Identification of genes involved in Salmonella intestinal colonization and persistence in chicken : a dissertation /
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| Format: | Thesis Book |
| Language: | English |
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[College Station, Tex.] :
[Texas A&M University System Health Science Center],
[2013]
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| Subjects: |
| Abstract: | ABSTRACT: Contaminated chicken or egg products are major sources of human salmonellosis. We used pools of mutants to identify novel genes that are important for colonization and persistence of S. Typhimurium in chickens. To minimize the complexity of the pools and avoid bottlenecks during infection we used a two-step procedure. First, we constructed a library of mutants containing targeted deletion of groups of contiguous genes that contains 182 mutants with a total of 2069 genes deleted. These multi-gene deletion (MGD) mutants were pooled and delivered to four-day old chicks. Regions under selection after 1, 3, and 9 days of infection were identified by comparing the representation of each mutant in the input and output pools. Eleven regions were under selection at all time points after infection ('colonization' mutants), and fifteen regions were under selection only at day 9 post-infection ('persistence' mutants). Next, we assembled deletion mutants in single genes (SGD) in nine of the regions under selection. We identified twelve novel genes under selection. Nine of these candidate genes were retested and six genes were confirmed to be under selection in chicks via competitive infections and complementation analysis; STM0580, STM01295, STM1297, STM3612, STM3615, and STM3734 appeared to be important for colonization of Salmonella Typhimurium in the chick model. Among mutants under selection from the second screening was a gene encoding orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10), pyrE (STM3733), involved in the fifth step of de novo pyrimidine synthetic pathway. We confirm that [delta] pyrE mutant poorly colonizes 4-day old chicks in competitive infection with isogenic wild type, and that the reduced colonization of this mutant could be restored by providing a copy of pyrE in trans. We further show that our [delta]pyrE mutant grows poorly in low-nutrient conditions and that the ability of this mutant to grow was restored when precursors to the pyrimidine salvage pathway were provided. Finally, we show that the colonization defect of a [delta]pyrE mutant during infection is restricted to chickens. The addition of uracil to the chick diet restores the ability of our [delta]pyrE mutant to grow normally in chicks. Thus, we show that de novo pyrimidine synthesis is necessary for colonization of Salmonella Typhimurium in the chick model, and that the salvage pathway is not used in these condidtions. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". "Submitted to the Office of Research and Graduate Studies The Texas A&M University System Health Science Center In partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2013." Approved as to style and content by: Helene Andrews-Polymenis, Jonathan T. Skare, James E. Samuel, Huaijun Zhou |
| Physical Description: | xiv, 128 leaves : illustrations (mostly color) ; 28 cm. |
| Bibliography: | Includes bibliographical references (leaves 93-111). |