Transcriptional profiles of Burkholderia pseudomallei reveal the direct and indirect roles of Sigma E under oxidative stress conditions

This is the final version of the article. Available from BioMed Central via the DOI in this record.BACKGROUND: Burkholderia pseudomallei, the causative agent of melioidosis, is a Gram-negative bacterium widely distributed in soil and water in endemic areas. This soil saprophyte can survive harsh environmental conditions, even in soils where herbicides (containing superoxide generators) are abundant. Sigma factor E (σE) is a key regulator of extra-cytoplasmic stress response in Gram-negative bacteria. In this study, we identified the B. pseudomallei σE regulon and characterized the indirect role that σE plays in the regulation of spermidine, contributing to the successful survival of B. pseudomallei in stressful environments. RESULTS: Changes in the global transcriptional profiles of B. pseudomallei wild type and σE mutant under physiological and oxidative stress (hydrogen peroxide) conditions were determined. We identified 307 up-regulated genes under oxidative stress condition. Comparison of the transcriptional profiles of B. pseudomallei wild type and σE mutant under control or oxidative stress conditions identified 85 oxidative-responsive genes regulated by σE, including genes involved in cell membrane repair, maintenance of protein folding and oxidative stress response and potential virulence factors such as a type VI secretion system (T6SS). Importantly, we identified that the speG gene, encoding spermidine-acetyltransferase, is a novel member of the B. pseudomallei σE regulon. The expression of speG was regulated by σE, implying that σE plays an indirect role in the regulation of physiological level of spermidine to protect the bacteria during oxidative stress. CONCLUSION: This study identified B. pseudomallei genes directly regulated by σE in response to oxidative stress and revealed the indirect role of σE in the regulation of the polyamine spermidine (via regulation of speG) for bacterial cell protection during oxidative stress. This study provides new insights into the regulatory mechanisms by which σE contributes to the survival of B. pseudomallei under stressful conditions.This work was supported by the National Science and Technology Development Agency and Siriraj Grant for Research and Development. S. Jitprasutwit was supported by the Royal Golden Jubilee Ph. D. Program (PHD0270/2551)

Analysis of the role of the QseBC two-component sensory system in epinephrine-induced motility and intracellular replication of Burkholderia pseudomallei

Burkholderia pseudomallei is a facultative intracellular bacterial pathogen that causes melioidosis, a severe invasive disease of humans. We previously reported that the stress-related catecholamine hormone epinephrine enhances motility of B. pseudomallei, transcription of flagellar genes and the production of flagellin. It has been reported that the QseBC two-component sensory system regulates motility and virulence-associated genes in other Gram-negative bacteria in response to stress-related catecholamines, albeit disparities between studies exist. We constructed and whole-genome sequenced a mutant of B. pseudomallei with a deletion spanning the predicted qseBC homologues (bpsl0806 and bpsl0807). The ΔqseBC mutant exhibited significantly reduced swimming and swarming motility and reduced transcription of fliC. It also exhibited a defect in biofilm formation and net intracellular survival in J774A.1 murine macrophage-like cells. While epinephrine enhanced bacterial motility and fliC transcription, no further reduction in these phenotypes was observed with the ΔqseBC mutant in the presence of epinephrine. Plasmid-mediated expression of qseBC suppressed bacterial growth, complicating attempts to trans-complement mutant phenotypes. Our data support a role for QseBC in motility, biofilm formation and net intracellular survival of B. pseudomallei, but indicate that it is not essential for epinephrine-induced motility per se

Identification of Burkholderia pseudomallei Genes Induced During Infection of Macrophages by Differential Fluorescence Induction

This is the final version. Available on open access from Frontiers media via the DOI in this recordData Availability Statement: The datasets GENERATED for this study can be found in the SNBI SRA accession PRJNA599542.Burkholderia pseudomallei, the causative agent of melioidosis, can survive and replicate in macrophages. Little is known about B. pseudomallei genes that are induced during macrophage infection. We constructed a B. pseudomallei K96243 promoter trap library with genomic DNA fragments fused to the 5′ end of a plasmid-borne gene encoding enhanced green fluorescent protein (eGFP). Microarray analysis showed that the library spanned 88% of the B. pseudomallei genome. The recombinant plasmids were introduced into Burkholderia thailandensis E264, and promoter fusions active during in vitro culture were removed. J774A.1 murine macrophages were infected with the promoter trap library, and J774A.1 cells containing fluorescent bacteria carrying plasmids with active promoters were isolated using flow cytometric-based cell sorting. Candidate macrophage-induced B. pseudomallei genes were identified from the location of the insertions containing an active promoter activity. A proportion of the 138 genes identified in this way have been previously reported to be involved in metabolism and transport, virulence, or adaptation. Novel macrophage-induced B. pseudomallei genes were also identified. Quantitative reverse-transcription PCR analysis of 13 selected genes confirmed gene induction during macrophage infection. Deletion mutants of two macrophage-induced genes from this study were attenuated in Galleria mellonella larvae, suggesting roles in virulence. B. pseudomallei genes activated during macrophage infection may contribute to intracellular life and pathogenesis and merit further investigation toward control strategies for melioidosis.Mahidol University, ThailandThailand Research Fun

