Secretion of cHtrA into host cell cytosol by most chlamydial organisms tested. HeLa cells infected with C. trachomatis serovars A, B, Ba, C, D, E, F, H, I, K, L1, L2, L3, C. muridarum Nigg strain, C. caviae GPIC, C. penumonaie AR39 isolate &C. psiitaci 6BC organisms (as indicated in each panel) were processed at 40 h (all C. trachomatis serovars), 24 h (Nigg, GPIC & 6BC) or 72 h (AR39) after infection. (A) The processed samples were detected for cHtrA using the mouse anti-cHtrA fusion protein polyclonal antibody (red) in an immunofluorescence assay. The chlamydial organisms were visualized using a rabbit anti-CT395 fusion protein antibody (green) while the DNA was labeled with Hoechst dye (blue). Note that cHtrA was consistently detected in both the lumen of chlamydial inclusion (red arrowheads) and cytosol (red arrows) of cells infected with all C. trachomatis serovars and C. muridarum and C. caviae isolates. However, the cytosolic labeling of cHtrA was not clear in cells infected with C. pneumoniae AR39 and C. psittaci 6BC organisms which were reexamined by co-staining with either anti-organisms (B, panels a-c) or anti-IncA (panels d-f) antibodies. Note that cytosolic cHtrA was detected in cells infected with C. pneumoniae AR39 (panels b & e) but not C. psittaci 6BC organisms (c & f).