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)

Detection and differentiation of Burkholderia species with pathogenic potential in environmental soil samples

The Burkholderia pseudomallei phylogenetic cluster includes B. pseudomallei, B. mallei, B. thailandensis, B. oklahomensis, B. humptydooensis and B. singularis. Regarded as the only pathogenic members of this group, B. pseudomallei and B. mallei cause the diseases melioidosis and glanders, respectively. Additionally, variant strains of B. pseudomallei and B. thailandensis exist that include the geographically restricted B. pseudomallei that express a B. mallei-like BimA protein (BPBM), and B. thailandensis that express a B. pseudomallei-like capsular polysaccharide (BTCV). To establish a PCR-based assay for the detection of pathogenic Burkholderia species or their variants, five PCR primers were designed to amplify species-specific sequences within the bimA (Burkholderia intracellular motility A) gene. Our multiplex PCR assay could distinguish pathogenic B. pseudomallei and BPBM from the non-pathogenic B. thailandensis and the BTCV strains. A second singleplex PCR successfully discriminated the BTCV from B. thailandensis. Apart from B. humptydooensis, specificity testing against other Burkholderia spp., as well as other Gram-negative and Gram-positive bacteria produced a negative result. The detection limit of the multiplex PCR in soil samples artificially spiked with known quantities of B. pseudomallei and B. thailandensis were 5 and 6 CFU/g soil, respectively. Furthermore, comparison between standard bacterial culture and the multiplex PCR to detect B. pseudomallei from 34 soil samples, collected from an endemic area of melioidosis, showed high sensitivity and specificity. This robust, sensitive, and specific PCR assay will be a useful tool for epidemiological study of B. pseudomallei and closely related members with pathogenic potential in soil

The condition-dependent transcriptional landscape of Burkholderia pseudomallei

This is the final version of the article. Available from the publisher via the DOI in this record.Burkholderia pseudomallei (Bp), the causative agent of the often-deadly infectious disease melioidosis, contains one of the largest prokaryotic genomes sequenced to date, at 7.2 Mb with two large circular chromosomes (1 and 2). To comprehensively delineate the Bp transcriptome, we integrated whole-genome tiling array expression data of Bp exposed to >80 diverse physical, chemical, and biological conditions. Our results provide direct experimental support for the strand-specific expression of 5,467 Sanger protein-coding genes, 1,041 operons, and 766 non-coding RNAs. A large proportion of these transcripts displayed condition-dependent expression, consistent with them playing functional roles. The two Bp chromosomes exhibited dramatically different transcriptional landscapes--Chr 1 genes were highly and constitutively expressed, while Chr 2 genes exhibited mosaic expression where distinct subsets were expressed in a strongly condition-dependent manner. We identified dozens of cis-regulatory motifs associated with specific condition-dependent expression programs, and used the condition compendium to elucidate key biological processes associated with two complex pathogen phenotypes--quorum sensing and in vivo infection. Our results demonstrate the utility of a Bp condition-compendium as a community resource for biological discovery. Moreover, the observation that significant portions of the Bp virulence machinery can be activated by specific in vitro cues provides insights into Bp's capacity as an "accidental pathogen", where genetic pathways used by the bacterium to survive in environmental niches may have also facilitated its ability to colonize human hosts.This work was funded by a core grant provided by the Agency for Science, Technology and Research to the Genome Institute of Singapore, and funding from the Defence Medical and Environmental Research Institute, Singapore. This work was supported in part through NIAID contract HHSN266200400035C to BWS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Analysis of the prevalence, secretion and function of a cell cycle-inhibiting factor in the melioidosis pathogen Burkholderia pseudomallei

Enteropathogenic and enterohaemorrhagic Escherichia coli express a cell cycle-inhibiting factor (Cif), that is injected into host cells via a Type III secretion system (T3SS) leading to arrest of cell division, delayed apoptosis and cytoskeletal rearrangements. A homologue of Cif has been identified in Burkholderia pseudomallei (CHBP; Cif homologue in B. pseudomallei; BPSS1385), which shares catalytic activity, but its prevalence, secretion and function are ill-defined. Among 43 available B. pseudomallei genome sequences, 33 genomes (76.7%) harbor the gene encoding CHBP. Western blot analysis using antiserum raised to a synthetic CHBP peptide detected CHBP in 46.6% (7/15) of clinical B. pseudomallei isolates from the endemic area. Secretion of CHBP into bacterial culture supernatant could not be detected under conditions where a known effector (BopE) was secreted in a manner dependent on the Bsa T3SS. In contrast, CHBP could be detected in U937 cells infected with B. pseudomallei by immunofluorescence microscopy and Western blotting in a manner dependent on bsaQ. Unlike E. coli Cif, CHBP was localized within the cytoplasm of B. pseudomallei-infected cells. A B. pseudomallei chbP insertion mutant showed a significant reduction in cytotoxicity and plaque formation compared to the wild-type strain that could be restored by plasmid-mediated trans-complementation. However, there was no defect in actin-based motility or multinucleated giant cell formation by the chbP mutant. The data suggest that the level or timing of CHBP secretion differs from a known Bsa-secreted effector and that CHBP is required for selected virulence-associated phenotypes in vitro

Genomic Islands as a Marker to Differentiate between Clinical and Environmental Burkholderia pseudomallei