The Monofunctional Catalase KatE of Xanthomonas axonopodis pv. citri Is Required for Full Virulence in Citrus Plants

BACKGROUND: Xanthomonas axonopodis pv. citri (Xac) is an obligate aerobic phytopathogen constantly exposed to hydrogen peroxide produced by normal aerobic respiration and by the plant defense response during plant-pathogen interactions. Four putative catalase genes have been identified in silico in the Xac genome, designated as katE, catB, srpA (monofunctional catalases) and katG (bifunctional catalase). METHODOLOGY/PRINCIPAL FINDINGS: Xac catalase activity was analyzed using native gel electrophoresis and semi-quantitative RT-PCR. We demonstrated that the catalase activity pattern was regulated in different growth stages displaying the highest levels during the stationary phase. KatE was the most active catalase in this phase of growth. At this stage cells were more resistant to hydrogen peroxide as was determined by the analysis of CFU after the exposition to different H(2)O(2) concentrations. In addition, Xac exhibited an adaptive response to hydrogen peroxide, displaying higher levels of catalase activity and H(2)O(2) resistance after treatment with sub-lethal concentrations of the oxidant. In the plant-like medium XVM2 the expression of KatE was strongly induced and in this medium Xac was more resistant to H(2)O(2). A XackatE mutant strain was constructed by insertional mutagenesis. We observed that catalase induction in stationary phase was lost meanwhile the adaptive response to peroxide was maintained in this mutant. Finally, the XackatE strain was assayed in planta during host plant interaction rendering a less aggressive phenotype with a minor canker formation. CONCLUSIONS: Our results confirmed that in contrast to other Xanthomonas species, Xac catalase-specific activity is induced during the stationary phase of growth in parallel with the bacterial resistance to peroxide challenge. Moreover, Xac catalases expression pattern is modified in response to any stimuli associated with the plant or the microenvironment it provides. The catalase KatE has been shown to have an important function for the colonization and survival of the bacterium in the citrus plant during the pathogenic process. Our work provides the first genetic evidence to support a monofunctional catalase as a virulence factor in Xac

Methionine Sulfoxide Reductase A (MsrA) Deficient Mycoplasma genitalium Shows Decreased Interactions with Host Cells

Mycoplasma genitalium is an important sexually transmitted pathogen that affects both men and women. In genital-mucosal tissues, it initiates colonization of epithelial cells by attaching itself to host cells via several identified bacterial ligands and host cell surface receptors. We have previously shown that a mutant form of M. genitalium lacking methionine sulfoxide reductase A (MsrA), an antioxidant enzyme which converts oxidized methionine (Met(O)) into methionine (Met), shows decreased viability in infected animals. To gain more insights into the mechanisms by which MsrA controls M. genitalium virulence, we compared the wild-type M. genitalium strain (G37) with an msrA mutant (MS5) strain for their ability to interact with target cervical epithelial cell lines (HeLa and C33A) and THP-1 monocytic cells. Infection of epithelial cell lines with both strains revealed that MS5 was less cytotoxic to HeLa and C33A cell lines than the G37 strain. Also, the MS5 strain was more susceptible to phagocytosis by THP-1 cells than wild type strain (G37). Further, MS5 was less able to induce aggregation and differentiation in THP-1 cells than the wild type strain, as determined by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the cells, followed by counting of cells attached to the culture dish using image analysis. Finally, MS5 was observed to induce less proinflammatory cytokine TNF-α by THP-1 cells than wild type G37 strain. These results indicate that MsrA affects the virulence properties of M. genitalium by modulating its interaction with host cells

Transcriptional profiles of Burkholderia pseudomallei reveal the direct and indirect roles of Sigma E under oxidative stress conditions

10.1186/1471-2164-15-787BMC Genomics15178