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Methods Enzymol. 2000;326:73-96.

IVET and RIVET: use of gene fusions to identify bacterial virulence factors specifically induced in host tissues.

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1
Department of Microbiology, University of Illinois, Urbana 61801, USA.

Abstract

IVET was designed to identify those bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes encode virulence factors, products specifically required for the infection process. The paradigm IVET system is based on complementation of an attenuating auxotrophic mutation by gene fusion and is designed to be of use in a wide variety of pathogenic organisms. In S. typhimurium, we have used this system successfully to identify a number of genes that are induced in a BALB/c mouse and that, when mutated, confer a virulence defect. The RIVET system is based on recombinase gene fusions, which, on induction during infection, mediate a site-specific recombination, the product of which can be screened for after recovery of bacteria from host tissues. In V. cholerae, we have used this system successfully to identify genes that are induced transcriptionally during infection of the gastrointestinal tract of infant mice. RIVET is also uniquely designed for postidentification analysis of in vivo-induced genes: (1) it has been used to analyze the temporal and spatial patterns of virulence gene induction during infection and (2) it has been used to dissect the regulatory requirements of in vivo induction with respect to both bacterial regulatory factors and host-inducing environments. The IVET system has several applications in the area of vaccine and antimicrobial drug development. This technique was designed for the identification of virulence factors and thus may lead to the discovery of new antigens useful as vaccine components. The IVET system facilitates the isolation of mutations in genes involved in virulence and, therefore, should aid in the construction of live-attenuated vaccines. In addition, the identification of promoters that are expressed optimally in animal tissues provides a means of establishing in vivo-regulated expression of heterologous antigens in live vaccines, an area that has been problematic previously. Finally, we expect that our methodology will uncover many biosynthetic, catabolic, and regulatory genes that are required for growth of microbes in animal tissues. The elucidation of these gene products should provide new targets for antimicrobial drug development.

PMID:
11036635
[Indexed for MEDLINE]

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