Elucidation of the regulon and cis-acting regulatory element of HrpB, the AraC-type regulator of a plant pathogen-like type III secretion system in Burkholderia pseudomallei.

The human pathogen Burkholderia pseudomallei possesses multiple type III secretion system (T3SS) gene clusters. One of these, the B. pseudomallei T3SS2 (T3SS2(bp)) gene cluster, which apparently plays no role in animal virulence, is also found in six additional Burkholderia spp. and is very similar to T3SSs found in phytopathogenic Xanthomonas spp. and Ralstonia solanacearum. The T3SS2(bp) gene cluster also encodes an AraC-type regulatory protein (HrpB(bp)) that is an ortholog of HrpB, the master regulator of the R. solanacearum T3SS (T3SS(rso)) and its secreted effectors. Transcriptome analysis showed that HrpB(bp) activates the expression of T3SS2(bp) genes, as well as their orthologs in R. solanacearum. In addition to activating T3SS2(bp), HrpB(bp) also upregulates the expression of ~30 additional B. pseudomallei genes, including some that may confer production of adhesive pili, a polyketide toxin, several putative T3SS2(bp)-secreted effectors, and components of a regulatory cascade. T3SS2(bp) promoter regions were found to contain a conserved DNA motif (p2(bp) box) identical in sequence and position to the hrp(II) box required for HrpB-dependent T3SS(rso) transcription activation. The p2(bp) box is also present in the promoter regions of the essentially identical T3SS found in the very closely related species Burkholderia thailandensis (T3SS2(bt)). Analysis of p2(bp) box mutants showed that it is essential for HrpB(bp)-mediated transcription activation in both species. Although it has been suggested that T3SS2(bp) and T3SS2(bt) may function in phytopathogenicity, we were unable to demonstrate a phytopathogenic phenotype for B. thailandensis in three different plant hosts.

Comparative genomics and an insect model rapidly identify novel virulence genes of Burkholderia mallei.

Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei cause the potentially fatal human diseases melioidosis and glanders, respectively. The antibiotic resistance profile and ability to infect via aerosol of these organisms and the absence of protective vaccines have led to their classification as major biothreats and select agents. Although documented infections by these bacteria date back over 100 years, relatively little is known about their virulence and pathogenicity mechanisms. We used in silico genomic subtraction to generate their virulome, a set of 650 putative virulence-related genes shared by B. pseudomallei and B. mallei but not present in five closely related nonpathogenic Burkholderia species. Although most of these genes are clustered in putative operons, the number of targets for mutant construction and verification of reduced virulence in animal models is formidable. Therefore, Galleria mellonella (wax moth) larvae were evaluated as a surrogate host; we found that B. pseudomallei and B. mallei, but not other phylogenetically related bacteria, were highly pathogenic for this insect. More importantly, four previously characterized B. mallei mutants with reduced virulence in hamsters or mice had similarly reduced virulence in G. mellonella larvae. Site-specific inactivation of selected genes in the computationally derived virulome identified three new potential virulence genes, each of which was required for rapid and efficient killing of larvae. Thus, this approach may provide a means to quickly identify high-probability virulence genes in B. pseudomallei, B. mallei, and other pathogens.

Type VI secretion is a major virulence determinant in Burkholderia mallei.

Burkholderia mallei is a host-adapted pathogen and a category B biothreat agent. Although the B. mallei VirAG two-component regulatory system is required for virulence in hamsters, the virulence genes it regulates are unknown. Here we show with expression profiling that overexpression of virAG resulted in transcriptional activation of approximately 60 genes, including some involved in capsule production, actin-based intracellular motility, and type VI secretion (T6S). The 15 genes encoding the major sugar component of the homopolymeric capsule were up-expressed > 2.5-fold, but capsule was still produced in the absence of virAG. Actin tail formation required virAG as well as bimB, bimC and bimE, three previously uncharacterized genes that were activated four- to 15-fold when VirAG was overproduced. Surprisingly, actin polymerization was found to be dispensable for virulence in hamsters. In contrast, genes encoding a T6S system were up-expressed as much as 30-fold and mutations in this T6S gene cluster resulted in strains that were avirulent in hamsters. SDS-PAGE and mass spectrometry demonstrated that BMAA0742 was secreted by the T6S system when virAG was overexpressed. Purified His-tagged BMAA0742 was recognized by glanders antiserum from a horse, a human and mice, indicating that this Hcp-family protein is produced in vivo during infection.