Investigating the Impact of Phosphate Acquisition and Homeostasis on Staphylococcus Aureus Pathogenesis

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Investigating the Impact of Phosphate Acquisition and Homeostasis on Staphylococcus Aureus Pathogenesis INVESTIGATING THE IMPACT OF PHOSPHATE ACQUISITION AND HOMEOSTASIS ON STAPHYLOCOCCUS AUREUS PATHOGENESIS BY JESSICA L. KELLIHER DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology in the Graduate College of the University of Illinois at Urbana-Champaign, 2019 Urbana, Illinois Doctoral Committee: Assistant Professor Thomas E. Kehl-Fie, Chair Professor William W. Metcalf Professor James M. Slauch Professor Brenda A. Wilson ABSTRACT Phosphate is an essential nutrient for all organisms. Therefore, transporters and regulatory systems in bacterial pathogens enabling phosphate acquisition within the host are important for virulence. However, the contribution of phosphate homeostasis to infection by the ubiquitous pathogen Staphylococcus aureus has not been evaluated. Bioinformatic analysis revealed that S. aureus encodes three inorganic phosphate (Pi) transporters: PstSCAB, PitA, and NptA. Each transporter imports Pi optimally in distinct environments. Interestingly, although loss of PstSCAB results in decreased virulence of several well-studied pathogens, a ΔpstSCAB mutant of S. aureus was not attenuated. However, these studies establish an important role for NptA in the pathogenesis of S. aureus. Although NptA has been sparsely characterized in bacteria, NptA homologs are widespread, suggesting that this type of Pi transporter may broadly contribute to pathogenesis. To regulate phosphate acquisition and homeostasis, bacteria contain a conserved, Pi-responsive two-component system named PhoPR in Gram-positives. In the model organism Escherichia coli and many others, the PhoPR homologs interact with PstSCAB and an accessory protein named PhoU to sense Pi, and mutation of PstSCAB or PhoU results in constitutive PhoPR activation. In contrast, deleting pstSCAB or phoU does not lead to dysregulated PhoPR activation in S. aureus, indicating that Pi sensing in this organism is fundamentally different from the current paradigm established in E. coli. In S. aureus, PhoPR is required for staphylococcal growth during Pi starvation and for virulence. Interestingly, the subset of PhoPR-regulated genes that promotes infection varies depending on other environmental factors. This is exemplified by the observation ii that in the liver, PhoPR is necessary for expression of Pi transporters PstSCAB and NptA, while PhoPR-regulated factors other than Pi transporters are required for infection of the heart. Cumulatively, the findings herein establish an essential role for Pi acquisition and homeostasis in staphylococcal pathogenesis and suggest that these processes are markedly different in S. aureus than in established models. iii ACKNOWLEDGMENTS My journey at the University of Illinois at Urbana-Champaign has been a long one, equally transformative and treasured. I am full of gratitude towards the many people at this institution who have profoundly impacted my career and my life. Thank you to the many faculty members in the Department of Microbiology who have fostered my passion for microbes and discovery. I am grateful to have trained with faculty renowned not just for their wisdom, but also for their open doors. In particular, I thank Drs. Rachel Whitaker and Carin Vanderpool for seeing past my abysmal grade point average as a lost undergraduate student to the budding scientist within. Without your support, graduate school would not have been a possibility. Thank you to my fantastic committee members for offering excellent advice, scientific and otherwise, at every juncture. I thank Dr. James Morrissey for serving as a temporary committee member. I thank Dr. James Imlay for serving as another mentor during graduate school, through lecture, joint lab meetings, and other impromptu chats. Thank you to my delightful labmates. You made the days move easy. I thank the two exceptional undergraduate students who worked with me, Kevin Grudzinski and Aleeza Leder Macek, for having patience with me as I grow as a scientist and a mentor. I thank my advisor Dr. Thomas Kehl-Fie for expecting nothing but the very best from me. Thank you to my friends and especially my loving family, who have supported me every step of the way. You have kept me grounded, audacious, and happy these last five years, for which I am eternally grateful. This chapter is ours. iv TABLE OF CONTENTS CHAPTER 1: Introduction .................................................................................................1 1.1 Staphylococcus aureus ..............................................................................................1 1.2 Phosphate ..................................................................................................................8 1.3 Aims of this study ...................................................................................................23 1.4 References ...............................................................................................................24 CHAPTER 2: Acquisition of the phosphate transporter NptA enhances Staphylococcus aureus pathogenesis by improving phosphate uptake in divergent environments .....................................................................................................................38 2.1 Abstract ...................................................................................................................38 2.2 Introduction .............................................................................................................39 2.3 Results .....................................................................................................................42 2.4 Discussion ...............................................................................................................48 2.5 Materials and methods ............................................................................................53 2.6 Figures and tables ....................................................................................................58 2.7 References ...............................................................................................................73 CHAPTER 3: PhoPR contributes to Staphylococcus aureus growth during phosphate starvation and pathogenesis in an environment-specific manner ....................78 3.1 Abstract ...................................................................................................................78 3.2 Introduction .............................................................................................................79 3.3 Results .....................................................................................................................83 3.4 Discussion ...............................................................................................................91 3.5 Materials and methods ............................................................................................96 3.6 Figures and tables ..................................................................................................100 3.7 References .............................................................................................................108 CHAPTER 4: Conclusions and future directions ..........................................................114 4.1 Conclusions ...........................................................................................................114 4.2 Future directions ....................................................................................................115 4.3 Figures ...................................................................................................................119 4.4 References .............................................................................................................121 v CHAPTER 1 Introduction 1.1 STAPHYLOCOCCUS AUREUS Epidemiology Staphylococcus aureus, first described in the 1880s, is a Gram-positive, coccoid bacterium that forms grape-like clusters resulting from perpendicular rotation of the cell division plane (103, 144). S. aureus is exquisitely evolved to life on and in mammals, colonizing the anterior nares of approximately one third of the human population at any given time (50). Carriage of S. aureus is asymptomatic but is a major predictor for the development of disease (73, 74). Once it breaches the epithelial barrier, typically through cuts, abrasions, burns, use of devices like catheters, or surgery, S. aureus can infect every tissue in the host. Accordingly, S. aureus causes a wide range of diseases, from mild, self-limiting skin infections like boils to much more serious conditions like osteomyelitis, pneumonia, and more (85). S. aureus is a leading cause of infective endocarditis, bacteremia, and skin and soft tissue infections, among others (42, 85, 138, 143). Infections caused by S. aureus result in approximately 700,000 hospitalizations and $14 billion in associated costs per year in the US (138). Additionally, S. aureus is consistently among the most common nosocomial pathogens and is thus a significant cause of morbidity and mortality in healthcare settings (156). 1 Staphylococcal infections: a (re-)emerging threat to human health Upon its introduction into the clinic in the 1940s, penicillin was hailed as a miracle drug in the battle against infectious disease, including that caused by S. aureus (38). Comparison of deaths caused by infectious disease between the first and second World Wars demonstrates that usage of antibiotics decreased the wartime
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