Biofilm dynamics and the response to N-oxides in Burkholderia pseudomallei

2019 Summer.Includes bibliographical references.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

Nitrate Sensing and Metabolism Inhibit Biofilm Formation in the Opportunistic Pathogen Burkholderia pseudomallei by Reducing the Intracellular Concentration of c-di-GMP

The opportunistic pathogen Burkholderia pseudomallei is a saprophytic bacterium and the causative agent of melioidosis, an emerging infectious disease associated with high morbidity and mortality. Although melioidosis is most prevalent during the rainy season in endemic areas, domestic gardens and farms can also serve as a reservoir for B. pseudomallei during the dry season, in part due to irrigation and fertilizer use. In the environment, B. pseudomallei forms biofilms and persists in soil near plant root zones. Biofilms are dynamic bacterial communities whose formation is regulated by extracellular cues and corresponding changes in the nearly universal secondary messenger cyclic dimeric GMP. Recent studies suggest B. pseudomallei loads are increased by irrigation and the addition of nitrate-rich fertilizers, whereby such nutrient imbalances may be linked to the transmission epidemiology of this important pathogen. We hypothesized that exogenous nitrate inhibits B. pseudomallei biofilms by reducing the intracellular concentration of c-di-GMP. Bioinformatics analyses revealed B. pseudomallei 1026b has the coding capacity for nitrate sensing, metabolism, and transport distributed on both chromosomes. Using a sequence-defined library of B. pseudomallei 1026b transposon insertion mutants, we characterized the role of denitrification genes in biofilm formation in response to nitrate. Our results indicate that the denitrification pathway is implicated in B. pseudomallei biofilm growth dynamics and biofilm formation is inhibited by exogenous addition of sodium nitrate. Genomics analysis identified transposon insertional mutants in a predicted two-component system (narX/narL), a nitrate reductase (narGH), and a nitrate transporter (narK-1) required to sense nitrate and alter biofilm formation. Additionally, the results presented here show that exogenous nitrate reduces intracellular levels of the bacterial second messenger c-di-GMP. These results implicate the role of nitrate sensing in the regulation of a c-di-GMP phosphodiesterase and the corresponding effects on c-di-GMP levels and biofilm formation in B. pseudomallei 1026b

Genome-scale analysis of the genes that contribute to <i>Burkholderia pseudomallei</i> biofilm formation identifies a crucial exopolysaccharide biosynthesis gene cluster

<div><p><i>Burkholderia pseudomallei</i>, the causative agent of melioidosis, is an important public health threat due to limited therapeutic options for treatment. Efforts to improve therapeutics for <i>B</i>. <i>pseudomallei</i> infections are dependent on the need to understand the role of <i>B</i>. <i>pseudomallei</i> biofilm formation and its contribution to antibiotic tolerance and persistence as these are bacterial traits that prevent effective therapy. In order to reveal the genes that regulate and/or contribute to <i>B</i>. <i>pseudomallei</i> 1026b biofilm formation, we screened a sequence defined two-allele transposon library and identified 118 transposon insertion mutants that were deficient in biofilm formation. These mutants include transposon insertions in genes predicted to encode flagella, fimbriae, transcriptional regulators, polysaccharides, and hypothetical proteins. Polysaccharides are key constituents of biofilms and <i>B</i>. <i>pseudomallei</i> has the capacity to produce a diversity of polysaccharides, thus there is a critical need to link these biosynthetic genes with the polysaccharides they produce to better understand their biological role during infection. An allelic exchange deletion mutant of the entire <i>B</i>. <i>pseudomallei</i> biofilm-associated exopolysaccharide biosynthetic cluster was decreased in biofilm formation and produced a smooth colony morphology suggestive of the loss of exopolysaccharide production. Conversely, deletion of the previously defined capsule I polysaccharide biosynthesis gene cluster increased biofilm formation. Bioinformatics analyses combined with immunoblot analysis and glycosyl composition studies of the partially purified exopolysaccharide indicate that the biofilm-associated exopolysaccharide is neither cepacian nor the previously described acidic exopolysaccharide. The biofilm-associated exopolysaccharide described here is also specific to the <i>B</i>. <i>pseudomallei</i> complex of bacteria. Since this novel exopolysaccharide biosynthesis cluster is retained in <i>B</i>. <i>mallei</i>, it is predicted to have a role in colonization and infection of the host. These findings will facilitate further advances in understanding the pathogenesis of <i>B</i>. <i>pseudomallei</i> and improve diagnostics and therapeutic treatment strategies.</p></div

Summary of phenotypes (biofilm, motility, growth, and colony morphology) along with genetic annotations for selected biofilm-deficient transposon insertion mutants.

