DISSERTATION BIOFILM DYNAMICS AND THE RESPONSE TO N-OXIDES IN Burkholderia pseudomallei Submitted by: Mihnea R. Mangalea Department of Microbiology, Immunology and Pathology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Summer 2019 Doctoral Committee: Advisor: Bradley R. Borlee Richard A. Slayden Richard A. Bowen Mark D. Stenglein Amy O. Charkowski Copyright by Mihnea R. Mangalea 2019 All Rights Reserved ABSTRACT BIOFILM DYNAMICS AND THE RESPONSE TO N-OXIDES IN Burkholderia pseudomallei Burkholderia pseudomallei is a saprophytic bacterium inhabiting wet soils in tropical regions and is the causative agent of melioidosis, an emerging infectious disease of high mortality. Although the incidence of melioidosis is more prevalent in the monsoonal wet season in Southeast Asia and Northern Australia, gardens and farms also serve as a reservoir for B. pseudomallei infection in the dry season, due to anthropogenic disturbances including irrigation and application of nitrogen (N)-based fertilizer use. Melioidosis is historically associated with rice farming in rural regions of the tropics where rain-fed lowland environments predominate and planting fields are often managed by the addition of N-based fertilizers to keep up with the demand for global rice consumption. In these oxygen-limiting environments, B. pseudomallei is a facultative anaerobic organism capable of growth in anoxic conditions by substituting nitrate - (NO3 ) as a terminal electron acceptor. B. pseudomallei is capable of complete denitrification, a step-wise enzymatic reaction that is carried out by four individual enzyme complexes or - reductases, that reduce NO3 to N2. Denitrification among proteobacteria is regulated by sensing systems that depend on both the presence of substrate and hypoxic conditions, however little is known about this ecological and physiological phenomenon in B. pseudomallei. In hosts infected with B. pseudomallei, similar oxygen tensions are experienced by the organisms in abscesses, lesions, and during intracellular growth; however, little is known regarding the extent of anaerobic metabolism and defense from host-associated reactive nitrogen intermediates in B. pseudomallei. This study examines the predicted nitrate sensing and metabolism genes in a clinical isolate, B. pseudomallei 1026b, and specifically their role in regulating biofilm dynamics. We hypothesized that nitrate sensing and metabolism negatively regulate biofilm formation and aimed to describe the genetic and metabolic determinants of this phenotype in B. pseudomallei. ii In Aim I of this study, we characterized a dose-dependent biofilm inhibition model that responds to increasing concentrations of sodium nitrate and sodium nitrite, donors of the - - inorganic anions NO3 and NO2 , respectively. Based on in silico analyses of predicted nitrate sensing and metabolism loci, we screened transposon insertional mutants to identify candidates involved in the biofilm inhibitory response. We identified five mutants that no longer respond to nitrate-mediated biofilm inhibition in genes predicted to comprise key components of the denitrification pathway: the alpha and beta subunits of the dissimilatory nitrate reductase narGHJI-1, the narX-narL two-component regulatory system, and the nitrate/nitrite extrusion gene narK-1. Using LC-MS/MS, we quantified the intracellular concentration of the secondary metabolite cyclic-di-GMP, and observed a significant decrease of this key biofilm-associated molecule in response to sodium nitrate treatment. Furthermore, we evaluated the expression of cyclic-di-GMP regulatory enzymes to propose a mechanism for the nitrate-dependent biofilm inhibition phenotype in B. pseudomallei. In Aim II, we examined the functions of NarX and NarL in response to exogenous sodium nitrate and sodium nitrite and the biofilm inhibition model using separate in-frame deletion mutants. We characterized a disparity in biofilm inhibition that is dependent on nitrate but not nitrite in this two-component sensing system, before analyzing the global transcriptome of these mutants relative to the wild type in growth conditions supplemented with either N-oxide. Differential expression analysis of RNA sequencing reads revealed significant transcriptomic shifts in several gene clusters associated with biofilm formation, nitrate metabolism, general metabolism, antibiotic resistance, virulence, and secondary metabolite biosynthesis that - - responded similarly to both NO3 and NO2 supplementation. Additionally, we demonstrated that narX and narL mutants are deficient in intracellular survival in murine macrophages, providing a link between nitrate sensing and metabolism and B. pseudomallei host-pathogen interactions. These data