In addition to detection by the inflammasome machinery, Yersinia[13] and Salmonella[14] can be detected by NFκB in a Toll-like receptor (TLR) and MyD88 independent manner that is reliant on T3SS, revealing another possible mechanism whereby T3SS can be detected by host Selonsertib molecular weight epithelial cells which lack inflammasome machinery. Using human embryonic kidney cells (HEK293T), which are epithelial cells that lack TLR 2, 4 and 9 expression but expresses low levels of TLR5 and 7 [15, 16], we have previously shown that Staurosporine in vivo B. pseudomallei stimulates NFκB independently

of TLRs and MyD88, leading to the production of IL-8. NFκB activation required bacterial internalization and a functional T3SS3 [17]. However, it is unclear whether NFκB activation is triggered by T3SS3 effector proteins, by components of the T3SS secretion apparatus itself, or indirectly via additional T3SS3-mediated processes. Our goal is to determine how T3SS3 contributes to NFκB activation www.selleckchem.com/JAK.html in the absence of TLR, MyD88 and inflammasome signalling using HEK293T epithelial cells as a model system.

We show that T3SS3-mediated endosome escape is required for NFκB activation and occurs independently of known T3SS3 effector proteins. Using a photothermal nanoblade to directly place bacteria into the cytoplasm, we show that cytosolic localization is sufficient to activate NFκB. Thus, B. pseudomallei T3SS3 is not directly detected by the host NFκB pathway but is instead responsible for bacterial escape from vacuolar compartments subsequently leading to the activation of cytosolic sensors. Results TLR-independent NFκB activation by B. pseudomallei is dependent on the activity of T3SS3 but not known T3SS3 effector proteins We had previously shown that activation of NFκB in HEK293T cells by B. pseudomallei was not dependent on host TLR and MyD88 signalling next but required a functional bacterial T3SS3 [17]. Here, we first investigate whether B. pseudomallei T3SS1 and T3SS2 contribute to NFκB activation, or if it is a specific consequence of T3SS3 activity. Derivatives of B. pseudomallei strain KHW containing deletions of the entire T3SS3, T3SS2 or T3SS1 gene clusters were constructed by allelic exchange. HEK293T

cells that were transiently transfected with the NFκB-SEAP (secreted embryonic alkaline phosphatase) reporter system were infected with wildtype KHW or mutant strain, and assayed for NFκB activation 6 hr. later. As shown in Figure 1A, infection with the ΔT3SS3 strain showed reduced NFκB activation in contrast to the ΔT3SS1 and ΔT3SS2 mutant derivatives, which led to robust activation comparable to wildtype bacteria. As the ΔT3SS3 mutant was unable to replicate as well as wildtype KHW and the other mutants (Figure 1B), the lack of NFκB activation could be due to lower bacterial numbers. Furthermore, it is known that complete deletion of T3SS3 also inactivates T6SS1 due to removal of T6SS1 regulatory loci located in the T3SS3 gene cluster [18].