Figure 3 TLR independent NFκB activation by B. pseudomallei is not dependent on T3SS3 effectors. HEK293T cells were cotransfected with pNFκB-SEAP and mammalian expression vectors encoding genes for BopA (A) BopC (B) and BopE (C) for 24 hr. Supernatants were collected for SEAP assay (left panels). Total RNA

was isolated for measuring of expression of effector genes (right panels) by real-time PCR. D) Cells transfected with BopE plasmid were lysed and analysed by Western blot with anti-BopE antibody. SopE was used as a positive control. Lazertinib in vitro Asterisks * and ** indicate significant differences of p < 0.05 and p < 0.01 between empty vector and plasmid expressing T3SS effector gene respectively. T3SS3 mutants activate NFκB when they gain access to the host cytosol It is known that T3SS3 facilitates escape from phagosomal or endosomal compartments into the host cell cytosol [8, 24],

although B. pseudomallei T3SS3 mutants have been observed to exhibit delayed escape via an unidentified mechanism [8]. A time-course of NFκB activation shows that the T3SS3 VX 809 mutant ∆bsaM was unable to activate NFκB at 6 hr. after infection, although it was increasingly able to do so when the incubation was extended to 24 hr. (Figure 4A), where levels became comparable to infection with wildtype KHW. In Figure 2C, Blasticidin S we had shown that ∆bsaM mutant was unable to form MNGCs Adenosine triphosphate at 12 hr., corresponding to their inability to activate NFκB at early time-points. By 18 hr., both wildtype KHW and ∆bsaM mutant induced the formation of MNGCs (Figure 4B). On the basis of these observations, we hypothesized that T3SS-independent escape from endosomes is responsible for NFκB activation by the ∆bsaM mutant at later time points, and the critical event required for NFκB activation is bacterial entry into the cytosol. Figure 4 T3SS3 mutants activate NFκB at late time-points corresponding to escape into cytosol. A) HEK293T cells were transfected with pNFκB-SEAP for 24 hr.

The transfected cells were infected with wildtype KHW and ΔbsaM at MOI of 10:1. Supernatants were collected at respective time points for SEAP assay. B) HEK293T cells were infected with wildtype KHW and ΔbsaM at MOI of 10:1 for 18 hr. The infected cells were fixed, stained with Giemsa and visualized under 10x magnification on a light microscope. If NFκB activation at early time points results from rapid escape from the endosome, then direct placement of bacteria into the cytosol should obviate the need for T3SS-mediated escape. This was tested using a photothermal nanoblade, which allows us to bypass the need for invasion and endosome escape altogether [24, 26]. The photothermal nanoblade utilizes a 6 ns pulse from a 540 nm laser to excite a titanium coating on glass micropipettes that are brought into contact with mammalian cell membranes.