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Evolutionary analysis of Burkholderia pseudomallei identifies putative novel virulence genes, including a microbial regulator of host cell autophagy.

ABSTRACT: Burkholderia pseudomallei, the causative agent of melioidosis, contains a large pathogen genome (7.2 Mb) with ?2,000 genes of putative or unknown function. Interactions with potential hosts and environmental factors may induce rapid adaptations in these B. pseudomallei genes, which can be discerned through evolutionary analysis of multiple B. pseudomallei genomes. Here we show that several previously uncharacterized B. pseudomallei genes bearing genetic signatures of rapid adaptation (positive selection) can induce diverse cellular phenotypes when expressed in mammalian cells. Notably, several of these phenotypes are plausibly related to virulence, including multinuclear giant cell formation, apoptosis, and autophagy induction. Specifically, we show that BPSS0180, a type VI cluster-associated gene, is capable of inducing autophagy in both phagocytic and nonphagocytic mammalian cells. Following infection of macrophages, a B. pseudomallei mutant disrupted in BPSS0180 exhibited significantly decreased colocalization with LC3 and impaired intracellular survival; these phenotypes were rescued by introduction of an intact BPSS0180 gene. The results suggest that BPSS0180 may be a novel inducer of host cell autophagy that contributes to B. pseudomallei intracellular growth. More generally, our study highlights the utility of applying evolutionary principles to microbial genomes to identify novel virulence genes.

SUBMITTER: Singh AP

PROVIDER: S-EPMC3889600 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Evolutionary analysis of Burkholderia pseudomallei identifies putative novel virulence genes, including a microbial regulator of host cell autophagy.

Singh Arvind Pratap AP Lai Shu-chin SC Nandi Tannistha T Chua Hui Hoon HH Ooi Wen Fong WF Ong Catherine C Boyce John D JD Adler Ben B Devenish Rodney J RJ Tan Patrick P

Journal of bacteriology 20131004 24

Burkholderia pseudomallei, the causative agent of melioidosis, contains a large pathogen genome (7.2 Mb) with ∼2,000 genes of putative or unknown function. Interactions with potential hosts and environmental factors may induce rapid adaptations in these B. pseudomallei genes, which can be discerned through evolutionary analysis of multiple B. pseudomallei genomes. Here we show that several previously uncharacterized B. pseudomallei genes bearing genetic signatures of rapid adaptation (positive s ...[more]

PMID: 24097950

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Project description:Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, often occupies environmental niches and infection sites characterized by limited concentrations of oxygen. Versatile genomic features enable this pathogen to maintain its physiology and virulence under hypoxia, but the crucial regulatory networks employed to switch from oxygen dependent respiration to alternative terminal electron acceptors (TEA) like nitrate, remains poorly understood. Here, we combined a Tn5 transposon mutagenesis screen and an anaerobic growth screen to identify a two-component signal transduction system with homology to RegAB. We show that RegAB is not only essential for anaerobic growth, but also for full virulence in cell lines and a mouse infection model. Further investigations of the RegAB regulon, using a global transcriptomic approach, identified 20 additional regulators under transcriptional control of RegAB, indicating a superordinate role of RegAB in the B. pseudomallei anaerobiosis regulatory network. Of the 20 identified regulators, NarX/L and a FNR homolog were selected for further analyses and a role in adaptation to anaerobic conditions was demonstrated. Growth experiments identified nitrate and intermediates of the denitrification process as the likely signal activateing RegAB, NarX/L, and probably of the downstream regulators Dnr or NsrR homologs. While deletions of individual genes involved in the denitrification process demonstrated their important role in anaerobic fitness, they showed no effect on virulence. This further highlights the central role of RegAB as the master regulator of anaerobic metabolism in B. pseudomallei and that the complete RegAB-mediated response is required to achieve full virulence. In summary, our analysis of the RegAB-dependent modulon and its interconnected regulons revealed a key role for RegAB of B. pseudomallei in the coordination of the response to hypoxic conditions and virulence, in the environment and the host.

