The broad, long-term objectives of the studies presented in this application are to define the contributions of Campylobacter jejuni potential virulence factors to disease. C. jejuni is a frequent cause of human diarrheal disease. Different C. jejuni isolates have been shown to possess various combinations of potential virulence factors: an enterotoxin, several apparently different cytotoxins, and invasiveness. None of these potential virulence properties are well-defined.
The specific aims of the research proposed here are to characterize two of the cytotoxins produced by C. jejuni and to examine their role in disease. Some strains of C. jejuni produce a Shiga-like toxin (SLT). DNA isolated from C. jejuni strains hybridizes to an E. coli SLT oligonucleotide probe. Experiments designed to clone the SLT genes from a C. jejuni strain are described. The SLT genes will be cloned into E. coli and will be examined by subcloning and sequencing. Methods of expressing the genes in E. coli using expression vectors will be tried. A mutant in the cytotoxin gene will be made and returned to C. jejuni so that isogeneic mutant and parental strains can be tested in an animal model and thereby define the role of this SLT in C. jejuni disease. Also, a strain will be made that produces enough SLT to make purification feasible. This toxin can then be reliably characterized and compared to other C. jejuni cytotoxins. A recently described type of cytotoxin that caused cultured cells to become distended prior to killing them is also produced by many C. jejuni strains. Since this toxin, called cytolethal distending toxin, (CLDT), appears to be produced in quantities that will allow purification, experiments are proposed for purifying CLDT and subsequently characterizing it. Other experiments outlining methods for cloning the CLDT gene(s) are proposed. Mutants in the cloned CLDT gene(s) will be made and returned to a parental C. jejuni strain which will then be tested, along with the parental strain, in an animal model. Hybridization studies are also proposed to test the prevalence and homogeneity of CLDT genes in other C. jejuni strains and in related species.
