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To test further the hypothesis that three unique RpoN-dependent PTS permeases are important for host colonization by S. Typhimurium, I propose to: 1) Individually characterize the roles of the RpoN-dependent PTS permeases in S. Typhimurium's colonization of the avian intestinal tract. Given my preliminary data that shows that knocking out all three RpoN-dependent PTS permeases has a significant effect on colonization of the chick gastrointestinal tract, I expect that one or more of the PTS permeases will be shown to be important for efficient colonization. Such a finding would contribute significantly to our understanding of how S. Typhimurium's physiology affects its ability to establish a niche within the avian gastrointestinal tract. <br/>2)To determine if the RpoN-dependent PTS permeases are important in the colonization of a mammalian animal model. I expect that one or more of the PTS permease mutants will be unable to colonize the mouse model since the macrophage phagosome, where S. Typhimurium resides in the liver and spleen, is considered to be nutrient-poor. If the RpoN-dependent PTS permeases are found to be needed for either virulence or colonization, this would be a significant finding since the PTS permeases could be potential targets for therapeutic agents. <br/>3) Identify the substrates transported by the RpoN-dependent PTS permeases. I expect to identify most (if not all) of the substrates for the RpoN-dependent PTS permeases. The results of these studies are imperative for understanding the roles of these PTS permeases in S. Typhimurium, and how they might assist S. Typhimurium in establishing a niche within the avian or mammalian gastrointestinal tract. Such results could lead to new strategies for controlling S. Typhimurium in broiler chickens and other food animals and thus have a significant impact on food safety.
Non-Technical Summary:<br/>
Salmonella enterica serovar Typhimurium (S. Typhimurium) is a source of food contamination and a leading cause of food-borne illness. S. Typhimurium possesses three sugar phosphotransferase (PTS) permeases which are absent in most strains of closely related bacteria, and whose expression is controlled by the transcription factor RpoN. The goal of the proposed studies is to examine the function of these PTS permeases in S. Typhimurium to test the hypothesis that they have important roles in Salmonella's ability to colonize poultry or mammals. This hypothesis will be tested by assessing the roles of the PTS permeases in Salmonella's colonization of the avian and mammalian gastrointestinal tract, examining the regulation of the genes encoding the permeases, and identifying the substrates transported by the permeases. In preliminary studies, I found that a strain in which I inactivated all three of the PTS permeases is altered in its ability to colonize the avian ileum and ceca, suggesting the importance of one or more of these PTS permeases for efficient avian colonization by S. Typhimurium. Characterization of the RpoN-dependent PTS permeases may lead to new strategies for controlling Salmonella in poultry and mammalian food animals.
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Approach:<br/>
To characterize the roles of the RpoN-dependent PTS permeases in S. Typhimurium's colonization of the avian intestinal tract, colonization assays in chickens will be carried out using strains in which each of the RpoN-dependent PTS permeases has been knocked out individually to identify which of these permeases is required for efficient colonization. A 2-day old White Leghorn broiler chick model will be used for these studies. Chicks will be challenged with an oral inoculation of 2 x 108 CFU of a 1:1 mixture of PTS mutant and wild-type strains. Chicks will be sacrificed at different time points following inoculation, and the ileum and ceca will be removed so that the ratio of PTS permease mutant to wild type S. Typhimurium SL1344 can be assessed in each location at different times during colonization. To determine if the RpoN-dependent PTS permeases are important in the colonization of a mammalian model, infection assays will be carried out in BALB/c mice. Five week-old female BALB/c mice orally with 1 x 106 cells of either the PTS permease mutant or wild-type S. Typhimurium SL1344. The number of CFUs isolated from mice containing the wild-type strain or the PTS mutant strain, will be determined separately. Data will be interpreted using the Wilcoxon signed-rank test which has been used to show significance in scatter plot data. To identify the substrates for these three RpoN-dependent PTS permeases, I assess the expression and growth phenotypes of the PTS permease mutants on a variety of sugars and use a metabolomics approach to identify phosphorylated sugars that accumulate in mutant strains. I will examine growth phenotypes of the mutants with different carbohydrates under several conditions, including aerobic, microaerobic, and anaerobic conditions. Given that the areas of the host gastrointestinal tract which S. Typhimurium would encounter are limited in oxygen, it is not unreasonable to speculate that these PTS permeases may be turned on only during oxygen depleted or oxygen limited conditions. In order to identify the substrates, I will use a metabolomics approach. I will try to trap the phosphorylated forms of the substrates transported by the PTS permeases by knocking out the last two genes of these operons. The products of these genes contain predicted amidotransferase and phosphosugar isomerase domains, and I postulate these enzymes are required for the catabolism of the phosphosugars once they have been transported into the cell.