University of Central Florida STARS Electronic Theses and Dissertations, 2004-2019 2014 Host and Bacterial Determinants of Staphylococcus aureus Nasal Colonization in Humans Gowrishankar Muthukrishnan University of Central Florida Part of the Medical Sciences Commons Find similar works at: https://stars.library.ucf.edu/etd University of Central Florida Libraries http://library.ucf.edu This Doctoral Dissertation (Open Access) is brought to you for free and open access by STARS. It has been accepted for inclusion in Electronic Theses and Dissertations, 2004-2019 by an authorized administrator of STARS. For more information, please contact [email protected]. STARS Citation Muthukrishnan, Gowrishankar, "Host and Bacterial Determinants of Staphylococcus aureus Nasal Colonization in Humans" (2014). Electronic Theses and Dissertations, 2004-2019. 1289. https://stars.library.ucf.edu/etd/1289 HOST AND BACTERIAL DETERMINANTS OF STAPHYLOCOCCUS AUREUS NASAL COLONIZATION IN HUMANS by GOWRISHANKAR MUTHUKRISHNAN M.E. Birla Institute of Technology and Science, Pilani, India, 2007 M.S. University of Central Florida, United States of America, 2010 A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Burnett School of Biomedical Sciences in the College of Medicine at the University of Central Florida Orlando, Florida Summer Term 2014 Major Professor: Alexander M. Cole © 2014 Gowrishankar Muthukrishnan ii ABSTRACT Staphylococcus aureus (SA), an opportunistic pathogen colonizing the anterior nares in approximately 30% of the human population, causes severe hospital-associated and community-acquired infections. SA nasal carriage plays a critical role in the pathogenesis of staphylococcal infections and SA eradication from the nares has proven to be effective in reducing endogenous infections. To understand SA nasal colonization and its relation with consequent disease, assessment of nasal carriage dynamics among a diverse population and determining factors responsible for SA nasal carriage have become major imperatives. Here, we report on an extensive longitudinal monitoring of SA nasal carriage in 109 healthy individuals over a period of up to three years to assess nasal carriage dynamics. Phylogenetic analyses of SA housekeeping genes and hypervariable virulence genes revealed that not only were SA strains colonizing intermittent and persistent nasal carriers genetically similar, but no preferential colonization of specific SA strains in these carriers was observed over time. These results indicated that other non-SA factors could be involved in determining specific carriage states. Therefore, to elucidate host responses during SA nasal carriage, we performed human SA nasal recolonization in a subset of SA nasal carriers within our cohort. In these studies, SA colonization levels were determined, and nasal secretions were collected and analyzed for host immune factors responsible for SA nasal carriage. Interestingly, we observed that stimulation of host immune responses lead to clearance of SA while sustained SA colonization was observed in hosts that did not mount a response during carriage. Further, iii analysis of nasal secretions from hosts revealed that proinflammatory cytokines and chemokines were significantly induced during SA nasal clearance suggesting that innate immune effectors influence carriage. SA utilizes a repertoire of surface and secreted proteins to evade host immune response and successfully colonize the nose. Analysis of the most abundant immunoevasive proteins in the exoproteome of SA nasal carrier strains revealed that expression levels of Staphylococcal protein A (SPA) produced by SA nasal carrier strains in vitro corresponded to the level of persistence of SA in the human nose. To determine if SPA is involved in modulating the host’s response to SA colonization, a subset of participants in our cohort was nasally recolonized with equal concentrations of both wild-type (WT) and spa-disrupted (Δspa) autologous strains of SA. Interestingly, Δspa strains were eliminated from the nares significantly faster than WT when the host mounted an immune response, suggesting that the immunoevasive role of SPA is a determinant of carriage persistence. Collectively, this report augments our understanding of SA nasal carriage dynamics, in addition to identifying important host and microbial determinants that influence SA nasal colonization in humans. Better understanding of this phenomenon can lead to improved preventative strategies to thwart carriage-associated SA infections. iv Dedicated to my parents v ACKNOWLEDGMENTS I wish to express my sincere gratitude to numerous people who supported me throughout my graduate school. First and foremost, I would like to thank my dissertation mentor Dr. Alexander Cole for his excellent guidance and