title: The Periplasmic Chaperone Network ofCampylobacter 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 pathogenCampylobacter jejunicontains 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 inC. 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, andcj1289) 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-proylcis/transisomerase (PPIase) domains. Mutants in eithercj1289orcj0694showed 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 isogenicpeb4mutant. 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 T1and 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 inC. jejuni.
authors.affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield Sheffield, UK.
authors.forename: Aidan J
authors.initials: AJ
authors.lastname: Taylor
authors.affiliation: Faculty of Medicine, King Abdulaziz University Jeddah, Saudi Arabia.
authors.forename: Shadi A I
authors.initials: SA
authors.lastname: Zakai
authors.affiliation: Department of Molecular Biology and Biotechnology, The University of Sheffield Sheffield, UK.
authors.forename: David J
authors.initials: DJ
authors.lastname: Kelly
keywords: Campylobacter; HtrA; PEB4; PpiD; SurA; VirK; outer membrane; periplasmic chaperone