The Periplasmic Chaperone Network of Campylobacter jejuni: Evidence that SalC (Cj1289) and PpiD (Cj0694) Are Involved in Maintaining Outer Membrane Integrity

Abstract

The outer membrane (OM) of Gram-negative pathogenic bacteria is a key structure in host–pathogen interactions that contains a plethora of proteins, performing a range of functions including adhesion, nutrient uptake, export of effectors and interaction with innate and adaptive components of the immune system. In addition, the OM can exclude drugs and thus contribute to antimicrobial resistance. The OM of the food-borne pathogen Campylobacter jejuni contains porins, adhesins and other virulence factors that must be specifically localized to this membrane, but the protein sorting mechanisms involved are only partially understood. In particular, chaperones are required to ferry OM proteins across the periplasm after they emerge from the Sec translocation system. The SurA-related chaperone PEB4 (Cj0596) is the only protein with a proven role in OM biogenesis and integrity in C. jejuni. In this work, we have constructed a set of isogenic deletion mutants in genes encoding both known and predicted chaperones (cj0596, cj0694, cj1069, cj1228c, and cj1289) using NCTC 11168H as the parental strain. These mutants were characterized using a range of assays to determine effects on growth, agglutination, biofilm formation, membrane permeability and hydrophobicity. We focused on Cj1289 and Cj0694, which our previous work suggested possessed both chaperone and peptidyl-proyl cis/trans isomerase (PPIase) domains. Mutants in either cj1289 or cj0694 showed growth defects, increased motility, agglutination and biofilm formation and severe OM permeability defects as measured by a lysozyme accessibility assay, that were comparable to those exhibited by the isogenic peb4 mutant. 2D-gel comparisons showed a general decrease in OM proteins in these mutants. We heterologously overproduced and purified Cj0694 and obtained evidence that this protein was an active PPIase, as judged by its acceleration of the refolding rate of reduced and alkylated ribonuclease T1 and that it also possessed holdase-type chaperone activity. Cj0694 is most similar to the PpiD class of chaperones but is unusual in possessing PPIase activity. Taken together, our data show that in addition to PEB4, Cj1289 (SalC; SurA-like chaperone) and Cj0694 (PpiD) are also key proteins involved in OM biogenesis and integrity in C. jejuni.

Quantitative Analyses Reveal Novel Roles for N- Glycosylation in a Major Enteric Bacterial Pathogen

“…1C). Among these chaperones, we detected GroL, a periplasmic chaperone that binds to unfolded and partially folded proteins promoting folding (13), HtpG, a chaperone involved in binding to aggregated proteins and protecting the cell against environmental stresses (14), and Cj0694, a peptidyl-prolyl- cis / trans isomerase (PPIase) that interacts with the Sec translocon and promotes folding of polypeptides emerging from the translocon (15, 16). Protein degradation pathway proteins were also significantly highly abundant, notably HtrA, which is a multifunctional protein that plays a central role in proteolytically cleaving misfolded proteins as well as preventing aggregation of nonnative proteins (15, 17).…”

Sodium Taurocholate Stimulates Campylobacter jejuni Outer Membrane Vesicle Production via Down-Regulation of the Maintenance of Lipid Asymmetry Pathway

“…The C. jejuni 11168 mlaA gene was inactivated by deletion of the reading frame by homologous cross-over and replacement with a kanamycin resistance cassette. The mutation vector was created using the Gibson isothermal assembly method as previously described (Taylor et al, 2017). The C. jejuni 11168 wild-type strain was transformed by electroporation and mutant clones selected for kanamycin resistance.…”

“…Complementation of the C. jejuni 11168 mlaA mutant was performed using the pRRA system to generate a complementation vector as previously described (Taylor et al, 2017). The 11168 mlaA mutant was transformed with the complementation vector by electroporation and clones selected for apramycin resistance.…”

Review on Stress Tolerance in Campylobacter jejuni

“…We have defined an ATR in C. jejuni previously. The mediation of acid and oxygen concentration, makes them to adopt improved survival mechanism against lethal pH ( Taylor et al, 2017 ). De novo protein synthesis was necessary for the initiation of ATR in C.jejuni , which implies enhanced protein synthesis occurred during the induction phase.…”