The pathogenesis of salmonellosis involves intestinal colonization, epithelial cell invasion, and toxin production. The precise role of the Salmonella enterotoxin and cytotoxin in contributing to fluid secretion and tissue damage is not clear. The overall objective of the proposed research is to explain more fully the pathogenesis of salmonellosis, particularly the involvement of these protein toxins. Research during the initial period of support demonstrated that crude, cell-free lysates of Salmonella, exhibiting these toxic activities, caused tissue damage when instilled into the lumen of rabbit small intestinal loops. Furthermore, this intestinal damage was accompanied by increased mucosal levels of cyclic AMP and diminished capacity to incorporate H3-leucine into protein. A major breakthrough has occurred in our efforts to purify the cytotoxin responsible for inhibiting H3-leucine incorporation (cytotoxic activity). We have purified to homogeneity a low molecular weight peptide (3400) that is cytotoxic in vitro (cell rounding and detachment) for Chinese hamster ovary cells (greater than 10 mug/ml). While H3-leucine incorporation in these cells is inhibited by microgram doses of the purified cytotoxin, nanogram concentrations stimulate H3-leucine incorporation and elevate cyclic AMP levels. The cytotoxin eluded purification earlier because at neutral pH, it binds firmly to several large protein molecules while retaining toxicity. We will continue to refine purification methods to provide sufficient quantities of purified Salmonella cytotoxin for use in the proposed studies. The chemical, biological, and antigenic characteristics of purified Salmonella cytotoxin will be determined. The molecular mechanism of action of Salmonella cytotoxin on eukaryotic cells will be studied using cellular and in vitro protein synthesis systems. Using polyclonal antiserum to purified Salmonella cytotoxin, an ELISA and a colony blot assay will be developed to facilitate quantitation of the toxin. Rabbits immunized with the purified Salmonella cytotoxin will be challenged with Salmonella to assess the extent of protection conferred against fluid exsorption, epithelial cell invasion, and tissue damage. We will continue efforts to purify the Salmonella enterotoxin using specifically purified cholera antitoxin, immunoaffinity chromatography. Genetic approaches to the study of the Salmonella enterotoxin will be continued with the aid of the cholera toxin gene probe.
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