Identification of a
            <i>Campylobacter jejuni</i>
            Protein That Cross-Reacts with Cholera Toxin

Albert, M. John; Haridas, Shilpa; Steer, David; Dhaunsi, Gursev S.; Smith, Allan E.; Adler, Ben

doi:10.1128/iai.00139-07

Cited by 14 publications

(18 citation statements)

References 28 publications

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“…Due to the fact that PorA (MOMP, Cj1259) is the major OMP of C. jejuni (Albert et al , 2007; Clark et al , 2007), it was an obvious choice as an outer-membrane marker (antibodies against MOMP were a kind gift from Qijing Zhang, Iowa State University). This protein has been well documented in the literature as an OMP.…”

Section: Resultsmentioning

confidence: 99%

Evaluation of procedures for outer membrane isolation from Campylobacter jejuni

et al. 2009

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Although infection with Campylobacter jejuni is one of the leading causes of gastroenteritis worldwide, relatively little is known about the factors that are required to elicit a protective immune response. The need for a vaccine against this pathogen is well recognized and a number of vaccine candidates have been tested with varying degrees of success; however, there is still a lack of a suitable vaccine. To gain a better understanding of the outer-membrane protein components of this organism, a 'gold standard' method to purify the outer membrane is needed. Therefore, we attempted to develop a robust and reliable method which resulted in a pure outermembrane fraction. A total of nine methodologies were examined and analysed by SDS-PAGE and immunoblotting using subcellular markers for the cytoplasm, cytoplasmic membrane and outer membrane. We found that glycine extraction, differential detergent extraction using Triton X-100, serial extraction using 1 M Tris pH 7, spheroplasting by lysozyme and sonication, and carbonate extraction did not produce pure outer-membrane preparations. However, we identified three methods that provided outer-membrane fractions free from subcellular contamination. Isopycnic centrifugation using a 30-60 % sucrose gradient produced seven fractions free from cytoplasmic or cytoplasmic membrane contamination; however, these fractions did not correspond as well as expected with the typical outer-membrane-associated peak (e.g. Escherichia coli or Salmonella). The spheroplast method using lysozyme alone also resulted in pure outer-membrane fraction, as did carbonate washing of this sample. The extraction of outer membranes using N-lauroylsarcosine (Sarkosyl) produced the purest and most reproducible sample. These outer-membrane preparations will be useful for future studies aimed at identifying C. jejuni surface proteins as vaccine components.

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Section: Resultsmentioning

confidence: 99%

Evaluation of procedures for outer membrane isolation from Campylobacter jejuni

et al. 2009

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“…There is uncertainty on the production of CTLT by C. jejuni . Our recent work indicated that the major outer membrane protein (MOMP-PorA) of C. jejuni cross-reacts with cholera toxin (CT) which would likely have misled investigators that C. jejuni produces a CTLT [5]. It is believed that the cytotoxin(s) may mediate inflammatory diarrhoea that is characteristic of infection in individuals in developed countries [6].…”

Section: Introductionmentioning

confidence: 99%

Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni

et al. 2012

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BackgroundCampylobater jejuni, a major foodborne diarrhoeal pathogen is reported to produce a number of cytotoxins of which only a cytolethal distending toxin (CDT) has been characterised so far. One or more additional cytotoxins other than CDT, including a Chinese hamster ovary (CHO) cell active, Vero cell inactive cytotoxin, may mediate inflammatory diarrhoea. Our objective was to develop a method to enrich and thus partially characterise this cytotoxin, as a pathway to the eventual identification and characterisation of the toxin.ResultsA number of biochemical methods including cation- and anion-exchange chromatography were evaluated to enrich the cytotoxin from a cell lysate of a known cytotoxin-producing C. jejuni, C31. The cytotoxin in crude lysate was initially prepared by size-exclusion desalting and then subjected to high pressure liquid chromatography (HPLC) ion-exchange fractionation. One pooled fraction (pool B) was cytotoxic for CHO cells equivalent to crude toxin (tissue culture infectivity dose 50 [TCID50] of 1–2 μg/ml). The proteins of pool B were identified by mass spectrometry (MS) after separation by SDS-PAGE and trypsin digestion. Also, pool B was directly digested with trypsin and then subjected to liquid chromatography tandem mass spectrometry (LCMS) analysis for identification of lesser abundant proteins in the fraction. A total of 41 proteins were found in the fraction, which included enzymes involved in metabolic and transport functions. Eighteen non-cytoplasmic proteins including 2 major antigenic peptide proteins (PEB2 and PEB3) and 3 proteins of unknown function were also identified in the screen. Cytotoxicity in pool B was trypsin-sensitive indicating its protein nature. The cytotoxic activity was heat-stable to 50°C, and partially inactivated at 60-70°C. The pool B fraction also induced fluid accumulation in the adult rabbit ileal loop assay with cytotoxicity for mucosa confirming the presence of the cytotoxin.ConclusionsWe report the enrichment and partial purification of C. jejuni cytotoxin by HPLC ion-exchange chromatography. Further purification may be achieved using additional complementary chromatographic techniques. A short-list of six candidate cytotoxin proteins was identified using an LCMS screen of pool B. Successful isolation of the cytotoxin will initiate steps for the determination of the role of this cytotoxin in the pathogenesis of C. jejuni diarrhoea.

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“…However, the ileal loop fluids did not cause CHO cell elongation. In the GM1 ELISA, CAYE filtrates from two Swedish strains, the Australian strain and 16 Kuwaiti strains were positive7.…”

Section: Resultsmentioning

confidence: 99%

“…However, a rabbit polyclonal antibody raised against the GST-PorA failed to react with-CT in the immunoblot. This suggested that the reaction was a one-way reaction7.…”

Section: Resultsmentioning

confidence: 99%

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Major Outer Membrane Proteins from Many Campylobacter Species Cross-React with Cholera Toxin

Albert

Haridas

Adler

2008

Clin Vaccine Immunol

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We have previously shown that Campylobacter jejuni strains do not produce a functional cholera toxin-like toxin (CTLT) detectable in a Chinese hamster ovary cell assay. Instead, the 53-kDa major outer membrane protein (OMP) of C. jejuni, PorA, reacts with cholera toxin (CT) antibody on immunoblots. Here, we have extended this observation to other species of Campylobacter, including C. coli, C. lari, C. fetus, C. hyointestinalis, and C. upsaliensis, the common 53-kDa OMP of which reacted with CT antibody in immunoblotting assays. There were additional reactive bands for C. fetus. As with C. jejuni, this finding may lead to the erroneous conclusion that these additional species produce a functional CTLT. However, this common cross-reactive OMP can be explored as a vaccine candidate to prevent campylobacteriosis.

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Identification of a Campylobacter jejuni Protein That Cross-Reacts with Cholera Toxin

Cited by 14 publications

References 28 publications

Evaluation of procedures for outer membrane isolation from Campylobacter jejuni

Evaluation of procedures for outer membrane isolation from Campylobacter jejuni

Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni

Major Outer Membrane Proteins from Many Campylobacter Species Cross-React with Cholera Toxin

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