title: Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni.
abstract: BACKGROUND
  Campylobater 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.
  
  
  RESULTS
  A 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 [TCID(50)] 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.
  
  
  CONCLUSIONS
  We 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.
authors.affiliation: Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria, Australia.
authors.forename: Xenia
authors.initials: X
authors.lastname: Gatsos
authors.forename: David L
authors.initials: DL
authors.lastname: Steer
authors.forename: Thamradeen A
authors.initials: TA
authors.lastname: Junaid
authors.forename: A Ian
authors.initials: AI
authors.lastname: Smith
authors.forename: Ben
authors.initials: B
authors.lastname: Adler
authors.forename: M John
authors.initials: MJ
authors.lastname: Albert
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D016466:CHO Cells; D016123:Campylobacter jejuni / Q000737:chemistry* / Q000472:pathogenicity*; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D003603:Cytotoxins / Q000737:chemistry / Q000302:isolation & purification* / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D007082:Ileum / Q000187:drug effects / Q000473:pathology; D020128:Inhibitory Concentration 50; D007413:Intestinal Mucosa / Q000187:drug effects / Q000473:pathology; D013058:Mass Spectrometry; D055550:Protein Stability; D011817:Rabbits; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins