INTERBACTERIAL COMPETITION IN THE BURKHOLDERIA CEPACIA COMPLEX Andrew Irving Perault A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor in Philosophy in the Department of Microbiology and Immunology in the School of Medicine. Chapel Hill 2020 Approved by: Peggy A. Cotter Matthew C. Wolfgang Brian P. Conlon Elizabeth A. Shank Alecia N. Septer Scott H. Donaldson Ó 2020 Andrew Irving Perault ALL RIGHTS RESERVED ii ABSTRACT Andrew Irving Perault: Interbacterial Competition in the Burkholderia cepacia Complex (Under the direction of Peggy A. Cotter) Bacteria often live in multicellular communities composed of multiple species. Bacteria interact with one another during intimate cell-cell contact within such environments, and these interactions influence the ecology and evolution of multicellular communities. Polymicrobial communities reside within the respiratory tracts of individuals with the genetic disorder cystic fibrosis (CF). Polymicrobial respiratory infections are characteristic of CF disease, and canonical pathogens can dominate the airway communities during infections. Intriguingly, pathogenic species of the Burkholderia cepacia complex (Bcc) can establish infections within polymicrobial CF respiratory tracts, even those dominated by the prevalent pathogen Pseudomonas aeruginosa, though these infections do not occur in young individuals and are limited to teenage and adult CF patients. Here we describe two mechanisms of cell contact-dependent interbacterial competition that provide Bcc pathogens ecological advantages: contact-dependent growth inhibition (CDI) and the type VI secretion system (T6SS). We show the Bcc CF pathogen Burkholderia dolosa produces three CDI systems that facilitate killing of competitor cells during co-culture experiments. These systems deliver unique polymorphic toxins to target cells, and production of immunity proteins cognate to these toxins rescues target cells from killing. We show the genes encoding one B. dolosa CDI system are present in multiple sequenced Bcc strains, suggesting this mechanism of interbacterial competition may be prevalent among Bcc pathogens. Using the Bcc pathogen Burkholderia cenocepacia, we show that the T6SS of Bcc species mediates strong competition against a iii variety of target bacteria. We describe the striking ability for this pathogen to use its T6SS to outcompete P. aeruginosa isolates from teenage and adult CF patients, but not P. aeruginosa isolates from young patients. Whole genome sequencing identified mutations in the susceptible P. aeruginosa isolates that abrogate activity by their own T6SSs, thus inhibiting their retaliation to attack from B. cenocepacia and promoting their elimination from co-cultures. These competitive dynamics may explain why Bcc infections are limited to teenage and adult CF patients. Altogether CDI and T6S likely provide Bcc pathogens competitive advantages within the polymicrobial communities of the CF respiratory tract, promoting their colonization and infection potential, and thus may be attractive targets for therapies treating these infections. iv ACKNOWLEDGEMENTS I am fortunate to have wonderful support circles, both professionally and non- professionally, and therefore a plethora of individuals deserve my gratitude. For the sake of brevity, I will try to keep this brief… First, I would like to thank my mentor Peggy A. Cotter. Academia can be a welcoming and supportive community, and graduate student trainees can be blessed by having terrific mentors; however, no one is luckier than me and the fellow students to be trained by Peggy. Forget her passion for science, her excitement to discuss data, her love for helping trainees become great scientists, her ability to let loose; Peggy is just a great person. To get that and all the professional benefits with a mentor, what else could you ask for? I was lucky to have a similarly great mentor, Victor J. DiRita, before starting my graduate studies at UNC, and I thank him as well. I also would like to thank my thesis committee members. Although we only have formal meetings once a year, your mentorship and assistance in progressing my thesis research certainly extends beyond that. I also want to thank my unofficial mentors within the UNC Microbiology & Immunology community and all the amazing individuals and support staff we have in our department. I, of course, also want to thank my family and friends. At the very least, my family has been proud of me during my studies. This may seem minimal, but it is quite the driving force to continue. My family has also been curious about my research, excited for my next steps, and supportive in many other ways. I also need to thank Uncle Bill for putting a roof over my head during my graduate studies and regularly asking how my “sea monkeys” are doing. I find v great fun in scientific research, but I have also been lucky to have great fun outside of lab, thanks to absolutely wonderful friends I have made over the years. My friends in North Carolina, including my graduate school colleagues, non-scientist friends, and all those who were members of the Cotter Lab, have made it so I will always cherish my time in graduate school, and I am excited to continue our friendships. My friends from before starting graduate school have brought me tremendous joy for many years, and I am lucky they will continue to do so long into my future. Also, I believe they still have not called me “nerd”! vi TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................... ix LIST OF TABLES ........................................................................................................................ xi LIST OF ABBREVIATIONS ....................................................................................................... xii CHAPTER 1 – IntroduCtion ........................................................................................................ 1 1.1 Microbial Communities ........................................................................................................ 1 1.2 Microbial Cooperation within Communities ......................................................................... 2 1.3 Microbial Competition within Communities ......................................................................... 2 1.4 Contact-Dependent Growth Inhibition ................................................................................. 3 1.5 Type VI Secretion Systems ................................................................................................. 7 1.6 Polymicrobial Communities within the Cystic Fibrosis Respiratory Tract ......................... 10 1.7 Pseudomonas aeruginosa ................................................................................................ 12 1.8 The Burkholderia cepacia Complex .................................................................................. 15 1.9 Objectives of this Dissertation ........................................................................................... 18 1.10 References ...................................................................................................................... 21 CHAPTER 2 – Three Distinct ContaCt-Dependent Growth Inhibition Systems Mediate InterbaCterial Competition by the CystiC Fibrosis Pathogen Burkholderia dolosa ................................................................................................ 40 2.1 Summary ........................................................................................................................... 40 2.2 Importance ........................................................................................................................ 41 2.3 Introduction ....................................................................................................................... 41 2.4 Materials and Methods ...................................................................................................... 43 2.5 Results .............................................................................................................................. 48 2.6 Discussion ......................................................................................................................... 56 2.7 References ........................................................................................................................ 73 CHAPTER 3 – Host Adaptation Predisposes Pseudomonas aeruginosa to Type VI SeCretion System-Mediated Predation by the Burkholderia cepacia Complex ...................................................................................................................... 78 vii 3.1 Summary ........................................................................................................................... 78 3.2 Introduction ....................................................................................................................... 78 3.3 Results .............................................................................................................................. 80 3.4 Discussion ......................................................................................................................... 88 3.5 Method Details .................................................................................................................. 94 3.6 References ....................................................................................................................