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Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus Aureus Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus aureus The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Coe, Kathryn Ann. 2019. Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus aureus. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42013046 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus aureus A dissertation presented by Kathryn Ann Coe to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University, Cambridge, Massachusetts June 2019 © 2019 Kathryn Ann Coe All rights reserved. Dissertation Advisors: Professor Suzanne Walker Kathryn Ann Coe Professor Yonatan Grad Using Transposon Sequencing to Identify Vulnerabilities in Staphylococcus aureus Abstract Antibiotic resistant infections cost thousands of lives in the United States every year. Resistance exists for every known antibiotic, making the development new antibiotics crucial to human health. One technique that has become instrumental in prioritizing targets for antibiotic development is transposon sequencing (Tn-Seq). Tn-Seq is a powerful high-throughput technology that connects bacterial genes with phenotypes and can be used to identify genes that are essential for bacterial survival. In the early years of Tn-Seq, results from a single representative strain were largely assumed to reflect the entire species. However, researchers are now showing that gene essentiality varies among members of a species and that this variability can have direct implications for antibiotic susceptibility. Here I describe the use of Tn- Seq to more thoroughly characterize Staphylococcus aureus gene reliance. We have generated a compendium of core essential genes shared by five strains from across the S. aureus phylogeny, including three strains of methicillin-resistant S. aureus (MRSA), a leading cause of antibiotic resistance-associated mortality in the United States. To better understand antibiotic resistance, we have also developed a new analytical approach that uses Tn-Seq data to identify genes whose overexpression confers a fitness advantage in a given condition. We applied this method to a range of data from antibiotic-treated samples and recovered many clinically- reported mechanisms of resistance as well as new mechanisms that may provide insights into how antibiotics work and how bacteria overcome them. Given the baseline differences in gene iii reliance between strains of S. aureus, we wondered whether antibiotic resistance factors would be shared across strains. We used daptomycin, a common antibiotic for the treatment of MRSA, as a case study to compare resistance factors and vulnerabilities across the five aforementioned strains of S. aureus. We found that the genes modulating antibiotic susceptibility were largely shared, even in cases where the strains relied on the genes to differing degrees in favorable growth conditions. Together, these studies provide the most nuanced characterization of S. aureus gene reliance to date and emphasize the utility of Tn-Seq for investigating antibiotic resistance. iv Table of Contents Chapter 1: Introduction to transposon sequencing and its growing import in Staphylococcus aureus bacteriology 1 Chapter 2: Comparative Tn-Seq reveals differences in gene essentiality between strains of Staphylococcus aureus 15 Chapter 3: Development of an analytical method to identify upregulation signatures in transposon sequencing data 46 Chapter 4: Comparative Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus despite strain-dependent differences in essentiality of shared cell envelope genes 72 Chapter 5: Conclusions and future directions 101 Appendix 108 Figures Figure 2.1: Tn-Seq can be used to identify essential genes. 19 Figure 2.2: Essential genes, while primarily shared, vary somewhat by strain. 32 Figure 2.3: The fitness of lipoteichoic acid pathway mutants varies by strain. 34 Figure 3.1: Adding an outward-facing promoter to a transposon makes insertion outcomes orientation-dependent. 48 Figure 3.2: Upregulation signatures in Tn-Seq data reveal fitness advantages through gene overexpression. 50 Figure 3.3: Single-strand tokens for HMM 2 were defined using read difference cutoffs. 56 Figure 3.4: Folate and DNA synthesis pathway genes have upregulation signatures in Tn-Seq data upon trimethoprim exposure. 63 Figure 4.1: Transposon sequencing supports previously reported daptomycin vulnerabilities in S. aureus and reveals new ones. 84 Figure 4.2: LTA loss sensitizes S. aureus to daptomycin. 