A Novel Approach for Functional Screening of Bacterial Genone for Virulence Genes

NON-TECHNICAL SUMMARY: Salmonellosis is one of the most common foodborne diseases in the United States. There is a tremendous need to apply molecular approaches to develop better and more specifically targeted control measures at the preharvest stage of food production. The main goal of this project is to use a novel approach for screening the genome of Salmonella typhmurium for the virulence genes that are required for survival within live host animals. We have devised a simple method, termed transposon footprinting, for functional screening of the bacterial genome. In this project, we will use mariner-based transposon mutants of S. typhimurium as a model system to take advantage of the feasibility of transposon footprinting for a thorough identification of the essential genes required for in vitro growth as well as the virulence genes required for in vivo survival. In addition, this approach will also be used to conduct a genome-wide comparison of the virulence genes for in vivo survival between different host animals or tissues. It is expected that our procedure would reveal the essential genes that are important in basic cellular processes and the virulence genes that are involved in various features of the bacterial pathogenesis including host-adaptation or tissue-tropism. These studies will provide insights into the molecular mechanisms governing the pathogenesis for foodborne S. typhimurium and also have practical food safety applications for the development of vaccine, antimicrobial agents, and preventive strategies.

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APPROACH: The idea here is to screen the genome of S. typhimurium for the virulence genes that are required for survival within live host animals using a novel approach. In this project we will use mariner-based transposon mutants of S. typhimurium as a model system to exploit the feasibility of footprinting in the comprehensive identification of essential genes that are required for in vitro growth as well as the virulence genes that are required for in vivo survival.

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PROGRESS: 2001/12 TO 2004/12<BR>
Salmonellosis is one of the most common foodborne diseases in the United States. There is a tremendous need to apply molecular approaches to develop better and more specifically targeted control measures at the preharvest stage of food production. The main goal of this project was to use a novel approach for screening the genome of Salmonella Typhmurium for the virulence genes that are required for survival within live host animals. We devised a simple method, termed transposon footprinting, for functional screening of the bacterial genome. In this project, we used transposon mutants of S. Typhimurium as a model system to take advantage of the feasibility of transposon footprinting for a thorough identification of the essential genes required for in vitro growth as well as the virulence genes required for in vivo survival. In addition, this approach can also be used to conduct a genome-wide comparison of the virulence genes for in vivo survival between different host animals or tissues. Our procedure has revealed some of genes that are potentially important in basic cellular processes and the virulence genes that are involved in various features of the bacterial pathogenesis including host-adaptation or tissue-tropism. These studies should provide insights into the molecular mechanisms governing the pathogenesis for foodborne S. Typhimurium and also have practical food safety applications for the development of vaccine, antimicrobial agents, and preventive strategies.
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IMPACT: 2001/12 TO 2004/12<BR>
Foodborne salmonellae continues to be difficult to limit in preharvest food production. New control strategies will require more extensive genetic analysis. Transposon footprinting is expected to facilitate the comprehensive functional analysis of bacterial genome for the genes that are required to increase fitness to a variety of selective conditions. When this technique is conducted with a transposon that harbors an inducible promoter facing outwardly, it would allow efficient identification of genes for Salmonella adherence and invasion. The elucidation of the proteins encoded by the genes identified in this study could provide new targets for novel vaccine development and preventative strategies. This approach is technically very simple and does not require any prior sequence information of the genome to be analyzed. Now that bacterial genome sequencing databases are available for S. Typhimurium this may help to facilitate identification of the genes that correspond to optimal targets to design vaccine strains.