The DH5a bacterial strain (Invitrogen, Carlsbad, CA) was used to express
the plasmids. The products from all the three plasmids (pFLAG-PhoA, pFLAG-’PhoA & pFLAG-HtrAss-’PhoA) contain a FLAG tag fused to the C-terminus of PhoA. For BCIP assay, bacterial cells were grown in LB supplemented with the corresponding selection antibiotics at 37°C overnight. The check details overnight cultures were streaked onto LB agar containing the same selection antibiotics and 50 μg/ml 5-bromo-4-chloro-3-indolyl phosphate (BCIP, cat# B6149, Sigma) and the plates were incubated at 30°C for 2 days. The bacterial colonies that are capable of exporting mature PhoA into periplasm turn blue while the colonies Palbociclib incapable of doing so remain white. Results 1. Chlamydial HtrA is localized in both chlamydial inclusion and host cell cytosol A mouse antiserum raised with GST-cHtrA fusion protein detected the endogenous cHtrA protein both inside and outside of the chlamydial inclusions in C. trachomatis-infected HeLa cells (Figure 1A). The amount of intra-inclusion labeling appeared to be greater since the labeling in the host cell cytosol (outside inclusions) disappeared first as the dilution of the antiserum increased. Interestingly, some of the cHtrA-positive
PF 2341066 intra-inclusion granules appeared to be distinct from C. trachomatis organisms,
suggesting that a portion Sodium butyrate of cHtrA may be secreted out of the organisms but still trapped inside the inclusions. Both the intra-inclusion and cytosolic distribution of cHtrA were confirmed with a mAb against cHtrA (Figure 1B). Similar intra-inclusion stainings that are free of organisms were reported previously [15, 57, 58]. In contrast, most CPAF molecules were secreted out of the inclusions without obvious intra-inclusion accumulation. As expected, most of the chlamydial HSP60 molecules co-localized with the chlamydial organisms. The secretion of cHtrA into host cell cytosol became more obvious when the chlamydial inclusion membrane was counter-labeled using an anti-inclusion membrane protein antibody (Figure 1C). The cHtrA molecules were detected both inside and outside the inclusion membrane. The above observations together suggested that cHtrA might be secreted into both intra-inclusion space and the host cell cytosol. Figure 1 Detection of cHtrA protease in the cytosol of C. trachomatis -infected cells. HeLa cells infected with C. trachomatis L2 organisms were processed for co-staining with mouse antibodies visualized with a goat anti-mouse IgG conjugated with Cy3 (red), rabbit antibodies visualized with a Cy2-conjugated goat anti-rabbit IgG (green) and the DNA dye Hoechst (blue).