Ivet

INFECTION AND IMMUNITY, Dec. 2002, p. 6518–6523 0019-9567/02/$04.00 0 DOI: 10.1128/IAI.70.12.6518–6523.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Vol. 70, No. 12

MINIREVIEW
In Vivo Expression Technology
Michael J. Angelichio and Andrew Camilli*
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts02111 The capacity to quickly and efficiently regulate gene expression has helped bacteria to colonize virtually every available niche in the biosphere, including dynamic and extreme ones. In accordance with this, bacterial populations must be ever ready to either take advantage of a favorable change or hunker down when the going gets tough. This proficiency is nicely demonstrated by infection ofhumans by facultative pathogens, for which up-regulation of genes necessary for survival and growth and down-regulation of genes deleterious to infectivity must occur on cue. To better understand this transition from ex vivo to in vivo conditions and to further our understanding of pathogenesis, it is necessary to identify genes that are specific to infection. Toward this end, in vivo expressiontechnology (IVET) was developed (27). The purpose of this short review is to update the reader on the many variations of IVET that have been developed, to discuss nuances of each method that may be helpful to investigators embarking on studies using this technology, and to discuss offshoot technologies of IVET as tools for studying regulation of virulence genes. The many individual microbial virulencefactors that have been identified using IVET are not reviewed here but have been recently reviewed by Mahan et al. (26). Because IVET is but one of several methods that can be used to screen for virulence genes induced during infection of cultured cells but is the only established method for accomplishing the same feat within infected animals, discussion herein is limited to reported uses of IVETin live animal hosts. GETTING CLOSER TO THE ACTION IVET was originally conceived upon the premise (now considered fact) that most virulence genes are transcriptionally induced at one or more times during infection (27). Although certain host environmental parameters can be mimicked in vitro to induce a subset of virulence genes, the full repertoire is only expressed in vivo. The beauty of IVET isthat a live host, with tissue barriers and immune system intact, is used to signal induction of virulence genes. Genetic trickery, the modus operandi of IVET, is then used to identify the in vivo-induced (ivi) genes. As is true of all genetic screens and selections, IVET does have its limitations. The most significant of these is that the relative level and timing of transcription of an ivi genelargely dictates whether the gene will be identified in a partic* Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-2144. Fax: (617) 636-0337. E-mail: Andrew.Camilli@Tufts.edu. 6518

ular IVET selection or screen. To date, there are four variations of IVET, and eachrelies on the generation of transcriptional fusions of genomic sequences to a reporter gene encoding an enzymatic activity. The variation in the four methods lies in the particular reporter gene utilized. In the original utilization of IVET (27), advantage was taken of the fact that purine auxotrophs (in this case purA) of Salmonella enterica serovar Typhimurium (referred to hereafter as Salmonella)are rapidly eliminated from the mouse unless they are complemented. To identify ivi genes, we cloned random genomic fragments directly upstream of a promoterless purA-lacZY synthetic operon present on a suicide vector (Fig. 1). This library was transferred by conjugation into purA Salmonella and was integrated by homologous recombination to form merodiploids. A particular advantage of generating...