Proteomics of Campylobacter jejuni Growth in Deoxycholate Reveals Cj0025c as a Cystine Transport Protein Required for Wild-type Human Infection Phenotypes

• Published in: Molecular & cellular proteomics Vol. 19; no. 8; pp. 1263 - 1280
• Main Authors: Man, Lok, Dale, Ashleigh L., Klare, William P., Cain, Joel A., Sumer-Bayraktar, Zeynep, Niewold, Paula, Solis, Nestor, Cordwell, Stuart J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2020; American Society for Biochemistry and Molecular Biology
• Subjects: Bacteria; Bacterial pathogenesis; Bacterial Proteins - metabolism; Campylobacter Infections - metabolism; Campylobacter Infections - microbiology; Campylobacter jejuni; Campylobacter jejuni - drug effects; Campylobacter jejuni - growth & development; Campylobacter jejuni - pathogenicity; Carbon - pharmacology; Carrier Proteins - metabolism; Cystine; Cystine - metabolism; Deoxycholic Acid - pharmacology; Humans; Mass Spectrometry; Metabolomics; Nutrient transport; Oxidative Stress - drug effects; Pathogens; Phenotype; Proteome - metabolism; Proteomics; Sulfur; Sulfur - deficiency; Virulence; Virulence - drug effects; Metabolomics; Pathogens; Bacterial pathogenesis; Nutrient transport; Virulence; Mass Spectrometry; Bacteria; Sulfur; Campylobacter jejuni; Cystine
• ISSN: 1535-9476; 1535-9484; 1535-9484
• DOI: 10.1074/mcp.RA120.002029

Growth in the bile salt component deoxycholate (DOC) results in a profound change in the Campylobacter jejuni proteome that is reflected in phenotypic alterations including increased virulence, antibiotic resistance, and nutrient transport. A significantly induced protein was Cj0025c, which shares sequence similarity with TcyP cystine transporters from other bacteria. We show that Cj0025c is required for cystine uptake and deletion of cj0025c results in a sulfur-starved proteome and loss of wild-type phenotypes including human cell invasion, motility and helical morphology. [Display omitted] Highlights •Growth in the bile salt deoxycholate (DOC) induces virulence proteins in C. jejuni.•A putative symporter Cj0025c is associated with DOC growth and cystine transport.•Deletion of cj0025c results in loss of cystine transport and a sulfur starved proteotype.•Cj0025c is required for wild-type virulence phenotypes including human cell invasion. Campylobacter jejuni is a major cause of food-borne gastroenteritis. Proteomics by label-based two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) identified proteins associated with growth in 0.1% sodium deoxycholate (DOC, a component of gut bile salts), and system-wide validation was performed by data-independent acquisition (DIA-SWATH-MS). LC-MS/MS quantified 1326 proteins (∼82% of the predicted C. jejuni proteome), of which 1104 were validated in additional biological replicates by DIA-SWATH-MS. DOC resulted in a profound proteome shift with 512 proteins showing significantly altered abundance. Induced proteins were associated with flagellar motility and antibiotic resistance; and these correlated with increased DOC motility and resistance to polymyxin B and ciprofloxacin. DOC also increased human Caco-2 cell adherence and invasion. Abundances of proteins involved in nutrient transport were altered by DOC and aligned with intracellular changes to their respective carbon sources. DOC increased intracellular levels of sulfur-containing amino acids (cysteine and methionine) and the dipeptide cystine (Cys-Cys), which also correlated with reduced resistance to oxidative stress. A DOC induced transport protein was Cj0025c, which has sequence similarity to bacterial Cys-Cys transporters. Deletion of cj0025c (Δcj0025c) resulted in proteome changes consistent with sulfur starvation, as well as attenuated invasion, reduced motility, atypical morphology, increased antimicrobial susceptibility and poor biofilm formation. Targeted metabolomics showed Δcj0025c could use known C. jejuni amino and organic acid substrates commensurate with wild-type. Medium Cys-Cys levels however, were maintained in Δcj0025c relative to wild-type. A toxic Cys-Cys mimic (selenocystine) inhibited wild-type growth, but not Δcj0025c. Provision of an alternate sulfur source (2 mm thiosulfate) restored Δcj0025c motility. Our data confirm that Cj0025c is a Cys-Cys transporter that we have named TcyP consistent with the nomenclature of homologous proteins in other species.