constant encouragement. His patience, mentoring and constant support has enabled me to mature as a scientist and changed the way I think about scientific problems and approach research. I will always remember and cherish the long scientific discussions with him and I value the life lessons that he has taught me. I would like to thank my dissertation committee members Dr. William Self, Dr. Sean Moore and Dr. Christopher Parkinson for their tremendous help throughout the dissertation. I am indebted to them for their invaluable suggestions and advice at committee meetings and otherwise. I would also like to extend a special thank you to Dr. Amy Cole. In addition to giving me hands-on training at the bench, she also provided valuable guidance to improve my experimental design and trouble-shooting skills. My fulfilling graduate school experience is filled wonderful memories that I will always cherish for the rest of my life. Thanks to the following people that made it possible: • My labmates past and present. Specifically Matthew Wood, Ryan Lamers, Colleen Eade, Nicole Cowan, Christine Chong, Nitya Venkataraman, Karthikeyan Sivaraman, Todd Penberthy, and Vanathy Paramanandam. I can never forget all the scientific and non- vi scientific bantering that we did in WackaTues, Natura, and other hangout places near UCF. • My non-Biomed UCF friends Apurva Jain, Devendra Salvi, Simranjeet Singh, Meghal Parikh, Karthika Nair and Jyoti Katoch. • Gerry Quinn (Dr. G) for his guidance and constant support that he extended during the initial years of my graduate school. I will fondly remember all the 2D PAGE and adhesion assays that we did together. • Thank you to all the undergraduate students that worked on my project – Austin Ellis, Allen Seba, Alana Persaud and John Deichen. Lastly, my heartfelt thanks to my family - Amma, Appa, Bala, Swetha, Naveen, Venkat, Shivani, Swati and Shruti. I would also like to extend a special thank you to the love of my life, my wife Anusha Naganathan. Without the constant support of my family especially during difficult times, this long and arduous graduate school journey would not have been possible. My acknowledgments would not be complete without this quote by Peter Griffin from Family Guy “I want to thank God and I want to thank the devil too because you know that is why God is there. He is minding the fence to make sure that guy never comes back. You know if it weren't for the devil God would have probably gone insane, blowing he's brains out from boredom.” vii TABLE OF CONTENTS LIST OF FIGURES ............................................................................................................................ xiii LIST OF TABLES .............................................................................................................................. xvi LIST OF ABBREVIATIONS ............................................................................................................. xviii CHAPTER 1: INTRODUCTION ........................................................................................................... 1 Staphylococcus aureus nasal carriage ......................................................................................... 1 Epidemiology and population dynamics of Staphylococcus aureus nasal carriage.................... 2 Host and microbial determinants that influence Staphylococcus aureus nasal carriage ........... 4 Host-related factors that determine Staphylococcus aureus nasal carriage ......................... 4 Bacterial determinants of Staphylococcus aureus nasal carriage .......................................... 6 Staphylococcus aureus nasal colonization models to study determinants of carriage .............. 9 CHAPTER 2: LONGITUDINAL GENETIC ANALYSES OF STAPHYLOCOCCUS AUREUS NASAL CARRIAGE DYNAMICS IN A DIVERSE POPULATION ....................................................................... 12 Introduction .............................................................................................................................. 12 Materials and Methods ............................................................................................................. 14 Ethics statement for collection of bacterial strains from donors ......................................... 14 Study population, design and bacterial strains .................................................................... 15 Multilocus sequence typing .................................................................................................. 16 viii Phylogenetic analyses of MLST data ..................................................................................... 17 eBURST analyses of MLST data ............................................................................................