Burkholderia pseudomallei, as a saprophytic bacterium that can cause a severe sepsis disease named melioidosis, has preserved several extra genes in its genome for survival. The sequenced genome of the organism showed high diversity contributed mainly from genomic islands (GIs). Comparative genome hybridization (CGH) of 3 clinical and 2 environmental isolates, using whole genome microarrays based on B. pseudomallei K96243 genes, revealed a difference in the presence of genomic islands between clinical and environmental isolates. The largest GI, GI8, of B. pseudomallei was observed as a 2 sub-GI named GIs8.1 and 8.2 with distinguishable %GC content and unequal presence in the genome. GIs8.1, 8.2 and 15 were found to be more common in clinical isolates. A new GI, GI16c, was detected on chromosome 2. Presences of GIs8.1, 8.2, 15 and 16c were evaluated in 70 environmental and 64 clinical isolates using PCR assays. A combination of GIs8.1 and 16c (positivity of either GI) was detected in 70% of clinical isolates and 11.4% of environmental isolates (P<0.001). Using BALB/c mice model, no significant difference of time to mortality was observed between K96243 isolate and three isolates without GIs under evaluation (P>0.05). Some virulence genes located in the absent GIs and the difference of GIs seems to contribute less to bacterial virulence. The PCR detection of 2 GIs could be used as a cost effective and rapid tool to detect potentially virulent isolates that were contaminated in soil

Identification of Motifs of <em>Burkholderia pseudomallei</em> BimA Required for Intracellular Motility, Actin Binding, and Actin Polymerization

Actin-based motility of the melioidosis pathogen Burkholderia pseudomallei requires BimA (Burkholderia intracellular motility A). The mechanism by which BimA mediates actin assembly at the bacterial pole is ill-defined. Toward an understanding of the regions of B. pseudomallei BimA required for intracellular motility and the binding and polymerization of actin, we constructed plasmid-borne bimA variants and glutathione-S-transferase fusion proteins with in-frame deletions of specific motifs. A 13-amino-acid direct repeat and IP(7) proline-rich motif were dispensable for actin binding and assembly in vitro, and expression of the mutated proteins in a B. pseudomallei bimA mutant restored actin-based motility in J774.2 murine macrophage-like cells. However, two WASP homology 2 (WH2) domains were found to be required for actin binding, actin assembly, and plaque formation. A tract of five PDASX direct repeats influenced the polymerization of pyrene-actin monomers in vitro and was required for actin-based motility and intercellular spread, but not actin binding. None of the mutations impaired surface expression or polar targeting of BimA. The number of PDASX repeats varied in natural isolates from two to seven. Such repeats acted additively to promote pyrene-actin polymerization in vitro, with stepwise increases in the rate of polymerization as the number of repeats was increased. No differences in the efficiency of actin tail formation could be discerned between strains expressing BimA variants with two, five, or seven PDASX repeats. The data provide valuable new insights into the role of conserved and variable motifs of BimA in actin-based motility and intercellular spread of B. pseudomallei

Identification of genes required for soil survival in Burkholderia thailandensis by transposon-directed insertion site sequencing.

Transposon-directed insertion site sequencing was used to identify genes required by Burkholderia thailandensis to survive in plant/soil microcosms. A total of 1,153 genetic loci fulfilled the criteria as being likely to encode survival characteristics. Of these, 203 (17.6 %) were associated with uptake and transport systems; 463 loci (40.1 %) coded for enzymatic properties, 99 of these (21.4 %) had reduction/oxidation functions; 117 (10.1 %) were gene regulation or sensory loci; 61 (5.3 %) encoded structural proteins found in the cell envelope or with enzymatic activities related to it, distinct from these, 46 (4.0 %) were involved in chemotaxis and flagellum, or pilus synthesis; 39 (3.4 %) were transposase enzymes or were bacteriophage-derived; and 30 (2.6 %) were involved in the production of antibiotics or siderophores. Two hundred and twenty genes (19.1 %) encoded hypothetical proteins or those of unknown function. Given the importance of motility and pilus formation in microcosm persistence the nature of the colonization of the rhizosphere was examined by confocal microscopy. Wild type B. thailandensis expressing red fluorescent protein was inoculated into microcosms. Even though the roots had been washed, the bacteria were still present but they were motile with no attachment having taken place, perhaps being retained in a biofilm

Identification of a predicted trimeric autotransporter adhesin required for biofilm formation of Burkholderia pseudomallei.

The autotransporters are a large and diverse family of bacterial secreted and outer membrane proteins, which are present in many Gram-negative bacterial pathogens and play a role in numerous environmental and virulence-associated interactions. As part of a larger systematic study on the autotransporters of Burkholderia pseudomallei, the causative agent of the severe tropical disease melioidosis, we have constructed an insertion mutant in the bpss1439 gene encoding an unstudied predicted trimeric autotransporter adhesin. The bpss1439 mutant demonstrated a significant reduction in biofilm formation at 48 hours in comparison to its parent 10276 wild-type strain. This phenotype was complemented to wild-type levels by the introduction of a full-length copy of the bpss1439 gene in trans. Examination of the wild-type and bpss1439 mutant strains under biofilm-inducing conditions by microscopy after 48 hours confirmed that the bpss1439 mutant produced less biofilm compared to wild-type. Additionally, it was observed that this phenotype was due to low levels of bacterial adhesion to the abiotic surface as well as reduced microcolony formation. In a murine melioidosis model, the bpss1439 mutant strain demonstrated a moderate attenuation for virulence compared to the wild-type strain. This attenuation was abrogated by in trans complementation, suggesting that bpss1439 plays a subtle role in the pathogenesis of B. pseudomallei. Taken together, these studies indicate that BPSS1439 is a novel predicted autotransporter involved in biofilm formation of B. pseudomallei; hence, this factor was named BbfA, Burkholderia biofilm factor A

Detection of IgM antibody against phase 1 flagelin for diagnosis of <em>Salmonella paratyphi</em> A infection

[no abstract available