Secretion of the chlamydial virulence factor CPAF requires the Sec-dependent pathway

Abstract

The chlamydial protease/proteasome-like activity factor (CPAF) is secreted into the host cytosol to degrade various host factors that benefit chlamydial intracellular survival. Although the full-length CPAF is predicted to contain a putative signal peptide at its N terminus, the secretion pathway of CPAF is still unknown. Here, we have provided experimental evidence that the N-terminal sequence covering the M1-G31 region was cleaved from CPAF during chlamydial infection. The CPAF N-terminal sequence, when expressed in a phoA gene fusion construct, was able to direct the export of the mature PhoA protein across the inner membrane of wild-type Escherichia coli. However, E. coli mutants deficient in SecB failed to support the CPAF signal-peptide-directed secretion of PhoA. Since native PhoA secretion was known to be independent of SecB, this SecB dependence must be rendered by the CPAF leader peptide. Furthermore, lack of SecY function also blocked the CPAF signal-peptide-directed secretion of PhoA. Most importantly, CPAF secretion into the host cell cytosol during chlamydial infection was selectively inhibited by an inhibitor specifically targeting type I signal peptidase but not by a type III secretion-system-specific inhibitor. Together, these observations have demonstrated that the chlamydial virulence factor CPAF relies on Sec-dependent transport for crossing the chlamydial inner membrane, which has provided essential information for further delineating the pathways of CPAF action and understanding chlamydial pathogenic mechanisms.

Fig. 1.

Prediction of CPAF signal peptide and N-terminal sequencing of CPAF. (a) The SignalP 3.0 program with both the NN and HMM algorithms (www.expasy.ch) was used to analyse the precursor CPAF sequence from C. trachomatis serovar D (http://stdgen.northwestern.edu/). The NN algorithm predicts a signal peptide covering a region from the methionine residue at position M1 to the histidine residue at position 26 (H26) while the HMM-predicted signal peptide extends to residue G31 (M1–G31). (b) Endogenous CPAF was immunoprecipitated from chlamydia-infected cell lysates using the mAb 100a; >95 % of the precipitate was resolved by using SDS-PAGE and was transferred onto a PVDF membrane for N-terminal sequencing using the Edman degradation method. The first six residues derived from the corresponding bands are listed to the right of the gel. The remaining <5 % of the precipitate was also resolved by using SDS-PAGE and analysed via Western blot detection of the N-terminus-containing CPAF bands with mAb 54b. P, Precursor CPAF; M, mature full-length processed CPAF; n, processed CPAFn.

Fig. 2.

The putative N-terminal signal peptide of CPAF is able to direct translocation of mature PhoA into the bacterial periplasmic space. (a) Constructs encoding CPAF N-terminal peptide–mature PhoA fusion protein (CPAFss-′PhoA; 1), mature PhoA (without a signal peptide, ′PhoA; 2) or full-length PhoA (with its intrinsic signal peptide; 3) were expressed in E. coli DH5α cells lacking endogenous PhoA. The transformed bacteria were incubated at 30 °C for 48 h on agar plates supplemented with BCIP. PhoA translocated into the periplasmic space is able to catalyse BCIP to produce a blue colour. Note that expression of the constructs encoding PhoA carrying either the native or CPAF signal peptide led to blue colonies, while the colonies expressing the mature PhoA alone remained white. (b) Bacterial transformants expressing the same three constructs were also fractionated into periplasmic (per) and cytosolic (cyt) fractions; these were separated by SDS-PAGE, transferred to a PVDF membrane by Western blot and detected with antibodies against a FLAG tag (anti-FLAG, i), MBP (anti-MBP, ii) and GroEL (anti-GroEL, iii). Note that mature PhoA was secreted into the periplasm of bacteria expressing either the full-length PhoA construct or CPAFss-′PhoA construct, while mature PhoA stayed in the cytoplasm of the bacteria expressing the mature PhoA alone construct.

Fig. 3.

The CPAF signal-peptide-directed translocation of mature PhoA is dependent on SecB. (a) The construct coding for the CPAF signal peptide–mature PhoA fusion protein (CPAFss-′PhoA-FLAG) was transformed into a mutant bacterial strain deficient in secB (DRS) or its wild-type control strain (DR473). The transformed bacteria were cultured for 48 h at 30 °C on agar plates supplemented with BCIP. Note that SecB-deficient bacteria remained white while the wild-type bacteria turned blue. (b) The parallel bacterial cultures were harvested for fractionation. The presence of PhoA in both the periplasmic (per) and cytosolic (cyt) fractions was analysed by Western blot as described in the legend to Fig. 2(b). The fusion construct was also tested in an independent SecB-deficient mutant strain JW3584-1 and its corresponding control, wild-type MC4100. Note that both SecB-deficient bacterial strains failed to support PhoA export into the periplasm.

Fig. 4.

The CPAF signal-peptide-directed export of mature PhoA also requires SecY. The construct coding for the CPAF signal peptide–mature PhoA fusion protein (CPAFss-′PhoA-FLAG) was transformed into bacterial strains that carry either a wild-type secY (secY^wt) or a temperature-sensitive mutant secY allele (secY^ts). The overnight culture incubated at 30 °C was diluted into fresh medium to reach OD₆₀₀=0.1. Half of the culture was grown at 30 °C and the other half was grown at 42 °C for another 4 h. The parallel cultures were harvested for fractionation. The presence of PhoA in both the periplasmic (per) and cytosolic (cyt) fractions was analysed by Western blot as described in the legend to Fig. 2(b). Note that at the permissive temperature, bacterial strains carrying either secY^wt or secY^ts exported mature PhoA into the periplasm while at the restrictive temperature, only the secY^wt strain but not the secY^ts strain exported mature PhoA into the periplasm.

Fig. 5.

CPAF secretion into the cytosol of chlamydia-infected cells is inhibited by arylomycin C16, an inhibitor targeting prokaryotic signal peptidase I. HeLa monolayers were infected with C. trachomatis L2 at an m.o.i. of 0.5 for 8 h. The cultures were treated with DMSO (a–c), 50 μM C1 compound (d–f; an inhibitor targeting the Gram-negative bacterial type III secretion system) or 10 μM C16 (g–i; an inhibitor targeting the signal peptidase I). Twenty-four hours after treatment, the cultures were processed for the immunofluorescence assay. The processed monolayers were immuno-labelled with antibodies against chlamydia (blue), IncA (green) and CPAF (red). Note that C1 selectively inhibits secretion of IncA but not CPAF, while C16 blocks secretion of CPAF but not IncA. White arrows indicate specific labelling while white arrowheads represent possible non-specific signals.