Biofilm dynamics and the response to N-oxides in Burkholderia pseudomallei
Date
2019
Authors
Mangalea, Mihnea R., author
Borlee, Bradley R., advisor
Slayden, Richard A., committee member
Bowen, Richard A., committee member
Stenglein, Mark D., committee member
Charkowski, Amy O., committee member
Journal Title
Journal ISSN
Volume Title
Abstract
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. 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.