However, we cannot rule out the possibility that the cytosolic presence of bacteria expose T3SS3 structural components to activate NFκB. The detection of endogenous

TAK1 activation in HEK293T cells following infection with wildtype, but not T3SS3 mutants, suggests the activation of the intracellular pattern recognition receptors (PRRs) NOD1 and NOD2, both of which signal through TAK1. B. pseudomallei is reportedly able to signal through NOD2 in RAW264.7 macrophages MK-4827 mouse to upregulate suppressor of cytokine signalling 3 (SOCS3) although it does not result in similar upregulation of the proinflammatory cytokines TNFα, IL-1β and IL-6 which depend on activation of NFκB [38]. Recently, it is reported that NOD2 plays a minor role in murine melioidosis and a human genetic polymorphism in NOD2 region is associated with melioidosis [39]. It is possible that NOD1 and NOD2, which sense bacterial peptidoglycan

derivatives IE-DAP and muramyl dipeptide respectively, may be the major cytosolic sensors responsible for NFκB activation. Conclusions Use of the HEK293T cells has allowed us to determine how Burkholderia T3SS3 contributes to NFκB activation in the absence of TLR and MyD88 signalling. We were able to discern that activation of NFκB does not occur as a direct consequence of Burkholderia T3SS3 secretion of effectors, but rather through cytosolic sensors that respond to the presence of bacteria in the cytosol following T3SS3-mediated escape from endocytic vesicles. CUDC-907 Our study serves as a model for future work to identify the new cytosolic sensors and the conditions leading to

NFκB activation. It is possible that NFκB is not triggered efficiently by surface or endosomal PRRs, whereupon cytosolic sensors become important in establishing recognition of bacterial pathogens and eventual protection. Alternatively, the activation of these cytosolic sensors may lead to a different gene expression program that provides a regulatory function distinct from the TLR response. Methods Cell-lines and bacterial strains Human embryonic kidney HEK293T (ATCC CRL-11268) cells were cultured in Dulbecco’s modified Eagle medium (Sigma-Aldrich) with 10% heat-inactivated fetal bovine serum (Life Technologies), 1X penicillin/streptomycin (Life Technologies) and 2 mM L-glutamine (Life Technologies) at 37°C with humidified atmosphere with 5% CO2. NFκB/293/GFP-Luc cell line was purchased from System Biosciences and cultured in the same medium as HEK293T cells. Bacterial strains used are listed in Table 1. Table 1 List of bacterial strains used in this study Strain Relevant characteristic(s) a Source or reference B. pseudomallei     KHW B.