A quantitative proteomic screen of the Campylobacter jejuni flagellar-dependent secretome

Abstract

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the world. A number of factors are believed to contribute to the ability of C. jejuni to cause disease within the human host including the secretion of non-flagellar proteins via the flagellar type III secretion system (FT3SS). Here for the first time we have utilised quantitative proteomics using stable isotope labelling by amino acids in cell culture (SILAC), and label-free liquid chromatography-mass spectrometry (LC/MS), to compare supernatant samples from C. jejuni M1 wild type and flagella-deficient (flgG mutant) strains to identify putative novel proteins secreted via the FT3SS. Genes encoding proteins that were candidates for flagellar secretion, derived from the LC/MS and SILAC datasets, were deleted. Infection of human CACO-2 tissue culture cells using these mutants resulted in the identification of novel genes required for interactions with these cells. This work has shown for the first time that both CJM1_0791 and CJM1_0395 are dependent on the flagellum for their presence in supernatants from C. jejuni stains M1 and 81-176.

Biological significance: This study provides the most complete description of the Campylobac er jejuni secretome to date. SILAC and label-free proteomics comparing mutants with or without flagella have resulted in the identification of two C. jejuni proteins that are dependent on flagella for their export from the bacterial cell.

Keywords: Campylobacter jejuni; Flagella; Mass spec; SILAC; Secretome; Type III secretion system.

Graphical abstract

Fig. S1

Generated M1 mutants display similar rates of growth and motility to that of M1 WT. (A, B) CFU counts of mutants generated, compared to M1 WT grown in MH broth. Cultures were equalized to an OD_600nm prior to incubation. (C) Motility of M1 mutants are comparable to that of M1 WT.

Fig. 1

Overview of M1 secretome analysis. After isotopic incorporation of C. jejuni M1 WT and flgG strains with either “light” or “heavy” arginine, respectively, (H = mutant labelled with “heavy” l-arginine, L = WT labelled with “light” arginine), bacterial cells were pelleted and supernatants were filtered, pooled and concentrated. After digestion, tryptic peptides were analysed by LC-MS/MS. Proteins secreted via the flagellum are characterized by enrichment of peptides containing “light” isotopes.

Fig. 2

Mean H/L ratios of proteins detected in culture supernatants from four biological replicates. Low H/L ratios represent proteins found at a lower abundance within flgG supernatants while high H/L are proteins found at increased abundance within flgG supernatants.

Fig. 3

Western blotting of FLAG-tagged proteins secreted from M1 WT and flgG strains identified at various H/L ratios within the SILAC experiment. Detection of FLAG-tag incorporated into C-terminus of proteins of interest. Immunoblotting exhibits the effect of an flgG mutant on protein abundance within supernatant and whole cell protein samples.

Fig. 4

Mutations in genes previously characterized to be secreted via the flagellum and proteins identified by SILAC and label-free LC/MS have altered rates of (A) adhesion and (B) invasion of CACO-2 cells. M1 WT cell interaction was set at 100%, * denotes strains with a P value < 0.05 compared to M1 WT.

Fig. 5

81–176 homologues of CJM1_0791 and CJM1_0395 are dependent on the flagellum for their presence in 81–176 supernatant. Immunoblotting of FLAG-tagged proteins exhibit the effect of an flgG mutant on protein abundance present in supernatant and whole cell protein samples obtained from strain 81–176.