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Mechanisms of Interaction Between Haemophilus Parainfluenzae and Streptococcus Mitis

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University of Rhode Island DigitalCommons@URI Open Access Dissertations 2021 MECHANISMS OF INTERACTION BETWEEN HAEMOPHILUS PARAINFLUENZAE AND STREPTOCOCCUS MITIS Dasith Perera Follow this and additional works at: https://digitalcommons.uri.edu/oa_diss MECHANISMS OF INTERACTION BETWEEN HAEMOPHILUS PARAINFLUENZAE AND STREPTOCOCCUS MITIS BY DASITH PERERA A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN CELL & MOLECULAR BIOLOGY UNIVERSITY OF RHODE ISLAND 2021 DOCTOR OF PHILOSOPHY DISSERTATION OF DASITH PERERA APPROVED: Thesis Committee: Matthew Ramsey, Major professor David Nelson David Rowley Brenton DeBoef DEAN OF THE GRADUATE SCHOOL UNIVERSITY OF RHODE ISLAND 2021 ABSTRACT The human oral cavity is a complex polymicrobial environment, home to an array of microbes that play roles in health and disease. Oral bacteria have been shown to cause an array of systemic diseases and are particularly concerning to type II diabetics (T2D) with numerous predispositions that exacerbate bacterial infection. In this dissertation, we investigated the serum of healthy subjects and T2D subjects to determine whether we see greater translocation of oral bacteria into the bloodstream of T2D indiviDuals. We didn’t observe any significant enrichment of oral taxa, however we detected the presence of an emerging pathogen, Acinetobacter baumannii that is also associated with impaired inflammation in T2D. While some are associated with disease, many oral taxa are important in the pre- vention of disease. In this dissertation we investigated the interactions between two abundant health-associated commensal microbes, Haemophilus parainfluenzae and Streptococcus mitis. We demonstrated that H. parainfluenzae typically exists adjacent to Mitis group streptococci in vivo in healthy subjects. We revealed that this co-occur- rence is density dependent and further influenced by H2O2 production. Additionally, Mitis group streptococci are likely the in vivo source of NAD for H. parainfluenzae further facilitating this co-occurrence. We also investigated H. parainfluenzae’s re- sponse to H2O2, which is composed of a redundant OxyR-controlled system. Further- more, we showed that H2O2 likely elicits the SOS response in H. parainfluenzae which may be a mechanism of generating genetic diversity within this species. These findings reveal mechanisms behind why these two highly abundant taxa coexist in healthy indi- viduals. ACKNOWLEDGMENTS I would like to thank Matthew Ramsey for the years of mentoring, advice, stimu- lating discussions, help and support. I would also like to thank my parents, brother and sister for the many decades of support and more things that could be written. I thank my wife, Phone who I know has made many sacrifices over the past few years and without whom I probably would not have finished this. I thank my in-laws for their help over the last few years and being there for Phone and me. I thank lab members Eric, Thais and Alex for being great lab members and the very interesting Friday talks. I thank Zach for the coffee runs to Dunkin and stimulating science discussions. I would also like to thank other grads students at URI that have helped maintain a great work environment, especially Eric, Thais, Alex, Zach, Kathleen, Tomali, Chris and Katie. iii PREFACE This dissertation has been written in manuscript format iv TABLE OF CONTENTS ABSTRACT .................................................................................................................. ii ACKNOWLEDGMENTS ............................................................................................ iii PREFACE ...................................................................................................................... iv TABLE OF CONTENTS ............................................................................................... v LIST OF TABLES ...................................................................................................... viii LIST OF FIGURES ........................................................................................................ x CHAPTER 1: Introduction ............................................................................................. 1 CHAPTER 2: Impaired host response and the presence of Acinetobacter baumannii in the serum microbiome of type-II diabetic patients. .................................................. 8 Summary .............................................................................................................................. 10 Introduction ......................................................................................................................... 10 Results .................................................................................................................................. 15 Patient demographics ...................................................................................................... 15 Microbiome analysis controls for low template sequencing ............................................ 20 V1-V3 16S microbiome diversity analyses reveal minor but significant differences between diabetic and nondiabetic groups ........................................................................ 21 MED and DADA2 analysis reveal the presence of Acinetobacter baumanii ................... 25 Validating the presence of A. baumanii in T2D subjects ................................................. 29 Host cytokine responses to T2D and T2DP vs Health ...................................................... 30 Host cytokine responses to specific microbiota ................................................................ 33 Discussion ............................................................................................................................ 36 References ........................................................................................................................... 42 Appendix .............................................................................................................................. 54 CHAPTER 3: Mechanisms underlying proximity between oral commensal bacteria . 82 Abstract ................................................................................................................................ 84 Introduction ......................................................................................................................... 85 v Results .................................................................................................................................. 87 H. parainfluenzae co-occurs with S. mitis and related streptococci in human supragingival plaque ........................................................................................................ 87 Species-specific FISH demonstrates frequent H. parainfluenzae - S. mitis co-proximity 88 S. mitis eliminates H. parainfluenzae via production of H2O2 ......................................... 89 Individual H2O2 sensitive genes do not affect the fitness of H. parainfluenzae in coculture with S. mitis ...................................................................................................................... 91 S. mitis and other Streptococcus sp. support H. parainfluenzae growth .......................... 93 In vivo transcriptional responses of H. parainfluenzae vs in vitro ................................... 96 Discussion ............................................................................................................................ 97 Materials and Methods ...................................................................................................... 101 Strains and Media .......................................................................................................... 102 Genomic and plasmid DNA isolation ............................................................................. 102 Genetic manipulation of H. parainfluenzae ................................................................... 102 Read abundance data ..................................................................................................... 103 Plaque Collection, Fixation, and Storage ...................................................................... 103 DNA FISH and Mounting ............................................................................................... 104 Imaging .......................................................................................................................... 104 Image Analysis ............................................................................................................... 105 Mono and Coculture Assays ........................................................................................... 105 Disk diffusion assays ...................................................................................................... 106 Coculture transcriptome sample preparation ................................................................ 106 Transcriptome analyses ................................................................................................. 107 Complementing Nicotinamide Adenine Dinucleotide (NAD) auxotrophy of H. parainfluenzae ................................................................................................................ 107 FiGures ................................................................................................................................ 108 References ........................................................................................................................
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