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.

Keywords: Campylobacter; HtrA; PEB4; PpiD; SurA; VirK; outer membrane; periplasmic chaperone.

FIGURE 1

Physiological phenotypes of chaperone mutants (blue bars) and isogenic parent strain C. jejuni 11168H (gray bars). (A) Growth measured as cell density at 600 nm at 12 h post-inoculation in BTS broth under standard microaerobic conditions. (B) Motility of strains determined by point inoculation of semi-solid agar plates and measuring the diameter of growth after 48 h. (C) Biofilm formation in BTS broth in 96-well plates after 24 h determined by crystal violet staining. (D) Autoagglutination (AAG) activity determined by the decrease in absorbance of cells in the aqueous phase of static cultures. (E) Cell surface hydrophobicity determined by the change in cell absorbance in the aqueous phase after mixing cells with the alkane hydrocarbon hexadecane. (F) Susceptibility to cell lysis by lysozyme digestion in the presence of 2.5 mM deoxycholate. Student t-test P-values are displayed as ^∗ < 0.05, ^∗∗ < 0.01, ^∗∗∗ < 0.001.

FIGURE 2

Genetic complementation of HΔcj0694 and HΔcj1289 strains. (A) Viability of mid-log BTS broth cultures diluted to an optical density at 600 nm of exactly 0.1, as determined by serial dilution and colony counts. (B) OM integrity as determined by susceptibility to cell lysis by lysozyme digestion. Student t-test P-values are displayed as ^∗ < 0.05, ^∗∗∗ < 0.001.

FIGURE 3

2D-PAGE analysis of the outer membrane and periplasmic proteins of the wild-type (colored orange) and cj0694 and cj1289 mutants (colored blue). Protein samples were prepared as described in Section “Materials and Methods” and separately resolved by 2D-PAGE. Orange spots represent proteins found in the wild-type fractions only, and absent in the mutant fractions. Blue spots represent proteins found in mutant fractions only, and absent in the wild-type fractions. Black spots represent proteins found in both the wild-type and mutant fractions. In (A,C), the circled numbered protein spots in the 2D-gels of the OMs were identified by mass spectrometry analysis. These are: 1, MOMP (PorA); 2, Cj0964 (Mascot score 88); 3, Cj0112 (TolB, Mascot score 2115); 4, Cj1228 (HtrA, Mascot score 2056). In (B,D), the circled numbered protein spots in the periplasms were correlated with those published in our previous study (Hitchcock et al., 2010). These are: 1, TorA; 2, MfrA; 3, Peb1A; 4, Cj0998; 5, Cj0715; 6, Cj1153; 7, Cj1659 (p19 protein).

FIGURE 4

Overproduction, purification and PPIase activity of Cj0694. (A) Lane 1; Cj0694 lacking the N-terminal membrane anchor and with a C-terminal his-tag initially purified by Ni-NTA affinity chromatography (monomer molecular weight 54.8 kDa). Lane M; molecular weight markers. (B) Further purification of Cj694 by DEAE anion-exchange chromatography with elution from 0 to 1 M NaCl. Lanes 1–7 are samples taken across the UV-absorbing peak eluted from the column and show a single major band on SDS-PAGE after staining with Coomassie Blue. Note that the migration of the protein is affected by the salt present in the elution buffer and the apparent molecular weight is higher than in (A). Lane M; molecular weight markers. (C) PPIase activity of Cj0694 demonstrated by refolding of RCM-RNaseT₁ in the presence of 4 M NaCl (see Materials and Methods) either without or with the addition of purified Cj0694 as shown. The purified periplasmic chaperone PEB4 was used as a positive control. The fluorimeter was set to zero at the time of dilution, so that the increase in fluorescence results from the uncatalyzed (blue progress curve) or chaperone catalyzed (green, orange, and red progress curves) refolding process. Results shown are a single representative experiment.

FIGURE 5

Inhibition of protein aggregation by Cj0694. Model proteins Lysozyme (A) or Rhodanese (B) were unfolded using guanidine-HCl and their aggregation due to renaturation at 25°C monitored in the absence (control) or presence of Cj0694 by light scattering at 320 nm. An additional control contained 1 μM bovine serum albumen (BSA) to ensure that the observed inhibition of aggregation was specifically catalyzed by Cj0694. The traces shown are typical of several experiments performed.

FIGURE 6

Model of the periplasmic chaperone network of C. jejuni. OM and periplasmic proteins emerging from the Sec export complex interact with PpiD (Cj0694) for initial folding and translocation to the SalC (Cj1289) or PEB4 (Cj0596) pathway. VirK (Cj1069) may interact with certain substrates in the cytoplasm prior to their entrance into the SEC complex, based on the localization of this protein determined by Novik et al. (2009). SalC and PEB4 are proposed to translocate partially folded OMPs across the periplasm to the BAM complex, where they are inserted into the OM. HtrA (Cj1228) may participate in folding various periplasmic proteins or possibly to rescue OMPs that dissociate from SurA or PEB4 before reaching the OM. HtrA is also secreted from the cell (Hoy et al., 2012) probably mediated by OM vesicles (Elmi et al., 2016).