Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host.

Abstract

We have recently described a genetic system, termed in vivo expression technology (IVET), that uses an animal as a selective medium to identify genes that pathogenic bacteria specifically express when infecting host tissues. Here, the potential utility of the IVET approach has been expanded with the development of a transcriptional-fusion vector, pIVET8, which uses antibiotic resistance as the basis for selection in host tissues. pIVET8 contains promoterless chloramphenicol acetyltransferase (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 intraperitoneal 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. These strains carry operon fusions to genes that are specifically induced in vivo (ivi genes). One of the ivi genes identified (fadB) encodes an enzyme involved in fatty acid oxidation, suggesting that this enzyme might contribute to the metabolism of bactericidal or proinflammatory host fatty acids. The pIVET8-based selection system was also used to identify S. typhimurium genes that are induced in cultured macrophages. The nature of ivi gene products will provide a more complete understanding of the metabolic, physiological, and genetic factors that contribute to the virulence of microbial pathogens.Defining the nature of microbial gene products that are produced during infection is essential to the understanding, treatment, and prevention of infectious disease (1-3). Previous genetic strategies for the identification of virulence factors have relied primarily on three approaches: brute force screens that identify potential virulence genes on the basis of mutational loss of a virulence phenotype in vitro (4, 5), cloning screens that identify new virulence genes on the basis of their ability to confer a virulence-associated phenotype on a heterologous bacterium in vitro (6), or regulatory screens that identify potential virulence genes on the basis of their coordinate expression with known virulence genes under defined laboratory conditions (7,8). These strategies have led to the identification of many virulence determinants (1, 2) but are often limited by their inability to reproduce accurately in vitro the complex and changing environments encountered by pathogens in their hosts.To overcome these limitations, we have recently developed an approach to the identification of bacterial genes involved in infection and pathogenesis, termed in vivo expression technology (IVET) (9). IVET does not rely on reproduction of environmental signals in the laboratory but rather uses the host as selective medium to enrich for gene fusions that are expressed in animal tissues. Among the genes identified were in vivo induced genes (ivi genes) that are poorly expressed on laboratory media but undergo elevated expression in host t...

The medium-/long-chain fatty acyl-CoA dehydrogenase (fadF) gene of Salmonella typhimurium is a phase 1 starvation-stress response (SSR) locus

“…B. Finlay, unpublished results), a utilizable sugar for Salmonella. A previous report, supporting our findings, has suggested that fad genes are specifically expressed during Salmonella pathogenesis (Mahan et al, 1995). It is clear, however, from these various studies that stationary phase, C-starvation and …”

“…This knowledge may provide insight into the conditions present in an intracellular environment as well as clues as to the potential importance of a locus in pathogenesis (Mahan et al, 1995;Valdivia & Falkow, 1997). T o this end, strains ST54 and ST64 were incubated in the presence of MDCK epithelial cells in tissue culture, and allowed to be internalized within these cells.…”

Environmentally regulated genes of Streptococcus suis: identification by the use of iron-restricted conditions in vitro and by experimental infection of piglets The GenBank accession numbers for the sequences determined in this work are AF302190–AF302207 (iri genes) and AF303226–303247 (ivs genes).

“…during iron restriction or during experimental infection. This system differs from the antibiotic-based IVET system described for Salmonella typhimurium (Mahan et al, 1995) in two ways. One is that the lacZ reporter gene fusion is omitted in our vector constructions because inclusion of the lacZ gene resulted in structural instability of the vector.…”

“…Recently, several approaches have been reported that allow the identification of genes that are specifically expressed in the host. Examples are signature-tagged mutagenesis (STM) and in vivo expression technology (IVET ; Mahan et al, 1993Mahan et al, , 1995Camilli & Mekalanos, 1995 ;Hensel et al, 1995 ;Mei et al, 1997 ;Young & Miller, 1997 ;Chiang & Mekalanos, 1998 ;Coulter et al, 1998 ;Lowe et al, 1998 ;Polissi et al, 1998 ;Camacho et al, 1999 ;Darwin & Miller, 1999 ;Edelstein et al, 1999 ;Fuller et al, 1999 ;Zhao et al, 1999). In addition, important virulence proteins could also be identified by the selection of genes specifically expressed under conditions mimicking in vivo conditions, for example by growth under ironrestricted conditions (Litwin & Calderwood, 1993 ;Martinez et al, 1990).…”

Host adapted serotypes of Salmonella enterica

“…A strategy which allows the detection of salmonella genes that are specifically induced in host tissues has been developed by Mahan and colleagues [236]. The strategy, named in i o expression technology (IVET), takes advantage of the fact that purine auxotrophy greatly attenuates growth of Typhimurium in the animal host ; this deficiency provides a strong selection for a PurA + phenotype in animal tissues.…”