cHtrA is secreted via a sec-dependent pathway. (A) The SignalP 3.0 program with both the Neural Networks (NN) and Hidden Markov Model (HMM) algorithms http://www.expasy.ch was used to analyze the precursor cHtrA sequence from C. trachomatis serovar D http://stdgen.northwestern.edu/. The NN algorithm predicts a signal peptide from the first methionine residue (M1) to a serine residue at position 16 (S16) while the HMM-predicted signal peptide is M1-S23. (B) The M1-S23 peptide of cHtrA (cHtrAss) directed translocation of PhoA into bacterial periplasmic space (cHtrAss-'PhoA, slot 1, blue). Expression of the positive control full-length PhoA construct also led to the translocation of mature PhoA (with its intrinsic signal peptide, slot 3, blue) but the negative control mature PhoA construct failed to do so (without a signal peptide, 'PhoA, slot 2, white). (C) Bacterial transformants expressing the same three constructs were fractionated into periplasmic (per) and cytosolic (cyto) fractions and the fractions were detected with antibodies against a FLAG tag (anti-Flag, panel a) and GroEL (anti-GroEL, panel b) on a Western blot. Mature PhoA was secreted into the periplasm of bacteria expressing either the full-length PhoA construct or HtrAss-PhoA construct while mature PhoA stayed in the cytoplasm of the bacteria expressing the mature PhoA alone construct. (D) cHtrA secretion into the cytosol of chlamydia-infected cells is not inhibited by the type III secretion inhibitor C1 compound. HeLa monolayers infected with C. trachomatis L2 for 6 hr were treated with DMSO (panels a, c & e) or 50 μM C1 (b, d & f). Thirty-six hours after treatment, the cultures were processed for triply labeling with antibodies against IncA (green) and cHtrA, CT621 or CPAF (red) and DAPI for DNA (blue). C1 inhibited secretion of IncA and CT621 but not cHtrA or CPAF. Red arrows indicate chlamydia proteins that are secreted into host cell cytosol.