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.

The structure of BPSL1549, a protein of unknown function from Burkholderia pseudomallei, reveals a similarity to Escherichia coli cytotoxic necrotizing factor 1. We found that BPSL1549 acted as a potent cytotoxin against eukaryotic cells and was lethal when administered to mice. Expression levels of bpsl1549 correlate with conditions expected to promote or suppress pathogenicity. BPSL1549 promotes deamidation of glutamine-339 of the translation initiation factor eIF4A, abolishing its helicase activity and inhibiting translation. We propose to name BPSL1549 Burkholderia lethal factor 1.

Rapid inactivation of Ebola virus (EBOV) is crucial for high-throughput testing of clinical samples in low-. In response to this outbreak, the international community has deployed an increasing number of Ebola diagnostic laboratories into the main West African countries affected (Guinea, Liberia, and Sierra Leone). Rapid diagnosis of EVD in humans is critical in the management of this disease in outbreak situations, as it allows prompt isolation and the chance to provide the best supportive care to patients, which helps reduce the overall infection rate and break the transmission chain.The preferred clinical sample for testing for Ebola virus (EBOV), an enveloped negative-sense single-strand RNA virus, is EDTA-blood, serum, or plasma with the primary diagnostic technology being real-time PCR (2). Other sample types, such as swabs or urine, may also be received by a laboratory. EBOV is designated in the United Kingdom by the Advisory Committee on Dangerous Pathogens (ACDP) as a hazard group 4 pathogen that must be handled under containment level (CL) 4 standards (biosafety level 4 [BSL4] in other countries). As such stringent laboratory infrastructure and containment procedures are required to handle viable EBOV material, only a few laboratories in Europe and elsewhere are suitably equipped (3). Within the timelines and budgets available, it has been impractical to create this laboratory infrastructure in West Africa, and therefore diagnostic laboratories have relied on methods that rapidly inactivate EBOV prior to routine processing and testing of samples by PCR.Laboratory methods of EBOV inactivation include gamma irradiation (4), nanoemulsion (5), photoinducible alkylating agents (6), and UV radiation (7), but these methods are primarily used for research purposes and may not be practicable in an outbreak situation that is likely to involve a high number of samples but reduced capability for handling and manipulation. In this context, any inactivation method must also be compatible with the EBOV PCR diagnostic approach.The CDC recommends Triton X-100 and heat treatment for 1 h for diagnostic samples containing hemorrhagic fever viruses (8), and this method has been adopted by many laboratories for handling of samples that may contain EBOV (9). Heating (alone or with acetic acid) for 1 h at 60°C has also been shown to reduce the titer of EBOV (10). Other guidelines can be nonspecific, specifying only the need for inactivation but not suggesting how (11) or suggesting generic use of denaturing/lysis buffers and/or heat (12). In the United Kingdom, the Advisory Committee on Dangerous Pathogens guidelines state that samples from confirmed cases may be processed in a containment level 2 laboratory using routine autoanalyzers if a containment level 4 laboratory is not available and provided specific procedures are followed (13). Within these guidelines, which encompass the application of multiple clinical tests, there is no specific requirement to inactivate EBOV (or other viral hemorrhagic fever agents) within a sa...

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The Gram-negative bacterium Burkholderia pseudomallei causes melioidosis and is a CDC category B bioterrorism agent. Toll-like receptor (TLR)-2 impairs host defense during pulmonary B.pseudomallei infection while TLR4 only has limited impact. We investigated the role of TLRs in B.pseudomallei-lipopolysaccharide (LPS) induced inflammation. Purified B.pseudomallei-LPS activated only TLR2-transfected-HEK-cells during short stimulation but both HEK-TLR2 and HEK-TLR4-cells after 24 h. In human blood, an additive effect of TLR2 on TLR4-mediated signalling induced by B.pseudomallei-LPS was observed. In contrast, murine peritoneal macrophages recognized B.pseudomallei-LPS solely through TLR4. Intranasal inoculation of B.pseudomallei-LPS showed that both TLR4-knockout(-/-) and TLR2x4-/-, but not TLR2-/- mice, displayed diminished cytokine responses and neutrophil influx compared to wild-type controls. These data suggest that B.pseudomallei-LPS signalling occurs solely through murine TLR4, while in human models TLR2 plays an additional role, highlighting important differences between specificity of human and murine models that may have important consequences for B.pseudomallei-LPS sensing by TLRs and subsequent susceptibility to melioidosis.