To analyse the possible role of BopC in B. pseudomallei-host cell interactions, we constructed a bpss1516 mutant and assessed its ability to invade epithelial cells. In a PLX4032 mouse first experiment, we assessed invasiveness of wild-type B. pseudomallei K96243, the isogenic bsaQ (an invasion-deficient control) (Muangsombut et al., 2008) and bopC mutant strains in epithelial A549 cells. The bopC mutant was less invasive than the wild-type strain (Fig. 5a). We then introduced a plasmid encoding the chaperone-BopC effector operon into the bopC mutant strain in trans. This resulted in the restoration of BopC secretion in vitro (Fig. 5b) and partial restoration

of the invasion defect in epithelial cells (Fig. 5c). The invasiveness of the trans-complemented strain could be boosted further by induction of the BopC expression with IPTG (Fig. 5c). The Bsa T3SS is an important virulence determinant of B. pseudomallei (Stevens et al., 2004), whose role in pathogenesis is expected to be mediated through the concerted actions of the multiple effector proteins delivered into host cell cytosol. However, only two Bsa effectors have GSK126 purchase been found and characterized.

To close this gap, we set out to identify new B. pseudomallei Bsa effectors. Our search criteria and experimental approaches to verify novel effector proteins were based on several well-established postulates: (1) effectors tend to be co-regulated with other T3SS-related genes; (2) at least some of the effector-encoding genes are located in the proximity to the T3SS clusters and often are linked with T3SS chaperone-encoding genes; (3) effectors can be secreted into culture supernatants via the T3SS; (4) many effectors can bind their T3SS chaperones in vitro; and (5)

the first 20–30 N-terminal amino acids of an effector can be sufficient to mediate its recognition by the native, or a heterologous, T3SS and its translocation from the bacteria across the host cell membrane into the host cell cytosol. Here, we identified BopC (BPSS1516) as a new Bsa effector. The work stemmed from the finding by Moore and colleagues (Moore et al., Ibrutinib manufacturer 2004) that bpss1516 and bpss1517 are co-regulated with other bsa T3SS genes. Furthermore, Panina et al. (2005) identified BPSS1517 as a putative T3SS chaperone and BPSS1516 as its putative binding partner. Based on this knowledge, we designed and performed a series of experiments to conclusively establish that BPSS1516 (BopC) is a Bsa T3SS effector of B. pseudomallei. We demonstrated that BopC interacts with its putative cognate chaperone BPSS1517 in vitro and showed that its first 20 N-terminal amino acids are sufficient to mediate the translocation of the reporter protein into host cells through the EPEC T3SS. To gain insight into the contribution of bopC to B. pseudomallei virulence, we created a specific bopC mutant and assessed it in an epithelial cell invasion assay.