Figure 2 Deletion of T3SS3 effector genes had little effect on TLR independent NFκB activation by B. pseudomallei . A) HEK293T cells were transfected with pNFκB-SEAP for 24 hr. The transfected cells were infected with wildtype KHW and mutants at MOI of 10:1 for 6 hr. Supernatants were collected for SEAP assay. B) HEK293T cells were infected with respective strains for 6 hr. Cells were lysed and plated for intracellular bacterial count. C) HEK293T cells were infected with respective strains for 12 hr. The infected Defactinib research buy cells were fixed, stained with Giemsa and visualized under 10x magnification on a light microscope. Asterisks * and ** indicate significant differences of p < 0.05 and p < 0.01

between B. pseudomallei wildtype and mutant strains respectively. T3SS3 does not facilitate invasion JQEZ5 of bacteria into cells but rather promotes their subsequent escape from endocytic vesicles

[24]. Therefore, defective endosome escape by mutants may provide an explanation for their reduced replication and inability to activate NFκB. Thus, we examined whether the ability of these mutants to activate NFκB correlate with their ability to escape from the endosome. The formation of multinucleated giant cells (MNGC) at 10–12 hr. following infection was utilized as a measure of endosome escape, since it requires the activity of T6SS1 and only occurs if bacteria have escaped from endocytic vesicles into the cytosol [18, 24]. We examined the formation of MNGC at 12 hr. post infection

of the single and triple effector mutants in comparison with wildtype KHW and the escape-deficient ΔbsaM (Figure 2C). All strains could induce MNGC at this time-point except for ΔbsaM, indicating that the ability to activate NFκB correlates Mannose-binding protein-associated serine protease with the ability to escape. ΔbopACE formed less MNGCs compared to the rest, likely reflecting its lower replication ability. Another possibility is that the ΔbsaM and ΔbopACE strains are defective in the secretion of T3SS3 effector proteins, which could be responsible for activating NFκB as has been reported for the T3SS effector PI3K targets proteins SopE and SipA from Salmonella[25]. This is unlikely given that our single effector mutants could still activate NFκB as well as wildtype bacteria. To confirm, BopA (Figure 3A), BopC (Figure 3B) or BopE (Figure 3C) were ectopically expressed in increasing plasmid concentrations in HEK293T cells. None of the Burkholderia effectors were able to activate NFκB significantly above background levels with the exception of BopE (Figure 3C), a homolog of Salmonella SopE, which showed only a slight activation. In contrast, expression of Salmonella SopE led to robust activation. We verified that the proteins were indeed expressed at the mRNA level (Figure 3A-C) as well as at the protein level for BopE (Figure 3D).