The Burkholderia pseudomallei K96243 genome encodes six type VI secretion systems (T6SSs), but little is known about the role of these systems in the biology of B. pseudomallei. In this study, we purified recombinant Hcp proteins from each T6SS and tested them as vaccine candidates in the BALB/c mouse model of melioidosis. Recombinant Hcp2 protected 80% of mice against a lethal challenge with K96243, while recombinant Hcp1, Hcp3, and Hcp6 protected 50% of mice against challenge. Hcp6 was the only Hcp constitutively produced by B. pseudomallei in vitro; however, it was not exported to the extracellular milieu. Hcp1, on the other hand, was produced and exported in vitro when the VirAG two-component regulatory system was overexpressed in trans. We also constructed six hcp deletion mutants (⌬hcp1 through ⌬hcp6) and tested them for virulence in the Syrian hamster model of infection. The 50% lethal doses (LD 50 s) for the ⌬hcp2 through ⌬hcp6 mutants were indistinguishable from K96243 (<10 bacteria), but the LD 50 for the ⌬hcp1 mutant was >10 3 bacteria. The hcp1 deletion mutant also exhibited a growth defect in RAW 264.7 macrophages and was unable to form multinucleated giant cells in this cell line. Unlike K96243, the ⌬hcp1 mutant was only weakly cytotoxic to RAW 264.7 macrophages 18 h after infection. The results suggest that the cluster 1 T6SS is essential for virulence and plays an important role in the intracellular lifestyle of B. pseudomallei.

Studies of inhalational melioidosis were undertaken in the common marmoset (Callithrix jacchus). Following exposure to an inhaled challenge with aerosolized Burkholderia pseudomallei, lethal infection was observed in marmosets challenged with doses below 10 cfu; a precise LD(50) determination was not possible. The model was further characterized using a target challenge dose of approximately 10(2) cfu. A separate pathogenesis time-course experiment was also conducted. All animals succumbed, between 27 and 78 h postchallenge. The challenge dose received and the time to the humane endpoint (1 °C below normal body temperature postfever) were correlated. The first indicator of disease was an increased core body temperature (T(c) ), at 22 h postchallenge. This coincided with bacteraemia and bacterial dissemination. Overt clinical signs were first observed 3-5 h later. A sharp decrease (typically within 3-6 h) in the T(c) was observed prior to humanely culling the animals in the lethality study. Pathology was noted in the lung, liver and spleen. Disease progression in the common marmoset appears to be consistent with human infection in terms of bacterial spread, pathology and physiology. The common marmoset can therefore be considered a suitable animal model for further studies of inhalational melioidosis.

Analysis of the genome of Francisella tularensis has revealed few regulatory systems, and how the organism adapts to conditions in different niches is poorly understood. The stringent response is a global stress response mediated by (p)ppGpp. The enzyme RelA has been shown to be involved in generation of this signal molecule in a range of bacterial species. We investigated the effect of inactivation of the relA gene in Francisella by generating a mutant in Francisella novicida. Under amino acid starvation conditions, the relA mutant was defective for (p)ppGpp production. Characterization showed the mutant to grow similarly to the wild-type, except that it entered stationary phase later than wild-type cultures, resulting in higher cell yields. The relA mutant showed increased biofilm formation, which may be linked to the delay in entering stationary phase, which in turn would result in higher cell numbers present in the biofilm and reduced resistance to in vitro stress. The mutant was attenuated in the J774A macrophage cell line and was shown to be attenuated in the mouse model of tularaemia, but was able to induce a protective immune response. Therefore, (p)ppGpp appears to be an important intracellular signal, integral to the pathogenesis of F. novicida.

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Burkholderia pseudomallei is the causative agent of melioidosis, a disease endemic to regions of Southeast Asia and Northern Australia. Both humans and a range of other animal species are susceptible to melioidosis, and the production of a group 3 polysaccharide capsule in B. pseudomallei is essential for virulence. B. pseudomallei capsular polysaccharide (CPS) I comprises unbranched manno-heptopyranose residues and is encoded by a 34.5-kb locus on chromosome 1. Despite the importance of this locus, the role of all of the genes within this region is unclear. We inactivated 18 of these genes and analyzed their phenotype using Western blotting and immunofluorescence staining. Furthermore, by combining this approach with bioinformatic analysis, we were able to develop a model for CPS I biosynthesis and export. We report that inactivating gmhA, wcbJ, and wcbN in B. pseudomallei K96243 retains the immunogenic integrity of the polysaccharide despite causing attenuation in the BALB/c murine infection model. Mice immunized with the B. pseudomallei K96243 mutants lacking a functional copy of either gmhA or wcbJ were afforded significant levels of protection against a wild-type B. pseudomallei K96243 challenge.