CRIS NUMBER: 0204714
SUBFILE: CRIS
PROJECT NUMBER: NCV-VMCG-0029
SPONSOR AGENCY: CSREES
PROJECT TYPE: NRI COMPETITIVE GRANT
PROJECT STATUS: TERMINATED
MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Sep 15, 2005
TERMINATION DATE: Aug 21, 2006
GRANT PROGRAM: ENSURING FOOD SAFETY
GRANT PROGRAM AREA: Nitrogen Fixation
CLASSIFICATION HEADINGS
KA712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins S4010 - Bacteria F1100 - Bacteriology G4.1 - Reduce Incidence of Foodborne Illnesses and Contaminants
RESEARCH EFFORT CATEGORIES
BASIC |
100% |
APPLIED |
(N/A)% |
DEVELOPMENTAL |
(N/A)% |
KEYWORDS: swine; disease carriers; bacterial diseases (animals); formic acid; genomes; gene analysis; salmonella; food contamination; zoonoses; salmonellosis; genes; gene expression; intestines; bacterial genetics; survival; shedding; colonization; host pathogen relations; signal transduction; gene regulation; disease transmission; human diseases
PROGRESS: Sep 15, 2005 TO Sep 14, 2008
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: 2005-09-15 TO 2008-09-14
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.
PUBLICATION INFORMATION: 2005-09-15 TO 2008-09-14
No publications reported this period
PROJECT CONTACT INFORMATION
NAME: |
Altier, C. |
PHONE: |
919-513-8284 |
FAX: |
919-513-6464 |
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