Project description:BackgroundBurkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while glanders caused by B. mallei infections are rare. Here we studied the proteomes of different B. mallei and B. pseudomallei isolates to determine species specific characteristics.MethodsThe expressed proteins of 5 B. mallei and 6 B. pseudomallei strains were characterized using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Subsequently, expression of potential resistance and virulence related characteristics were analyzed and compared.ResultsProteome analysis can be used for the identification of B. mallei and B. pseudomallei. Both species were identified based on >60 discriminative peptides. Expression of proteins potentially involved in antimicrobial resistance, AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, PenA as well as several other efflux pump related proteins and putative β-lactamases was demonstrated. Despite, the fact that efflux pump BpeAB-OprB was expressed in all isolates, no clear correlation with an antimicrobial phenotype and the efflux-pump could be established. Also consistent with the phenotypes, no amino acid mutations in PenA known to result in β-lactam resistance could be identified. In all studied isolates, the expression of virulence (related) factors Capsule-1 and T2SS was demonstrated. The expression of T6SS-1 was demonstrated in all 6 B. pseudomallei isolates and in 2 of the 5 B. mallei isolates. In all, except one B. pseudomallei isolate, poly-beta-1,6 N-acetyl-D-glucosamine export porin (Pga), important for biofilm formation, was detected, which were absent in the proteomes of B. mallei. Siderophores, iron binding proteins, malleobactin and malleilactone are possibly expressed in both species under standard laboratory growth conditions. Expression of multiple proteins from both the malleobactin and malleilactone polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters was demonstrated in both species. All B. pseudomallei expressed at least seven of the nine proteins of the bactobolin synthase cluster (bactobolin, is a ribosome targeting antibiotic), while only in one B. mallei isolate expression of two proteins of this synthase cluster was identified.ConclusionsAnalyzing the expressed proteomes revealed differences between B. mallei and B. pseudomallei but also between isolates from the same species. Proteome analysis can be used not only to identify B. mallei and B. pseudomallei but also to characterize the presence of important factors that putatively contribute to the pathogenesis of B. mallei and B. pseudomallei.

Project description:Burkholderia pseudomallei is a soil-dwelling bacterium able to survive not only under adverse environmental conditions, but also within various hosts which can lead to the disease melioidosis. The capability of B. pseudomallei to adapt to environmental changes is facilitated by the large number of regulatory proteins encoded by its genome. Among them are more than 60 uncharacterized LysR-type transcriptional regulators (LTTRs). Here we analyzed a B. pseudomallei mutant harboring a transposon in the gene BPSL0117 annotated as a LTTR, which we named gvmR (globally acting virulence and metabolism regulator). The gvmR mutant displayed a growth defect in minimal medium and macrophages in comparison with the wild type. Moreover, disruption of gvmR rendered B. pseudomallei avirulent in mice indicating a critical role of GvmR in infection. These defects of the mutant were rescued by ectopic expression of gvmR. To identify genes whose expression is modulated by GvmR, global transcriptome analysis of the B. pseudomallei wild type and gvmR mutant was performed using whole genome tiling microarrays. Transcript levels of 190 genes were upregulated and 141 genes were downregulated in the gvmR mutant relative to the wild type. Among the most downregulated genes in the gvmR mutant were important virulence factor genes (T3SS3, T6SS1, and T6SS2), which could explain the virulence defect of the gvmR mutant. In addition, expression of genes related to amino acid synthesis, glyoxylate shunt, iron-sulfur cluster assembly, and syrbactin metabolism (secondary metabolite) was decreased in the mutant. On the other hand, inactivation of GvmR increased expression of genes involved in pyruvate metabolism, ATP synthesis, malleobactin, and porin genes. Quantitative real-time PCR verified the differential expression of 27 selected genes. In summary, our data show that GvmR acts as an activating and repressing global regulator that is required to coordinate expression of a diverse set of metabolic and virulence genes essential for the survival in the animal host and under nutrient limitation.

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Evolutionary analysis of Burkholderia pseudomallei identifies putative novel virulence genes, including a microbial regulator of host cell autophagy.

Publications

Evolutionary analysis of Burkholderia pseudomallei identifies putative novel virulence genes, including a microbial regulator of host cell autophagy.

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