. Zhang, A. Nantel, and M. Whiteway. 2008. Analysis of PRA1 and Its Relationship to Candida albicans- Macrophage Interactions. Infection and Immunity 76: 4345–4358.

429. Swe, P. M., N. C. K. Heng, G. M. Cook, J. R. Tagg, and R. W. Jack. 2010. Identification of DysI, the Immunity Factor of the Streptococcal Bacteriocin Dysgalacticin. Applied and Environmental Microbiology 76: 7885–7889.

430. Duplantier, A. J., and M. L. van Hoek. 2013. The Human Cathelicidin Antimicrobial Peptide LL-37 as a Potential Treatment for Polymicrobial Infected Wounds. Front Immunol 4: 1–14.

431. Ernst, J. F., and J. Pla. 2011. Signaling the glycoshield: Maintenance of the Candida albicans cell wall. International Journal of Medical Microbiology 301: 378–383.

432. Hassan, M., M. Kjos, I. F. Nes, D. B. Diep, and F. Lotfipour. 2012. Natural antimicrobial peptides from bacteria: characteristics and potential applications to fight against antibiotic resistance. Journal of Applied Microbiology 113: 723–736.

433. Lohans, C. T., and J. C. Vederas. 2012. Development of Class IIa Bacteriocins as Therapeutic Agents. International Journal of Microbiology 2012: 1–13.

434. Dabour, N., A. Zihler, E. Kheadr, C. Lacroix, and I. Fliss. 2009. In vivo study on the effectiveness of pediocin PA-1 and Pediococcus acidilactici UL5 at inhibiting Listeria monocytogenes. International Journal of Food Microbiology 133: 225–233.

435. Ingham, A., M. Ford, R. J. Moore, and M. Tizard. 2003. The bacteriocin piscicolin 126 retains antilisterial activity in vivo. Journal of Antimicrobial Chemotherapy 51: 1365–1371.

436. Rihakova, J., J. M. Cappelier, I. Hue, K. Demnerova, M. Federighi, H. Prevost, and D.

163 Drider. 2009. In Vivo Activities of Recombinant Divercin V41 and Its Structural Variants against Listeria monocytogenes. Antimicrobial Agents and Chemotherapy 54: 563–564.

437. Fimland, G., L. Johnsen, L. Axelsson, M. B. Brurberg, I. F. Nes, V. G. Eijsink, and J. Nissen-Meyer. 2000. A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum. Journal of Bacteriology 182: 2643–2648.

438. Johnsen, L., G. Fimland, and J. Nissen-Meyer. 2005. The C-terminal domain of pediocin- like antimicrobial peptides (class IIa bacteriocins) is involved in specific recognition of the C- terminal part of cognate immunity proteins and in determining the antimicrobial spectrum. Journal of Biological Chemistry 280: 9243–9250.

439. O'Shea, E. F., P. M. O'Connor, P. D. Cotter, R. P. Ross, and C. Hill. 2010. Synthesis of trypsin-resistant variants of the Listeria-active bacteriocin salivaricin P. Applied and Environmental Microbiology 76: 5356–5362.

440. Oppegård, C., P. Rogne, P. E. Kristiansen, and J. Nissen-Meyer. 2010. Structure analysis of the two-peptide bacteriocin lactococcin G by introducing D-amino acid residues. Microbiology (Reading, Engl.) 156: 1883–1889.

441. Benech, R.-O., E. E. Kheadr, R. Laridi, C. Lacroix, and I. Fliss. 2002. Inhibition of Listeria innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture. Applied and Environmental Microbiology 68: 3683–3690.

442. Lilian M Were, Barry D Bruce, A. P Michael Davidson, Jochen Weiss. 2003. Size, Stability, and Entrapment Efficiency of Phospholipid Nanocapsules Containing Polypeptide Antimicrobials. J. Agric. Food Chem. 51: 8073–8079.

443. Were, L. M., B. Bruce, and P. M. Davidson. 2004. Encapsulation of nisin and lysozyme in liposomes enhances efficacy against Listeria monocytogenes. Journal of Food ….

444. Kazazic, M., J. Nissen-Meyer, and G. Fimland. 2002. Mutational analysis of the role of charged residues in target-cell binding, potency and specificity of the pediocin-like bacteriocin sakacin P. Microbiology 148: 2019–2027.

445. Tominaga, T., and Y. Hatakeyama. 2007. Development of innovative pediocin PA-1 by DNA shuffling among class IIa bacteriocins. Applied and Environmental Microbiology 73: 5292– 5299.

446. Jasniewski, J., C. Cailliez-Grimal, I. Chevalot, J.-B. Millière, and A.-M. Revol-Junelles. 2009. Interactions between two carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5 on target bacteria and Caco-2 cells. Food Chem. Toxicol. 47: 893–897.

447. 2008. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. 3: 1494–1500.

448. Santos, M. A., and M. F. Tuite. 1995. The CUG codon is decoded in vivo as serine and not leucine in Candida albicans. Nucleic Acids Res 23: 1481–1486.

449. Papon, N., V. Courdavault, M. Clastre, and R. J. Bennett. 2013. Emerging and Emerged Pathogenic Candida Species: Beyond the Candida albicans Paradigm. PLoS Pathog 9: e1003550–4.

164 450. Pfaller, M. A., and D. J. Diekema. 2010. Epidemiology of invasive mycoses in North America. Critical Reviews in Microbiology 36: 1–53.

451. Vylkova, S., W. S. Jang, W. Li, N. Nayyar, and M. Edgerton. 2007. Histatin 5 Initiates Osmotic Stress Response in Candida albicans via Activation of the Hog1 Mitogen-Activated Protein Kinase Pathway. Eukaryotic Cell 6: 1876–1888.

452. Argimon, S., S. Fanning, J. R. Blankenship, and A. P. Mitchell. 2011. Interaction between the Candida albicans High-Osmolarity Glycerol (HOG) Pathway and the Response to Human - Defensins 2 and 3. Eukaryotic Cell 10: 272–275.

453. Gomez-Raja, J., and D. A. Davis. 2012. The --Like Protein Rim8 Is Hyperphosphorylated and Complexes with Rim21 and Rim101 To Promote Adaptation to Neutral-Alkaline pH. Eukaryotic Cell 11: 683–693.

454. Fox, E. P., E. S. Cowley, C. J. Nobile, N. Hartooni, D. K. Newman, and A. D. Johnson. 2014. Anaerobic Bacteria Grow within Candida albicans Biofilms and Induce Biofilm Formation in Suspension Cultures. Current Biology 24: 2411–2416.

455. He, J., D. Kim, X. Zhou, S.-J. Ahn, R. A. Burne, V. P. Richards, and H. Koo. 2017. RNA- Seq Reveals Enhanced Metabolism in Streptococcus mutans Co-cultured with Candida albicans within Mixed-Species Biofilms. Front. Microbiol. 8: 175–15.

165 Vita

After Carrie Elaine Graham completed her coursework at Dumas High School, Dumas, Texas, she entered Texas Tech University in Lubbock, Texas. She received the degree of Bachelor of Science with a major in Biology from Texas Tech University in May, 2005. Then she went on to complete a Master of

Science in Microbiology where she completed her Masters Thesis titled: Structural and functional characterization of bacterial diversity in the rhizosphere of Gossypium hirsutum. She worked as a research assistant in the Department of Microbiolgy and Molecular Genetics at UTHealth for one year. In

August of 2012 she entered The University of Texas MD Anderson Cancer Center UTHealth Graduate

School of Biomedical Sciences.

Permanent address:

80 Behr Ave, Apt 301 San Francisco, California 94131

166