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ADHERENCE and ALKALINIZATION by Elizabeth Hwang

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ADHERENCE and ALKALINIZATION by Elizabeth Hwang TWO EARLY PROCESSES DURING INFECTION BY THE FUNGAL PATHOGEN CANDIDA GLABRATA: ADHERENCE AND ALKALINIZATION By Elizabeth Hwang-Wong A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland November, 2016 Abstract Candida glabrata is a yeast pathogen of increasing diagnostic incidence. Its intrinsic resistance to antifungal agents used in standard clinical settings compels a need to further characterize and understand the pathogenesis of this species. The ability of C. glabrata to adhere to both abiotic surfaces and host cells is an essential early step in establishment of infection. It is also postulated that the capability of this pathogen to externally alkalinize an acidic environment, such as that found within an immune effector’s phagolysosome, could provide an evasive mechanism to resist initial onslaught of an innate immune response. Members of a major class of adhesins encoded by the C. glabrata genome were previously described as Epithelial Adhesins (Epas). Earlier studies have demonstrated the existence of more than 20 members of this class, many of which are encoded in subtelomeric regions of the pathogen’s genome. A major sequencing project has now defined a total complement of 25 members, a newly described one of which is shown to function as a major adhesin across multiple host cell types. In fact, functional adherence of all putative adhesins encoded in the subtelomeres of C. glabrata has been tested, and with minor exception, all are EPAs. The ligand specificities of these functional adhesins were further tested utilizing glycan arrays, and revealed clues identifying a specific EPA responsible for mediating adherence to macrophages. Deletion of these adhesins was finally shown to abrogate colonization in a murine infection model. Previous experiments investigating external alkalinization mediated by fungal pathogens described only distant phylogenetic relatives of C. glabrata, like Candida albicans. Akin to its distant cousins, C. glabrata also alkalinizes its environment when grown with amino acids as its only available carbon source. Mediation of this phenomenon likely functions by the ability of the pathogen to import basic amino acids. Organ colonization by mutants impermeable to this subset of amino acids is decreased. ii PHD DISSERTATION REFEREES FOR ELIZABETH HWANG-WONG Brendan P. Cormack, PhD: Professor, Department of Molecular Biology and Genetics at Johns Hopkins School of Medicine (faculty sponsor) Jeffry Corden, PhD: Professor, Department of Molecular Biology and Genetics at Johns Hopkins School of Medicine (reader) iii Acknowledgements I cannot express enough thanks to one of the most intelligent individuals I have had the honor of knowing - my advisor, Dr. Brendan Cormack. He had the wisdom to recommend the project that forms the majority of this thesis, and his infectious curiosity is a driving force for the entire lab. I am indebted to my reader, Dr. Jeffry Corden, who reviewed my work at the eleventh hour, and was the most generous neighboring mentor any graduate student could ask for. Thank you also to my committee members, Drs. Ronald Schnaar, Fidel Zavala, and Peter Espenshade, who provided wise suggestions and counsel. I am blessed to have been part of a very supportive and collegial lab. Thank you to Dr. Brian Green, who is not the well-known theoretical physicist but is equally as brilliant. Brian spearheaded the sequencing project that forms the basis for the work in Chapters 2-5, and was an instrumental second mentor for much of my early work on Epa family members. Thank you also to Dr. Rebecca Zordan, who marvels at minds that are “like a steel trap!” while I marvel at her equally well-endowed intelligence. Her constructive analyses, both big and small, aided my work in immeasurable ways. I also need to thank previous graduate students in the lab. I never had the pleasure of meeting Dr. Margaret Zupancic, but the heterologous expression experiments and microarray analyses were an expansion of her work on previously characterized proteins. I was lucky enough to spend a few years with Dr. Shih-Jung Pan, who was the resident expert at most of the relevant assays performed in our lab. Thank you for teaching me countless techniques. Thank you to the rest of the lab as well, for providing moments of levity, kindness, and consideration. I have made some lifelong and irreplaceable bonds with fellow classmates, both within my program and without. I am indebted to my roommate Nina Rajpurohit, who is quite possibly the iv best person I have ever known. Thank you to the CVP crew – Cassie, Vy, Nina, YaWen, Hoku, Emily, Ouma, Sophie, Rob, Meredith, and Jose. They have been there