ANTIBIOTIC-BASED SELECTION FOR BACTERIAL GENES THAT ARE SPECIFICALLY INDUCED DURING INFECTION OF A HOST

We have recently described a genetic system, termed in vivo expression technology (IVET), that uses an animal as a selective medium to ident ify genes that pathogenic bacteria specifically express when infecting host tissues. Here, the potential utility of the IVET approach has be en expanded with the development of a transcriptional-fusion vector, p IVET8, which uses antibiotic resistance as the basis for selection in host tissues. pIVET8 contains promoterless chloramphenicol acetyltrans ferase (cat) and lacZY genes. A pool of Salmonella typhimurium clones carrying random cat-lac transcriptional fusions, produced with pIVET8, was used to infect BALB/c mice that were subsequently treated with in traperitoneal injections of chloramphenicol. Strains that survived the selection by expressing the cat gene in the animal were then screened for those that had low-level lacZY expression on laboratory medium. T hese strains carry operon fusions to genes that are specifically induc ed in vivo (ivi genes). One of the ivi genes identified (fadB) encodes an enzyme involved in fatty acid oxidation, suggesting that this enzy me might contribute to the metabolism of bactericidal or proinflammato ry host fatty acids. The pIVET8-based selection system was also used t o identify S. typhimurium genes that are induced in cultured macrophag es. The nature of ivi gene products will provide a more complete under standing of the metabolic, physiological, and genetic factors that con tribute to the virulence of microbial pathogens.