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

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.

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

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.

Effect of acidic pH on the invasion efficiency and the type III secretion system of Burkholderia thailandensis.

Burkholderia thailandensis is a close relative of Burkholderia pseudomallei. These organisms are very similar, but B. thailandensis is far less virulent than B. pseudomallei. Nucleotide sequencing and analysis of 14 B. thailandensis isolates revealed variation in the regions coding for the type III secreted BipD protein. The degree of B. thailandensis BipD sequence variation was greater than that found in B. pseudomallei. Western blot analysis indicated that, unlike B. pseudomallei, B. thailandensis type III secreted proteins including BipD and BopE could not be detected in the supernatant of culture medium unless induced by acidic conditions. In addition, culturing B. thailandensis under acidic growth conditions (pH 4.5) can induce the ability of this bacterium to invade human respiratory epithelial cells A549. The identification of an environmental stimulus that increases the invasion capability of B. thailandensis invasion is of value for those who would like to use this bacterium as a model to study B. pseudomallei virulence.