through the toughest and most joyous events of these past few years, both professional and personal, and often over an ethanol-imbued poison of choice. I hope that the tradition continues, and that distance only opens up more places for us to explore. I am also immensely grateful to the administrators of my program, specifically Dr. Carolyn Machamer and Dr. Arhonda Gogos, who continually go beyond their call of duty. This work would not have been possible without their professional, social, and emotional support. My first personal thanks go to my grandmother, Jang Soo Kwak. She was a woman of remarkable strength and character, who raised a family in a war-torn country and overcame challenges I will luckily never have to face. While many grandparents joke about marching to school a few miles uphill both ways, my grandmother was a woman who fled an invading army with three small children and an infant in tow (my mother). Years later, she raised me along with my two siblings in a strange but remarkable country. Thanks to my parents, Sook Hee Hwang and Joon Sik Hwang, who stressed the value of education, and reared three children who all reached for postgraduate degrees. Thank you to my sister, Dr. Jean Hwang, for the free medical advice and for shared laughter and tears. Thanks to my husband, Vinson Wong, for partnering with me in the greatest adventure of our lives – parenthood. Lastly and most importantly, I thank my two young children Zoey and Margaret. Their tenacious inquisitiveness reminds me every day that experimentation and delight at discovery is universal. v Table of Contents Section 1: Adhesins encoded by Candida Glabrata Chapter 1: Background and Significance The yeast cell wall Adhesin structure and function Subtelomeres in other microorganisms Subtelomeres in Candida glabrata Conclusion Chapter 2: Heterologous Expression of subtelomeric GPI-CWPs Defining the complement of subtelomeric GPI-CWPs Construction of expression vectors Expression test Adherence to Cells Experimental Procedures Conclusion Chapter 3: Transcriptional Profile of Subtelomeric GPI-CWPs Sir2 Deletion Experimental Procedures Conclusion Chapter 4: Ligand Specificity of Subtelomeric Adhesins Glycan arrays Multiple sequence alignment analyses Whole cell adherence to arrays Epa12 specificty and inhibition vi Experimental Procedures Conclusion Chapter 5: Deletion of all major Epas Adherence to cells Murine Infection Model Experimental Procedures Conclusion Section 2, Chapter 6: External alkalinization of C. glabrata Background and significance Candida glabrata alkalinizes in response to amino acids as a sole carbon source Mutants in putative alkalinization pathways Results Conclusion and Future Directions vii List of Figures Fig 1: The C. glabrata cell wall…………………………………………………………….page 3 Fig 2: Diagram of ALS gene structure…………………………………………….…….page 11 Fig 3: Structure of GPI-CWP Expression constructs……………………..……...page 32 Fig 4: Expression of GPI-CWP Fusion constructs…………………………...…...page 41 Fig 5: Adherence to cell lines………………………………………………………………page 43 Fig 6: Summary of Strongest Adhesins…………………………………………….…page 55 Fig 7: Pwp family binding to various cell lines………………………………….…page 56 Fig 8: Location of EPA genes in the C. glabrata genome……………….……page 66 Fig 9: Subtelomeric transcript abundance as a function of distance to first telomeric repeat in a Sir2Δ strain…………...……page 70 Fig 10: Multiple sequence alignments of Epa proteins……………………….page 84 Fig 11: Glycan Arrays for whole yeast hybridization of Epa1, Epa6, Epa7, Epa12, Epa15, Epa16, Epa23, Epa24, Epa25, and Epa26…………………………………………………………………………..…page 86 Fig 12: Inhibition of Epa12 binding to bone marrow-derived macrophages by sulfated GAGs…………………………………….………page 108 Fig 13: Adherence of Epa deletion mutants to Lec2 cells………….………..page 124 Fig 14: Adherence of Epa deletion mutants to various cell types………..page 126 Fig 15: Organ colonization of C. glabrata infected BALB/c mice by Epa deletion strains……………………………………………………………….page 131 Fig 16: Putative ammonia-production and extrusion pathways in C. glabrata………………………………………………………………………..page 147 Fig 17: Optical density and pH changes in response to viii different carbon sources………………………………………………………page 154 Fig 18: Growth of C. glabrata WT and permease mutant strains in the presence of canavanine…………………………………………………page 156 Fig 19: External alkalinization by deletion mutants in response to growth with amino acids……………………………………………………..page 158 Fig 20: Organ colonization of C. glabrata infected BALB/c mice by a quadruple amino acid permease deletion strain………….page 161 List of Tables Table 1: Heterologous expression srains……………………………………..……page 34 Table 2: Primer table for heterologous expression strains…………..…..page 36 Table 3: Subtelomeric GPI-CWP qRT-PCR primers……………………..….….page
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