Type VI secretion is a major virulence determinant in <i>Burkholderia mallei</i>

<jats:title>Summary</jats:title><jats:p><jats:italic>Burkholderia mallei</jats:italic> is a host‐adapted pathogen and a category B biothreat agent. Although the <jats:italic>B. mallei</jats:italic> 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 <jats:italic>virAG</jats:italic> resulted in transcriptional activation of ∼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 <jats:italic>virAG</jats:italic>. Actin tail formation required <jats:italic>virAG</jats:italic> as well as <jats:italic>bimB</jats:italic>, <jats:italic>bimC</jats:italic> and <jats:italic>bimE</jats:italic>, 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 <jats:italic>virAG</jats:italic> 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 <jats:italic>in vivo</jats:italic> during infection.</jats:p>