The Cluster 1 Type VI Secretion System Is a Major Virulence Determinant in Burkholderia pseudomallei

Abstract

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.

Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade

“…In our analysis, T6SS-1 was found to facilitate intercellular spread following infection or photothermal delivery. Consistent with recent reports (14,15,19), deletion of clpV1 resulted in a defect in plaque formation in HEK293 cells and a delay in the formation L.m. of MNGCs.…”

“…Of the multiple T6SSs encoded by Burkholderia species, T6SS-1 has been repeatedly linked to host-pathogen interactions (12)(13)(14)(15)19). These correlations with virulence led us to investigate the role of T6SS-1 following infection and nanoblade delivery.…”

“…Using the nomenclature of Schell et al (13), T6SS-1 (also referred to as T6SS-5) (14) is critical for virulence in the Bt murine model of acute melioidosis and contributes to the lethality of Bm in hamsters (13). Recently, T6SS-1 mutants in Bp were shown to be capable of endosome escape in RAW264 cells but were defective in MNGC formation (15).…”

EliA is required for inducing the stearyl alcohol-mediated expression of secretory proteins and production of polyester in Ralstonia sp. NT80

“…The Hcp identified in this study shared 95 and 71 % identity with the haemolysin-coregulated protein of Ralstonia solanacearum PSI07 and Burkholderia pseudomallei 1026b, respectively (Remenant et al, 2010;Hayden et al, 2012), whereas the Ndk identified in this study shared 97 and 86 % identity with the nucleoside diphosphate kinase of R. solanacearum PSI07 and B. pseudomallei 1026b, respectively (Remenant et al, 2010;Hayden et al, 2012). Of these two proteins, Hcp has been shown to be an extracellular part of the T6SS (Mougous et al, 2006;Records, 2011;Decoin et al, 2014), which has been reported to be involved in secreting virulence-related proteins either out of or directly into the cells of a eukaryotic host (Schell et al, 2007;Burtnick et al, 2011). Secretory Ndk, by contrast, has been shown to be exported via the T3SS and is known to be involved in the virulence of pathogenic bacteria such as Pseudomonas aeruginosa and Mycobacterium tuberculosis (Chakrabarty, 1998;Neeld et al, 2014).…”

Current biodefense vaccine programs and challenges

“…The capsular polysaccharide and lipopolysaccharide of B. mallei and B. pseudomallei were shown to induce robust antibody responses in animal models and clinical samples, with some evidence of protection associated with this response. 4 Recent work identified secretion system-related virulence factors such as Hcp1, BopE and BimA that are involved in evading the innate immune response, 5,6 and suggested that TssM modulates the adaptive immune response. 1 Additionally, cellular immunity including TNF-α and IFN-γ responses correlated with protection in animal models.…”