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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI018401-15
Application #
2671731
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1982-09-01
Project End
2000-07-31
Budget Start
1998-08-01
Budget End
2000-07-31
Support Year
15
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Peterson, J W; King, D; Ezell, E L et al. (2001) Cholera toxin-induced PGE(2) activity is reduced by chemical reaction with L-histidine. Biochim Biophys Acta 1537:27-41
Peterson, J W; Finkelstein, R A; Cantu, J et al. (1999) Cholera toxin B subunit activates arachidonic acid metabolism. Infect Immun 67:794-9
Chopra, A K; Huang, J H; Xu, X et al. (1999) Role of Salmonella enterotoxin in overall virulence of the organism. Microb Pathog 27:155-71
Peterson, J W; Boldogh, I; Popov, V L et al. (1998) Anti-inflammatory and antisecretory potential of histidine in Salmonella-challenged mouse small intestine. Lab Invest 78:523-34
Peterson, J W; Saini, S S; Dickey, W D et al. (1996) Cholera toxin induces synthesis of phospholipase A2-activating protein. Infect Immun 64:2137-43
Peterson, J W; Dickey, W D; Saini, S S et al. (1996) Phospholipase A2 activating protein and idiopathic inflammatory bowel disease. Gut 39:698-704
Chopra, A K; Brasier, A R; Das, M et al. (1994) Improved synthesis of Salmonella typhimurium enterotoxin using gene fusion expression systems. Gene 144:81-5
Chopra, A K; Peterson, J W; Houston, C W et al. (1991) Enterotoxin-associated DNA sequence homology between Salmonella species and Escherichia coli. FEMS Microbiol Lett 61:133-8
Chopra, A K; Peterson, J W; Prasad, R (1991) Cloning and sequence analysis of hydrogenase regulatory genes (hydHG) from Salmonella typhimurium. Biochim Biophys Acta 1129:115-8
Chopra, A K; Peterson, J W; Prasad, R (1991) Nucleotide sequence analysis of purH and purD genes from Salmonella typhimurium. Biochim Biophys Acta 1090:351-4

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