Chlamydia trachomatis outer membrane complex protein B (OmcB) is processed by the protease CPAF

Abstract

We previously reported that the Chlamydia trachomatis outer membrane complex protein B (OmcB) was partially processed in Chlamydia-infected cells. We have now confirmed that the OmcB processing occurred inside live cells during chlamydial infection and was not due to proteolysis during sample harvesting. OmcB processing was preceded by the generation of active CPAF, a serine protease known to be able to cross the inner membrane via a Sec-dependent pathway, suggesting that active CPAF is available for processing OmcB in the periplasm. In a cell-free system, CPAF activity is both necessary and sufficient for processing OmcB. Both depletion of CPAF from Chlamydia-infected cell lysates with a CPAF-specific antibody and blocking CPAF activity with a CPAF-specific inhibitory peptide removed the OmcB processing ability of the lysates. A highly purified wild-type CPAF but not a catalytic residue-substituted mutant CPAF was sufficient for processing OmcB. Most importantly, in chlamydial culture, inhibition of CPAF with a specific inhibitory peptide blocked OmcB processing and reduced the recovery of infectious organisms. Thus, we have identified OmcB as a novel authentic target for the putative chlamydial virulence factor CPAF, which should facilitate our understanding of the roles of CPAF in chlamydial biology and pathogenesis.

Fig 1

OmcB is processed during C. trachomatis infection. (A) HeLa cells either uninfected or infected with C. trachomatis were harvested at various time points after infection, as indicated on the figure, for Western blot detection of OmcB (a), CPAF (b), golgin-84 (c), vimentin (d), keratin-8 (e), the major outer membrane protein (MOMP) (f), and human HSP70 (g). The CPAFc fragment was first detected at 24 h, and processed OmcBc was detected at 28 h after infection. OmcB processing was detected regardless of the cell lysis methods while the degradation fragments (df) of previously reported CPAF substrates golgin-84, vimentin, and keratin-8 were detectable only in samples harvested with RIPA buffer and not 8 M urea. The question mark indicates a protein that may represent OmcB with incomplete reduction or an OmcB fragment. (B) The supernatants of the above cultures were harvested to measure the levels of LDH. No significant LDH was detected in any of the culture supernatants. The whole-cell lysate of a 36-h chlamydia-infected cell sample was used as a positive control. The data were from three independent experiments. P.I., postinfection; α, anti; MW, molecular weight (in thousands).

Fig 2

CPAF is sufficient for processing OmcB into OmcBc. (A) In a cell-free cleavage assay, a recombinant OmcB (reOmcB) was used as the substrate for cleavage by GST fusion proteins CPAF (lane 3), cHtrA (lane 4), or CT441 (lane 5). A lysate made from Chlamydia trachomatis-infected HeLa cells (Ct-HeLa lysate) (lane 2) was used as a positive control. Both C. trachomatis-infected HeLa cell lysate and GST-CPAF but not other GST fusion proteins cleaved reOmcB into OmcBc. (B) The recombinant His-CPAF wild-type or His-CPAF with an alanine substitution of the catalytic residue glutamic acid at position 558 (E558A) was used to cleave reOmcB. The wild-type CPAF (lane 4) but not the E558A mutant (lane 5) cleaved reOmcB to produce OmcBc. The positive-control C. trachomatis-infected HeLa cell lysate also cleaved reOmcB into OmcBc (lane 3). (C) The processing of reOmcB into OmcBc by CPAF was inhibited by a CPAF-specific inhibitory peptide. The inhibition was noticed at 7 μM and peaked at 700 μM. The question mark indicates a protein that may represent OmcB with incomplete reduction or OmcB fragments; the asterisk indicates preexisting OmcBc from the C. trachomatis-infected HeLa cell lysates.

Fig 3

CPAF activity is necessary for processing reOmcB. (A) Both C. trachomatis-infected (Ct-HeLa) and uninfected HeLa cell lysates were used to digest reOmcB in a cell-free system. The C. trachomatis-infected but not the uninfected HeLa cell lysate cleaved reOmcB to produce OmcBc. (B) C. trachomatis-infected HeLa cell lysate with or without precipitation into pellet and supernatant fractions with anti-CPAF or anti-cHtrA antibody-conjugated protein G agarose beads, respectively, was used to digest reOmcB. The anti-CPAF-precipitated pellet at 15 μl (out of the total of 20 μl) significantly cleaved reOmcB to produce OmcBc (lane 4) while 15 μl (out of the total of 20 μl) supernatant failed to do so (lane 6). In contrast, a similarly precipitated pellet with the anti-cHtrA antibody failed to process reOmcB (lane 7) while the supernatant retained reOmcB processing ability (lane 8). (C) The processing of reOmcB by C. trachomatis-infected HeLa cell lysate was significantly inhibited by a CPAF-inhibitory peptide. The question mark indicates a protein that may represent OmcB with incomplete reduction or OmcB fragments; the asterisk indicates preexisting OmcBc from the C. trachomatis-infected HeLa cell lysates.

Fig 4

CPAF activity is required for processing the endogenous OmcB during Chlamydia trachomatis infection. (A) HeLa cells infected with C. trachomatis organisms were treated with an inhibitory peptide targeting CPAF or a scrambled peptide as a control at the concentrations listed at the top of the figure. At 36 h after infection, all samples were harvested in 8 M urea for Western blot detection with antibodies recognizing OmcB (a), MOMP (b), CPAFc (c), or host β-actin as indicated. Treatment with the CPAF-inhibitory peptide blocked processing of OmcB starting at 5 μM and peaked at 9 μM while the control peptide failed to do so even at 10 μM. The total amounts of chlamydial proteins including the remaining OmcB, MOMP, and CPAFc were reduced by the CPAF-inhibitory peptide treatment while the host protein β-actin maintained equal amounts regardless of the treatments. The question mark indicates a protein that may represent OmcB with incomplete reduction or OmcB fragments. (B) HeLa cells infected with C. trachomatis organisms and treated with peptides as described above were harvested for titrating infectious organisms, and the results were expressed as infection-forming units (IFUs), as displayed along the y axis. The data were from three independent experiments. *, P < 0.05 (Student t test). MW, molecular weight in thousands.