suggest that denitrification is an important mechanism for biofilm dynamics and is also relevant to survival and pathogenicity in animal hosts during B. pseudomallei infection. iii ACKNOWLEDGEMENTS The work presented here would not have been possible without support, guidance, and inspiration from several people. First and foremost, I would like to thank Dr. Brad Borlee for his mentorship and for presenting me with challenging opportunities to do exciting research. Joining the Borlee Lab was a great experience and I consider myself a much better scientist today because of Brad’s training and direction through this entire process. I am thankful for his efforts to challenge me to think independently, take initiative with project collaborations, learn new techniques, improve my writing, and keep me focused. I have appreciated our almost weekly meetings and discussions of research progress, career options, conferences and seminars to attend, networking opportunities, and so much more. I am grateful for his encouragement and motivation to broaden my scope and expand my research capabilities. There are many other current and former members of the Borlee Lab to thank for putting me in a position to succeed. I would like to thank Dr. Grace Borlee for tremendous support in refining my ideas, goals, presentations, and manuscripts. I am thankful for our discussions on science, life, and everything in between. I have enjoyed working alongside Grace in the lab and on several publications and I appreciate her mentorship, friendship, and motivation to seek out opportunities that advance my career. In addition, I want to thank Brooke Plumley and Kevin Martin for being the best lab-mates, friends, and co-workers I could have asked for. Thank you for always actively listening to my presentations and offering helpful suggestions to improve my research and refine my experiments. Thank you for being my barrier buddies and keeping me sane after long hours in the confines of the BSL-3. Thank you to Ian McMillan, honorary member of the Borlee Lab from the University of Hawaii at Manoa, for all the support and thoughtful discussions on B. pseudomallei biology. Also, thank you to all the members of the Borlee Lab with whom I have interacted with during my time at CSU: Lily Filipowska, Maria Brock, Jordanne Lesher, Michelle Khale, Nick Bartolillo, Molly Newton, Reed Woyda, and Sam iv Golon. Each of you has helped make my time in the lab more enjoyable and I appreciate your friendship, support, and willingness to listen to my research updates. Thank you to my committee members for taking great interest in my research, offering your time and support to meet with me, and generally improving the quality of my work. I owe a great deal of gratitude to Drs. Ric Slayden, Dick Bowen, Mark Stenglein, and Amy Charkowski. Thank you all for challenging me to become a better scientist and answer difficult questions, and keeping me on course for a career in research. Thank you to Drs. Brendan Podell and Mark Zabel for allowing me to rotate in their labs at the beginning of my graduate studies and teaching me techniques in immunology that were fundamental to my development as a microbiologist. Thank you to all the following professors in MIP that have been a part of my education and professional development: Drs. Carol Wilusz, Brian Geiss, Jeff Wilusz, Gregg Dean, Zaid Abdo, Bob Ellis, among many others. Thank you to Heidi Runge for facilitating all the logistics of navigating the MIP graduate program. Thank you to all members of the GAUSSI program at CSU that considered me worthy of a year-long fellowship and the worthwhile training in computational biology. Being part of the GAUSSI program offered great learning opportunities to think outside the box of my specific research niche and to see bacteriology and infectious disease through the eyes of a programmer as well as a biologist. There are many more colleagues, friends, past advisors and mentors, that I should thank for helping me get to this monumental step in my career, and I appreciate everyone that has guided me along my research and academic path, from the University of North Carolina to Colorado State University. To the global melioidosis research community that seeks to mitigate the public health threat from B. pseudomallei infections, I am forever inspired and motivated by the impactful work being done worldwide and am grateful to consider myself part of this diverse research community. And to my love, Dr. Rachel West, thank you for the tremendous support that has kept me going through everything; I look forward to celebrating all future achievements, scientific or otherwise, with you. v DEDICATION To my mother, Smaranda, who sacrificed much to offer me