“Background Campylobacter jeuni is a foodborne pathogen and a major cause of bacterial diarrhoea worldwide , yet its pathogenicity is poorly understood. The virulence attributes of C. jejuni include cell culture adherence and invasion, flagella and motility, iron-acquisition capability and toxin production . Known toxins include a cytolethal distending toxin (CDT), a cholera toxin-like enterotoxin (CTLT), and a number
of cytotoxins . However, only the genes encoding the CDT have been identified so far . There is uncertainty on the production of CTLT by C. jejuni. Our recent work indicated that the major outer membrane protein (MOMP-PorA) selleck chemical of C. jejuni cross-reacts with cholera toxin (CT) which would likely have misled investigators that C. jejuni produces a CTLT . It is believed Proteasome purification that the cytotoxin(s) may
mediate inflammatory diarrhoea that is JNK-IN-8 clinical trial characteristic of infection in individuals in developed countries . One major cytotoxin is a protein-sized molecule that is active on a number of cell lines such as HeLa and Chinese hamster ovary (CHO), but is inactive on Vero cells . A previous report claimed that the MOMP of C. jejuni was responsible for cytotoxicity on mammalian cells . However, in our previous work, the expressed PorA protein from the cloned gene of a cytotoxin-producing C. jejuni strain did not have cytotoxic activity for mammalian cells and was also devoid of diarrhoeagenic activity in an animal model of infection . In our continuing efforts to characterise this unknown cytotoxin, we investigated a series of chromatographic methods to enrich the cytotoxin from a cytotoxic C. jejuni
strain. Using previously established methods of detection as well as further modifications to these protocols, we have attempted to isolate and purify the cytotoxin. The results of further characterisation studies confirm that the likely cytotoxin candidate is a protein. The results are reported in this communication. Results and discussion Cytotoxicity assay In this study, we have developed a methodology to detect and purify the toxin potentially involved in the diarrhoeagenic activity of C. jejuni, C31 strain. To detect and quantify cytotoxic activity during purification, we used an activity assay based on the lethal effects of the toxin on CHO cells. The TCID50 Demeclocycline of C31 strain for CHO cells was similar at 1–2 μg for a freshly prepared protein extract as well as a reconstituted form of the lyophilised extract as estimated by the visual method by direct microscopic observation of cytotoxic effect on cells  or by the indirect methyl thiazol tetrazolium (MTT) method by spectrophotometric measurement of formazin . The cytotoxic effect of C31 toxin on CHO cells is shown in Figure 1. The extract was devoid of any cytotoxic effect when tested on Vero cells as described previously . Figure 1 Effect of C. jejuni crude protein extract on CHO cells.