The overall objective continues to be the probing of the pathogenic mechanism of experimental salmonellosis to define the role of Salmonella enterotoxin (Stn) as a selected virulence factor in the intestinal phase of Salmonella infection. Our past research has enabled us to clone a fragment of Salmonella chromosomal DNA, encoding the stn gene. When expressed with the T7 RNA promoter/polymerase system, cell-free lysates of E. coil, containing Stn, evoked enterotoxic responses in rabbit intestinal loops. We determined the molecular structure of cloned Stn, based on predictions from the nucleotide sequence, which allowed us to localize the stn gene at approximately 90 min on the Salmonella chromosome opposite the hydHG operon. We used site-directed mutagenesis to identify the stn initiation codon, which was a """"""""TTG"""""""" rather than the typical """"""""ATG."""""""" Further, the unusually high pI of the protein (11.7) confers a strong negative charge on the toxin's surface, causing it to bind to positively charged molecules at pH 7.0, a property that has complicated Stn purification.
Our Specific Aims seek to construct an isogenic stn derivative of a virulent Salmonella strain(s) (e.g., TML- R66), for use in determining the precise role of Stn in the pathogenic mechanism (i.e., secretion and/or invasion). Precautions will be taken to avoid secondary alterations in hydHG function. Further, we are striving to improve gene expression and Stn solubility by subcloning the stn gene into selected fusion protein vectors. Total Stn antigen was, increased 64 fold by preparing two fusion proteins [glutathione S- transferase::Stn (Gst::Stn) and thioredoxin A::Stn (TrxA::Stn], and TrxA::Stn increased Stn solubility by 50 fold. With improvements in gene expression of Stn, we anticipate that purification to homogeneity is achievable. The purified protein will be used in experiments to study the molecular mechanism of the enterotoxin in elevating cAMP and PGE2 levels in intestinal cells, as well as to generate a polyclonal antiserum to Stn. Finally, stn genes from selected Salmonella isolates, that differ in intestinal virulence (e.g., fluid accumulation), will be amplified by PCR so that the nucleotide sequence of each can be examined and compared. The proposed studies should provide new and helpful information about the pathogenesis of salmonellosis.
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