However, our data do not exclude the possibility that cytotoxic effects may be mediated by a mixture of proteins. Guerrant et al. [16] reported that the cytotoxin is a periplasmic protein as it can be extracted by polymyxin B. However, in our hands, polymyxin B interfered with the CHO cell assay, as it produced cytotoxic effects similar to the C. jejuni cytotoxin (unpublished data). Conclusions Even though C. jejuni is a major foodborne diarrhoeal Torin 1 in vitro pathogen causing significant morbidity and mortality, its pathogenesis is poorly understood. It is important to MEK inhibitor purify and characterise its major

cytotoxin to define its role in pathogenesis. We have succeeded in developing a method (HPLC ion-exchange

purification method) for enriching VS-4718 solubility dmso and partially purifying the cytotoxin. Further studies are required for a complete purification of the cytotoxin. The cytotoxin may be highly active at very low concentrations, low enough to remain undetected by our current proteomics identification procedures, removing most of the contaminating proteins via sub-fractionation of the cell should increase the chances of isolating and identifying this cytotoxin. One other option is to purify the supernatant of broth culture of C. jejuni, although given its fastidious nature and slow growth rate, high levels of active cytotoxin may be difficult ID-8 to purify from the supernatant. In this paper, we present preliminary data in our attempt to isolate, purify and identify the protein involved in cytotoxic activity of C. jejuni. We have employed an activity assay based on the lethal effects of the toxin on CHO cells to rapidly screen for activity and used this assay to screen chromatographic fractions to locate the presence of the active protein. We have been unable to unequivocally identify the protein as the sample remains too complex although we have identified some previously uncharacterised non-cytoplasmic proteins which with further experimentation

potentially may be attributable to the cytotoxin. We will attempt further isolation of the protein so that we are then able to sequence and identify the protein. The activity of the toxin containing fraction was validated by performing the rabbit ileal loop assay. Methods Preparation of the cytotoxin and its detection The reference cytotoxin-positive C. jejuni strain, C31 used in our previous study was used in this study [8]. The organism was grown on 7% sheep blood agar in a microaerobic atmosphere generated with BBL gaspak (Becton Dickinson, Sparks, MD, USA) in a jar with catalyst at 42°C for 48 h. The bacterial growth was suspended in phosphate-buffered saline (PBS, pH, 7.2) to McFarland’s opacity of 10 (equivalent to 3 X 109 cells).