The pathogenesis of salmonellosis appears to involve a combination of virulence factors that contribute to either gastroenteritis or enteric fever. The initial intestinal phase of the infection includes both invasion of epithelial cells and elaboration of protein toxins. This research program will continue to define the virulence mechanisms that contribute to the diarrheal phase of this disease and to identify those factors allowing progression of the infection to the more severe septicemic form. The major advances in the study of this disease during the current project period have been the purification and characterization of a cholera toxin- related enterotoxin from Salmonella, made possible by our recent success in cloning the chromosomally encoded enterotoxin (stx) gene of Salmonella. A multi-directional approach has been designed to 1) sequence the cloned stx gene, 2) prepare isogenic (stx-) mutants of Salmonella, 3) evaluate the virulence of the Salmonella mutants, 4) Purify and characterize the cloned Salmonella enterotoxin from fermenter cultures of Escherichia coli (pCHP4) (expressing the enterotoxin) and compare with native Salmonella enterotoxin isolated by specific antibody affinity chromatography, 5) perform automated amino acid sequence analysis of the purified enterotoxin to confirm the nucleotide sequence, and 6) compare the purified enterotoxin with cholera toxin and E. coli LTI, using antigenic, biologic, and chemical methodologies. Additionally, sera and intestinal washings from salmonellosis patients will be assayed with a Salmonella enterotoxin-specific ELISA, as well as by toxin neutralization in rabbit skin and intestinal loops, to determine if these patients develop a rising antitoxin titer following infection. In addition, we will continue research efforts to clarify the role of a less-defined cytotoxin in the pathogenesis of salmonellosis. The purification scheme for the Salmonella cytotoxin will be refined further, and the purified cytotoxin will be tested for effects on the intestinal mucosa of adult rabbits by light and electron microscopy. Further studies will also be pursued to study the importance of the divalent cation, Ca , in enhancing the invasion of Salmonella in orally challenged mice leading to an increased rate of death. Search for Ca mediated alterations in cell envelope proteins that could account for the enhanced invasive capacity of Ca++-grown cells will continue. This program of research should continue to provide new information about the pathogenesis of salmonellosis.
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