The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol

Abstract

BackgroundThe periplasmic High Temperature Requirement protein A (HtrA) plays important roles in bacterial protein folding and stress responses. However, the role of chlamydial HtrA (cHtrA) in chlamydial pathogenesis is not clear.ResultsThe cHtrA was detected both inside and outside the chlamydial inclusions. The detection was specific since both polyclonal and monoclonal anti-cHtrA antibodies revealed similar intracellular labeling patterns that were only removed by absorption with cHtrA but not control fusion proteins. In a Western blot assay, the anti-cHtrA antibodies detected the endogenous cHtrA in Chlamydia-infected cells without cross-reacting with any other chlamydial or host cell antigens. Fractionation of the infected cells revealed cHtrA in the host cell cytosol fraction. The periplasmic cHtrA protein appeared to be actively secreted into host cell cytosol since no other chlamydial periplasmic proteins were detected in the host cell cytoplasm. Most chlamydial species secreted cHtrA into host cell cytosol and the secretion was not inhibitable by a type III secretion inhibitor.ConclusionSince it is hypothesized that chlamydial organisms possess a proteolysis strategy to manipulate host cell signaling pathways, secretion of the serine protease cHtrA into host cell cytosol suggests that the periplasmic cHtrA may also play an important role in chlamydial interactions with host cells.

Mutations in hemG Mediate Resistance to Salicylidene Acylhydrazides, Demonstrating a Novel Link between Protoporphyrinogen Oxidase (HemG) and Chlamydia trachomatis Infectivity

“…Subsequent studies indicate that SAHs block Chlamydia growth but not entry into cells (17)(18)(19)(20)(21), which supports the prevalent notion that the T3S is essential during the middle and late stages of the Chlamydia developmental cycle (22). Subsequent studies indicate that secretion or localization of predicted T3S effectors is altered by SAHs (19,(23)(24)(25)(26). Because the growth inhibition of Chlamydia by SAHs is reversed by exogenous iron, it has been postulated that iron chelation by SAHs may be responsible for their antichlamydial properties (27,28).…”

The Chlamydia-Secreted Protease CPAF Promotes Chlamydial Survival in the Mouse Lower Genital Tract

“…Pgp3 is an immunodominant antigen that is secreted into the cytosol of the infected cells (19)(20)(21). Many other C. trachomatis proteins, including CT311 (22,23), CT621/622 (24,25), CT795 (26), and cHtrA (27) as well as GlgA (28), all encoded by hypothetical ORFs, have also been localized in the cytosol of the infected cells. However, the functions of these proteins are largely unknown.…”

Chlamydia trachomatis Outer Membrane Complex Protein B (OmcB) Is Processed by the Protease CPAF

“…After centrifugation at 21,000 ϫ g for 15 min at 4°C, the remaining supernatant was collected as cell lysates for degradation assay. In some experiments, C. trachomatis-infected HeLa cell lysates were separated into pellet and supernatant fractions by using either anti-CPAF (clone 100a) or anti-cHtrA (MAb clone 6A2) antibody-mediated precipitation as described previously (32,41). Both the pellet and remaining supernatant were used as sources of enzyme in cell-free degradation assays.…”

“…A cell-free degradation assay was carried out as described previously (32). The following materials were used as the source of enzymes: fusion proteins with glutathione-S-transferase (GST) fused to the N terminus of chlamydial proteins, including GST-CPAF, GST-cHtrA (where cHtrA is chlamydial high-temperature requirement protein A), and GST-CT441 (19,40,41), or with a His tag fused to the C terminus of chlamydial proteins, including wild-type CPAF (designated CPAFwt) and the CPAF mutant with the 558 catalytic residue glutamic acid (E) replaced with alanine (A) [designated CPAF(E558A)] as described previously (34,42,43 20 M leupeptin (catalog number L2884), 1.6 M pepstatin A (catalog number P5318), 1.7 g of aprotinin/ml (catalog number A6279)]; all from Sigma). The resuspension mixtures were incubated on ice for 30 min.…”