85 v Tables Table 2.1: We created complex transposon libraries of similar coverage in five diverse S. aureus strains. 31 Table 3.1: Emission probabilities for HMM 1. 54 Table 3.2: Emission probabilities for HMM 2. 58 Table 3.3: Known hits in three test files recaptured by each proposed analysis method. 61 Table 4.1: Daptomycin-exposed samples included in the multi-strain comparison of depleted, enriched, and upregulated genes. 79 Table 4.2: Upregulation signatures identify genes previously linked to reduced susceptibility to daptomycin. 86 Table 4.3: Genes enriched in reads in the presence of daptomycin are often slow growing. 88 Appendix Supplemental Table 1: Primers used in these studies. 109 Supplemental Table 2: Bacterial strains used in these studies. 110 Supplemental Table 3: Plasmids used in these studies. 111 Supplemental Table 4: Comparison of essential genes in S. aureus strains. 112 Supplemental Table 5: Core essential genes in S. aureus. 119 Supplemental Table 6: Gene ontology analysis reveals central dogma pathways to be universally essential in S. aureus. 125 Supplemental Table 7: Genes with upregulation signatures in the presence of various antibiotic compounds. 129 Supplemental Table 8: Genes with upregulation signatures in the presence of various antibiotic compounds, sorted by compound. 136 Supplemental Table 9: The number of antibiotics in the presence of which each gene has an upregulation signature. 145 Supplemental Table 10: Genes depleted of reads in the presence of daptomycin. 151 Supplemental Table 11: Genes with upregulation signatures in the presence of daptomycin. 154 Supplemental Table 12: Genes enriched in reads in the presence of daptomycin. 158 Supplemental Table 13: SNPs present in daptomycin-nonsusceptible isolates not found in paired susceptible isolates. 161 vi Acknowledgements It takes a team to bring a dissertation to fruition. I am incredibly grateful for the mentorship Suzanne Walker has provided me throughout this process. She took me on even though I knew nothing about microbiology and has been a crucial advocate for me ever since. When I was floundering in my third year, an aspiring bioinformatician in a decidedly noncomputational lab, she connected me with Yonatan Grad, who became my second advisor. Yonatan has likewise been instrumental to my success here, encouraging me to approach all of my analyses with utmost rigor and making me a better scientist as a result. I also want to thank all of the people in Suzanne’s and Yonatan’s labs who have helped me along the way, providing both experimental and emotional support and making the experience enjoyable despite the toils. Their passion for science has been inspiring and infectious. I am especially grateful to Marina Santiago for all the work she did to set up the Tn-Seq analysis pipelines in the lab; Wonsik Lee for collaborating with me on all of my projects; Scott Olesen for his statistics and career mentorship; my bay-mates Anthony Hesser, Atsushi Taguchi, and Chris Vickery for providing so many laughs; Truc Do and Leigh Matano for helping me stay positive during rough patches; and Jake Muscato and Sarah Potter for fearlessly taking over ordering and social planning, two of the most thankless lab jobs, so that I could focus on my research. I would also like to thank Tim Meredith, Gloria Komazin-Meredith, Marina Santiago, and Leigh Matano for making the transposon libraries that were at the crux of all of my projects. I am appreciative of my preliminary qualifying exam committee (Ann Hochschild, Paula Watnick, and Marcia Goldberg), my dissertation advisory committee (Tom Bernhardt, Simon Dove, and David Hooper), and my dissertation examiners (Simon Dove, Tim van Opijnen, Steve Lory, and Dan Kahne) for their thoughtful advice and fruitful conversations and the Biological and Biomedical Sciences Program and Microbiology Department for providing a richly intellectual and incredibly collaborative environment where I could thrive as a student. vii Last and most, I would like to thank my family. My parents, Larry and Diane Coe, have been my constant cheerleaders throughout my time here, ready with a drink and a pep talk during the rocky parts and a drink and a high-five to celebrate every success. If I ever needed anything, whether it was someone to talk to, a printer, help with a broken faucet, etc., they were always there for me in an instant. You would be hard-pressed to find anyone more loving and supportive
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