- Altier, Craig
- North Carolina State University
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- End date
- Determine the importance of Salmonella genes specifically expressed in the lumen of the pig intestine to bacterial carriage and shedding. In this way, we will identify the bacterial determinants likely to be required for survival and growth.
- Investigate the global regulation of Salmonella genes by formate, an important signaling molecule of the ileum, the site of Salmonella colonization.
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- NON-TECHNICAL SUMMARY: Salmonellosis remains the leading cause of death in this country among foodborne diseases. Little is known about the mechanisms by which Salmonella is maintained in food-animal species that fail to show overt disease, including pigs. We plan to identify the genetic determinants of the bacteria and environmental cues of the intestinal tract that allow Salmonella survive and be carried by pigs.
1.) We will identify Salmonella genes specifically expressed within the pig as a means to find those required for survival in the animal. We will use an in vivo gene expression selection to identify these genes, then test them for their importance in infection by making mutations of the genes and testing the resulting mutant strains for survival and carriage in pigs.
2.) We will use genomic expression analysis to identify changes in gene expression due to the presence of formate. DNA microarrays will be used to identify changes in Salmonella gene transcription in response to formate, a short chain fatty acid in abundance in the ileum, the site of Salmonella colonization and invasion.
PROGRESS: 2005/09 TO 2008/09
We have sought to determine the importance of Salmonella genes specifically expressed in the lumen of the pig intestine to bacterial carriage and shedding and, in this way, to identify the bacterial determinants likely to be required for survival and growth in this animal host. We used an in vivo expression technology (IVET) system to identify genes specifically expressed in the lumen of the pig intestine. This system consists of a promoterless derivative of cre, the phage P1 recombinase, carried on a plasmid, and two chromosomal loxP sites, the targets of the Cre recombinase. The loxP sites flank npt, conferring kanamycin resistance, and sacB, which confers sensitivity to sucrose, allowing positive selection for both the presence and absence of this chromosomal cassette. Fusion of active promoters to cre induces recombination of the loxP sites and deletion of intervening DNA, allowing selection on media containing sucrose, while inactive promoters fail to induce recombination and so remain resistant to kanamycin. Using this system, we screened a library containing approximately genomic 10,000 fragments. We isolated sucrose-resistant, kanamycin-susceptible colonies that were expressed specifically when Salmonella resided within the pig after. Testing these isolates in pools demonstrated that 59 of them were reproducibly expressed within the pig. To characterize those genes, we have sequenced them and identified the regions by comparison to the completed S. typhimurium genome. We have identified several classes of genes that are induced in vivo. Among these are genes likely to be required for survival in an animal host, including those used for anaerobic growth, carbon metabolism, and production of flagella. It also appears that some of the induced genes are regulated by iron, an element known to be in low supply in animal tissues. We also found that some of the promoter fragments were expressed only in the tonsils, while others were induced in the intestinal tract. Currently, we are testing the expression of these genes to determine the signals required for their expression, using a number of environmental conditions likely to be found within the animal host. Project is being terminated at NCSU as of 8/21/06 - PI will have grant transferred to Cornell.
IMPACT: 2005/09 TO 2008/09
Pigs are a food-producing species that readily carry Salmonella but, in the great majority of cases, do not show clinical signs of disease. This high rate of unapparent infections makes pigs potential incubators of Salmonella, allowing the expansion of bacterial populations and threatening human health. It also makes pigs an important species for study in understanding infection with Salmonella. We therefore seek to determine the mechanisms by which Salmonella is maintained in pigs, a species that fails to show overt disease.
- Funding Source
- Nat'l. Inst. of Food and Agriculture
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- Bacterial Pathogens
- Meat, Poultry, Game