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To investigate the virulence mechanisms of a tetratripeptide repeat protein (TPR) of Salmonella. The protein has not been previously associated with virulence in Salmonella or other bacteria. STM20007 may be a heat stress protein. Roles of STM2007 and an accompanying apparent operon protein STM2008 will be studied in vitro and in pig infections. In addition, the independence of the virulence pathway of STM2007 from that of STM2008 and genes in the five known Salmonella pathogenicity islands will be determined.
NON-TECHNICAL SUMMARY: The studies will investigate virulence roles and mechanisms of a gene in Salmonella not previously known to be associated with bacterial virulence. The findings will extend beyond Salmonella work as the novel virulence gene encodes a repeat protein (TPR) and the repeat motif is found in proteins produced by other bacteria. The studies will extend the understanding of Salmonella infections and provide needed information of infections in a natural host. The knowledge and the attenuated mutants can be applied to a new generation of vaccines to control Salmonella infections.
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APPROACH: We will use cell cultures and tissue culture infections and experimental pig infections to investigate the virulence mechanism of Salmonella open reading frame STM2007 and STM2008. STM2007 encodes a putative tetratricopeptide repeat (TPR) protein in an apparent two gene operon with STM2008 encoding a putative periplasmic protein. Our first aim is to construct specific mutants of STM2007 (TPR) and adjacent STM2008 in Salmonella choleraesuis and Salmonella typhimurium by homologous recombination and confirm attenuation of the null mutants for colonization in pigs. The second aim is to investigate the in vitro phenotype and specific gene expression of the STM2007 (TPR) and STM2008 null mutants under stress growth conditions and screen for roles in adherence, invasion, proliferation, and survivability in cultured epithelial cells and macrophages. The final aim is to determine the independence status of STM2007 (TPR) and STM2008 virulence pathways and independence of STM2007 with that of the five Salmonella pathogenicity islands.
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PROGRESS: 2003/10 TO 2005/09<BR>
A novel TPR-like gene was identified as a possible virulence gene by screening signature tag mutants in Salmonella choleraesuis. The experimental infection was oral with about 5 x 10E7 cfu. Colonization was measured three days later by culturing the ileum and mesenteric lymph node. Colonization of systemic organs (liver, spleen, and lung) was too low to measure competitive infections. Some pigs developed a mild fever and most pigs were partially anorexic on day three. The signature tagged mutant was attenuated based on competitive infections with wild type S. choleraesuis in pigs. The TPR-like gene (STM2007) and STM2008, an adjacent gene apparently in a common operon, were reconstructed in S. typhimurium using a recombinase plasmid and a PCR-amplified kanamycin resistant cassette. Neither one of the S. typhimurium mutants were attenuated in oral infections in pigs based on competitive infections. This contrasts with the attenuation of S. choleraesuis signature tagged in STM2007. Reconstructed S. choleraesuis mutants are needed to confirm attenuation is gene specific; such mutants have been difficult to reconstruct. P22 transduction and transforming S. choleraesuis with P22 recognized LPS antigen was inadequate. Other methods of specific modification are in progress; if the reconstructed S. choleraesuis are attenuated in the competitive infections, the TPR-like gene or operon is a candidate to at least partially explain the host differences of the two salmonellae (S. choleraesuis, host restricted and S. typhimurium, broad host range).
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IMPACT: 2003/10 TO 2005/09<BR>
Understanding how a pathogen causes disease can lead to new means of disease treatment and prevention. Salmonella is the second most common cause of bacterial food borne disease and the top cause of death from such diseases.