<p>Summary of phenotypes (biofilm, motility, growth, and colony morphology) along with genetic annotations for selected biofilm-deficient transposon insertion mutants.</p

Deletion of biofilm exopolysaccharide gene cluster alters biofilm formation and growth on NAP-A plates, but not motility.

<p>(A) Biofilm formation of wild type, Δ<i>becA-R</i> (biofilm EPS deficient), Δ<i>wcbR-A</i> (CPSI deficient), and Δ<i>wcbR-A</i> Δ<i>becA-R</i> (CPSI and biofilm EPS deficient) strains after 24 h at 37°C. (B) Pellicle formation of wild type and deletion mutants after six days at 37°C. (C) Swim zone diameters (cm) of the wild type and deletion mutants after 24 h at 37°C. (D) Overnight cultures were spotted onto NAP-A plates and grown for two day at 37°C. Asterisks indicate a significant difference as obtained with a paired Student’s t-test utilizing for the biofilm data and the Mann-Whitney test for the swim motility data utilizing a p-value of 0.001. Error bars indicate standard error of the mean.</p

<i>B</i>. <i>pseudomallei</i> T24 transposon mutants impaired in biofilm formation.

<p>The wild type and 37 transposon mutants were grown statically for 24 h at 37°C in polystyrene plates. Biofilm formation was quantified using crystal violet. All transposon mutants exhibited at least a 40% decrease and were tested at least twice in replicates of six. Error bars indicate standard error of the mean.</p

Swimming motility of <i>B</i>. <i>pseudomallei</i> T24 transposon mutants.

<p>Overnight cultures of the wild type and transposon mutants were used to inoculate 0.3% agar plates, incubated at 37°C, and swim zone diameter was measured at 24 h. Asterisks indicate a significant difference as obtained with the Mann-Whitney test utilizing the Bonferroni correction (p = 0.001) to account for multiple comparisons (n = 37). All mutants were tested at least twice in triplicate. Error bars indicate standard error of the mean.</p

Biofilm-associated exopolysaccharide gene cluster from <i>B</i>. <i>pseudomallei</i> and comparative analysis with <i>B</i>. <i>cenocepacia</i>.

<p>The putative exopolysaccharide gene clusters from the sequenced genomes of <i>B</i>. <i>pseudomallei</i> 1026b (top) and <i>B</i>. <i>cenocepacia</i> J2315 (bottom). A total of 18 loci spanning Bp1026b_I2907-Bp1026b_I2927 (<i>becA</i>-<i>R</i>) on chromosome I of <i>B</i>. <i>pseudomallei</i> 1026b, not including a cluster of three pseudogenes, are aligned with 21 loci spanning BCAM1330-BCAM1350 on chromosome II of <i>B</i>. <i>cenocepacia</i> J2315. Coding sequences are depicted by arrows per positive or negative strand orientation. Sizes of genes and intergenic regions are to scale. The results of BLASTN annotations with minimum identity of 60% and threshold E-value of 1E-3 are aligned to regions of similarity. Red bars depict sequence inversions and blue bars depict direct homology in a color density gradient.</p

Complementation of I1954, I2907 (<i>becA</i>), and II2527 <i>FRT</i> mutants in the static biofilm assay and colony morphology on NAP-A agar plates.

<p>(A) Biofilm formation and (B) colony morphology of complemented I1954, I2907 (<i>becA</i>), and II2527 FRT mutants. EV indicates empty vector. Complementation was induced with 1mM IPTG. Asterisks indicate a significant difference as obtained with a pairwise Student’s t-test utilizing a p-value of 0.001. Error bars indicate standard error of the mean.</p

Summary of phenotypes (biofilm, motility, growth, and colony morphology) for selected transposon insertion and deletion mutants in previously published genes that contribute to biofilm formation.

<p>Summary of phenotypes (biofilm, motility, growth, and colony morphology) for selected transposon insertion and deletion mutants in previously published genes that contribute to biofilm formation.</p