Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment - PubMed (original) (raw)

Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment

Janine H Peterson et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Autotransporters are bacterial virulence factors consisting of an N-terminal "passenger domain" that is secreted in a C- to-N-terminal direction and a C-terminal "β domain" that resides in the outer membrane (OM). Although passenger domain secretion does not appear to use ATP, the energy source for this reaction is unknown. Here, we show that efficient secretion of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP requires the stable folding of a C-terminal ≈17-kDa passenger domain segment. We found that mutations that perturb the folding of this segment do not affect its translocation across the OM but impair the secretion of the remainder of the passenger domain. Interestingly, an examination of kinetic folding mutants strongly suggested that the ≈17-kDa segment folds in the extracellular space. By mutagenizing the ≈17-kDa segment, we also fortuitously isolated a unique translocation intermediate. Analysis of this intermediate suggests that a heterooligomer that facilitates the membrane integration of OM proteins (the Bam complex) also promotes the surface exposure of the ≈17-kDa segment. Our results provide direct evidence that protein folding can drive translocation and help to clarify the mechanism of autotransporter secretion.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

The mutation of C-terminal hydrophobic residues perturbs the folding of the EspP passenger domain. (A) Illustration of EspP showing the signal peptide (SP) (residues 1–55), the passenger domain (residues 56–1023), and the β domain (residues 1024–1300). ProEspP contains covalently linked passenger and β domains. The location of predicted structural elements is also shown. (B) Crystal structure of the C terminus of Hbp (2). The conserved residues that correspond to F963 and W990 in EspP are shown. (C) Refolding of the β helix of EspP, EspP(F963A), and EspP(W990A) was examined by monitoring Trp fluorescence after dilution of the polypeptides out of 4 M Gdn-HCl. Fluorescence intensity is in arbitrary units. (D) The β-helical domain of EspP, EspP(F963A), and EspP(W990A) was heated at the indicated temperature in sample buffer containing 0.1% SDS and resolved by SDS/PAGE.

Fig. 2.

Point mutations that impair the folding of the EspP stable core delay passenger domain secretion. AD202 transformed with pRLS5 (P_trc_-espP) or a plasmid encoding the indicated EspP mutant were subjected to pulse–chase labeling after the addition IPTG. Half of the cells were treated with PK, and EspP-containing polypeptides were immunoprecipitated using C-terminal (A) and N-terminal (B) anti-EspP antisera. The samples in B were resolved on 4–12% NuPage/MOPS gels (Invitrogen). The percent of the passenger domain that was surface exposed or cleaved from proEspP in A is plotted in C and D, and the percent of each protein that was converted to a ≈47- or ≈33-kDa C-terminal fragment by PK treatment is plotted in E and F.

Fig. 3.

A large C-terminal deletion traps the majority of the EspP passenger domain in the periplasm. (A) Illustration of EspP Δ891–988. (B) AD202 transformed with pJH104 (P_trc_-espPΔ891–988) were treated as described in Fig. 2, and EspP-containing polypeptides were immunoprecipitated using C- and N-terminal anti-EspP antisera. The samples in B Lower were resolved on a 4–12% NuPage/MOPS gel. The percent of the passenger domain that was surface exposed or cleaved from proEspP in B is shown in C and D.

Fig. 4.

An α-helical segment traverses the EspP β barrel before the completion of passenger domain translocation. AD202 transformed with a plasmid encoding the indicated derivative of EspP*Δ1 or EspP(F963A) were pulse-labeled with [35S]cysteine after the addition IPTG and subjected to a 1- or 5-min chase. Half of the cells were treated with PK, and EspP-containing polypeptides were immunoprecipitated using the C-terminal anti-EspP antisera.

Fig. 5.

Cross-linking of an early EspP secretion intermediate to BamA. AD202 were transformed with pDULEBpa and a derivative of pRI22 [P_lac_-_espP_] harboring the F963A mutation and an amber codon at the indicated position. Cells were pulse-labeled and subjected to a 1-min chase after the addition of IPTG. Half of each sample was UV-irradiated, and equal portions were used for immunoprecipitations with the N-terminal anti-EspP antiserum or anti-BamA or anti-SurA antisera. Truncated forms of EspP that resulted from translation termination at the amber codon are denoted (*).

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Secretion of a bacterial virulence factor is driven by the folding of a C-terminal segment - PubMed (original) (raw)