Differential proteomic analysis of Clostridium perfringens ATCC13124; identification of dominant, surface and structure associated proteins

Abstract

BackgroundClostridium perfringens is a medically important clostridial pathogen causing diseases in man and animals. To invade, multiply and colonize tissues of the host, a pathogen must be able to evade host immune system, and obtain nutrients essential for growth. The factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis. Many of the virulence determinants and putative vaccine candidates for bacterial pathogens are known to be surface localized.ResultsUsing 2-DE mass spectrometry strategy, we identified major surface (22) and cell envelope (10) proteins from Clostridium perfringens ATCC13124 and those differentially expressed (11) in cells grown on cooked meat medium (CMM) in comparison with cells grown in reference state (tryptose-yeast extract-glucose medium). Riboflavin biosynthesis protein, ornithine carbamoyltransferase, cystathionine beta-lyase, and threonine dehydratase were the predominant proteins that exhibited 2.19 to 8.5 fold increase in the expression level in cells growing on CMM.ConclusionOrnithine carbamoyltransferase and cystathionine beta-lyase were over-expressed in cells grown on cooked meat medium and also identified in the surface protein fraction and the former was immunogenic; making them potential vaccine candidates. Based upon bioinformatic analysis; choloylglycine hydrolase family protein, cell wall-associated serine proteinase, and rhomboid family protein were predicted as surface protein markers for specific detection of C. perfringens from the environment and food. Most of the proteins over-expressed in CMM were shown to have putative function in metabolism, of which seven were involved in amino acid transport and metabolism or lipid metabolism.

Selenium effects on the metabolism of a Se-metabolizingLactobacillus reuteri: analysis of envelope-enriched and extracellular proteomes

“…27 These results corroborate the suggestion of the need for the acidophilic L. reuteri to control the entire operon to attenuate NH 3 -induced toxicity, since it is meanwhile involved to cope with stress induced by selenium. Interestingly OTC was found in the envelope-enriched fraction as well as on the surface of Clostridium perfringens, 50 and the opportunistic Staphylococcus epidermidis. 52 If we consider the physiological role of ADI, we find that this route is coupled with an antiport system ensuring new arginine supply inside the cell by exchanging the OTC-generated ornithine.…”

“…48 CBAs have been suggested to repress bacterial growth in the small intestine by means of direct antimicrobial effects, up-regulation of host mucosal defenses, or synergistic action of both mechanisms. 49 Choloylglycine hydrolase has widely been reported to be exposed on the surface of several Gram positive bacteria, such as Clostridium perfringens 50 and Bifidobacterium lactis BI07, where it is responsible for Plg-binding. 14 The expression and up-regulation of this enzyme by L. reuteri Lb2 BM DSM 16143 have a great importance for its probiotic traits, suggesting both the ability of the strain to survive to CBAs action and also a positive role of selenium in enhancing bile-salt survival.…”

Comparative Proteomic Analysis of Extracellular Proteins of Clostridium perfringens Type A and Type C Strains

“…For MALDI-TOF mass spectrometry, 1 l of the digest was mixed with 2 l of the matrix solution (5 mg ␣-cyano-4-hydroxycinnamic acid in 80% [vol/vol] acetonitrile and 0.1% [wt/vol] trifluoroacetic acid [TFA]), and 1 l of this mixture was deposited onto the MALDI target. The spectrum was obtained in the mass range of 500 to 4,000 Da and was calibrated using a calibration mixture containing angiotensin I, Substance P, adrenocorticotropin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17) [ACTH(1-17)], ACTH , and somatostatin (28).…”

“…Extracellular proteins were immunoblotted using a standard method as described previously (2). Sera (anti-WC or anti-gangrene) obtained from mice were used at a 1:1,000 dilution (or as indicated otherwise) in blocking buffer.…”

“…The SPS agar was composed of tryptose (15 g), yeast extract (10 g), ferric citrate (0.5 g), sodium sulfite (0.5 g), sodium thioglycolate (0.1 g), polysorbate 80 (0.05 g), sulfadiazine (0.12 g), polymyxin B sulfate (0.01 g), agar (15 g), and distilled water (1,000 ml). The inoculation was carried out in an anaerobic workstation (Don Whitley Scientific Ltd., Shipley, England) operating at 30°C as described previously (2). After incubation, black colonies were anaerobically transferred to 3 ml of thioglycolate broth (TGB) containing 5 g of yeast extract, 15 g of tryptone, 5.5 g of glucose, 0.5 g of sodium thioglycolate, 2.5 g of sodium chloride, 0.5 g of L-cystine, 0.001 g of resazurin, 0.75 g of agar, and 1,000 ml of distilled water.…”