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Characterization of the Desiccation Tolerance and Potential for Fomite Spread of Streptococcus Pneumoniae

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Characterization of the Desiccation Tolerance and Potential for Fomite Spread of Streptococcus Pneumoniae Characterization of the Desiccation Tolerance and Potential for Fomite Spread of Streptococcus pneumoniae A thesis submitted by Rebecca L. Walsh In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Molecular Microbiology TUFTS UNIVERSITY Sackler School of Graduate Biomedical Sciences February, 2015 Adviser: Andrew Camilli ABSTRACT Streptococcus pneumoniae is the causative agent of diseases such as otitis media, pneumonia, and bacterial meningitis. Historically, published literature asserted that pneumococcal transmission occurs via direct contact with respiratory secretions from infected individuals, yet spread of other respiratory tract pathogens has been linked to fomites. As the only known reservoir of S. pneumoniae is the human upper respiratory tract, little work has investigated its capacity to survive in the environment. Environmental persistence often depends on an ability to withstand desiccation. A knowledge gap exists regarding the viability of vegetative cells, particularly those of major human pathogens, post dehydration. In an era where nosocomial infections are increasingly common, this has become an important area of study. The goal of this work is to characterize the ability of S. pneumoniae to withstand desiccation. We hypothesize that fomites serve as an alternate source of pneumococcal transmission. To test our hypothesis, we ask three central questions. Can S. pneumoniae survive desiccation? Is S. pneumoniae infectious after desiccation? And which genes or pathways are involved in desiccation tolerance? Here, we describe the development of a desiccation protocol using plate-grown S. pneumoniae. We show that pneumococci can survive at least 28 days of desiccation under ambient conditions, survival is independent of polysaccharide capsule, and desiccation is a more complicated phenomenon than starvation alone. Multiple serotypes survive desiccation, suggesting that this is a species-wide ability encoded by the core pneumococcal genome. Finally, S. pneumoniae retains infectivity in a mouse model of colonization, lending support to the fomite transmission hypothesis. ii To explore the pathways correlated with desiccation tolerance, we constructed a transposon insertion library, which was desiccated for two and four days, after which we performed the Tn-seq method, a high throughput sequencing and quantitation of the transposon junctions. By comparing the frequency of reads for each insertion in the pre- and post-desiccation samples, we determined the importance of each gene for S. pneumoniae’s desiccation survival. Of 12 genes selected for further study, mutants in genes encoding the UvrABC nucleotide excision repair complex, SpxB (pyruvate oxidase), SodA (superoxide dismutase), MscS (mechanosensitive channel of small conductance), and cardiolipin synthase all display phenotypes in stress assays. We demonstrate that S. pneumoniae’s desiccation tolerance is tied to its ability to withstand stresses, including those imposed by osmotic changes and reactive oxygen species, leading to a need for DNA damage repair. iii ACKNOWLEDGEMENTS I must begin by thanking my advisor, Andrew Camilli, who has been my mentor throughout this challenging, and also very rewarding, journey. He has at times been my teacher, my taskmaster, and my biggest champion. Without his insight, enthusiasm, understanding, and encouragement, I would not be at this point. His love for science is contagious, and a constant reminder to me to be passionate about my own work. He also recognizes the importance of perspective, and is very supportive of and interested in lab members’ non-scientific pursuits. Andy, you have made your lab a wonderful place for my graduate career. My graduate work has been improved significantly with input from my committee members Ralph Isberg, Carol Kumamoto, and Joan Mecsas. Thank you for your critical evaluations, your helpful suggestions, and your encouragement along the way. On a daily basis, the Camilli lab members inspired and supported me. In small group discussions, lab meetings, and practice talks, I always received valuable feedback and reassurances. Thank you to all the other S. pneumoniae researchers for your help (and sometimes your samples) – Tim van Opijnen, Katie Price, Revati Masilamani, Mara Shainheit, Chelsea Witte, Neil Greene, Ankur Dalia, and Ellie Fleming. Thank you to my various small group members – Ayman Ismail, Bharathi Patimalla, Kim Seed, Mimi Yen, Heather Kamp, Chelsea, Revati, and Ellie. Thank you to Dave Lazinksi for your suggestions and your crazy bank of knowledge. Thank you to Bharathi for your tireless help with the many mouse experiments. Thank you to lab managers Marc Kimball and Trish Winterfield for being ordering and organizing whizzes. Thank you to past and present members of the Lion Bay – Evan Bradley, Revati, Chelsea, and Mimi – for iv making our end of the lab a blast, whether with musical sing-alongs, Disney paraphernalia, venting sessions, or anything to do with our mascot Sir Percival Riggle. The members of the Molecular Microbiology Program and Department of Molecular Biology and Microbiology are outstanding; thank you to the students, faculty, post-docs, and staff for making it such a collaborative, nurturing, and fun environment. I also need to thank my classmates – Andrew Hempstead, Julia (Murphy) Keith, and Jessica Silverman. From studying together first year, to commiserating about research woes, to wrapping up our graduate work, I couldn’t imagine a better group for me to have experienced all of this with. To my close friends that I’ve made during my time here – Dana, Katie, Marc, Revati, EmilyKate, Stephanie, Julia, Erin, and Mimi – thank you for all of the great memories. Thank you to Greg Priebe for being a wonderful mentor in my years after college, and for pushing me to go after this. Thanks to friends, near and far, for being so positive and understanding – Shaunagh, Penny, Laura and Tom, Marc and Amy, and many more. Finally, and most importantly, thank you to my family who have been unwavering in their belief in me. Jill and Paul, thank you for your reminders that I would get through this. Special thanks to Bobbie, who would have been so excited for me. Mom and Dad, thank you for everything; there are no words. My parents instilled a love of learning and science in all of us, and have been my biggest cheerleaders in everything I do. Lisa and Michael, thank you for your emails, texts, and conversations (scientific and otherwise) that have helped me through this. Gavin, it’s been a long road, and your perspective, confidence, patience, and encouragement continue to amaze me. Thank you for your love and your support. v TABLE OF CONTENTS Abstract ........................................................................................................................... ii Acknowledgements ....................................................................................................... iv Table of Contents .......................................................................................................... vi List of Tables ................................................................................................................. ix List of Figures ..................................................................................................................x List of Abbreviations ................................................................................................... xii CHAPTER 1: Introduction ...........................................................................................1 1.1 Streptococcus pneumoniae ...............................................................................2 1.1.1 Capsule and disease .....................................................................................5 1.1.2 Cell membrane and cardiolipin ....................................................................7 1.2 Bacterial transmission via fomites ..................................................................8 1.3 Desiccation tolerance .....................................................................................13 1.3.1 Desiccation-tolerant bacteria .....................................................................13 1.3.2 Effect of relative humidity .........................................................................14 1.3.3 Effect of season ..........................................................................................17 1.3.4 Effect of cell density ..................................................................................18 1.3.5 Preventative responses to desiccation ........................................................18 1.3.6 Genes identified with roles in desiccation tolerance ..................................20 1.4 Stress response ...............................................................................................23 1.4.1 Oxidative stress response ...........................................................................25 1.4.2 DNA damage repair ...................................................................................28 1.4.3 Competence ................................................................................................31 1.4.4 Protein protection .......................................................................................32 1.4.5 Osmotic stress response .............................................................................33 1.5 Pneumococcal desiccation tolerance ............................................................36
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