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Infection and Immunity, February 2005, p. 972-980, Vol. 73, No. 2
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.2.972-980.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Second-Generation Recombination-Based In Vivo Expression Technology for Large-Scale Screening for Vibrio cholerae Genes Induced during Infection of the Mouse Small Intestine

C. G. Osorio,1 J. A. Crawford,2 J. Michalski,2 H. Martinez-Wilson,3 J. B. Kaper,2 and A. Camilli3*

Programa de Microbiología y Micología, Facultad de Medicina, Universidad de Chile, Santiago, Chile,1 Center for Vaccine Development and Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland,2 Department of Molecular Biology and Microbiology, Tufts University Medical School, Tufts University, Boston, Massachusetts3

Received 21 June 2004/ Returned for modification 7 September 2004/ Accepted 12 October 2004

We have constructed an improved recombination-based in vivo expression technology (RIVET) and used it as a screening method to identify Vibrio cholerae genes that are transcriptionally induced during infection of infant mice. The improvements include the introduction of modified substrate cassettes for resolvase that can be positively and negatively selected for, allowing selection of resolved strains from intestinal homogenates, and three different tnpR alleles that cover a range of translation initiation efficiencies, allowing identification of infection-induced genes that have low-to-moderate basal levels of transcription during growth in vitro. A transcriptional fusion library of 8,734 isolates of a V. cholerae El Tor strain that remain unresolved when the vibrios are grown in vitro was passed through infant mice, and 40 infection-induced genes were identified. Nine of these genes were inactivated by in-frame deletions, and their roles in growth in vitro and fitness during infection were measured by competition assays. Four mutant strains were attenuated >10-fold in vivo compared with the parental strain, demonstrating that infection-induced genes are enriched in genes essential for virulence.

* Corresponding author. Mailing address: Tufts University Medical School, 150 Harrison Ave., Boston, MA 02111. Phone: (617) 636-2144. Fax: (617) 636-0337. E-mail: Andrew.Camilli{at}TUFTS.EDU.

Editor: V. J. DiRita

Infection and Immunity, February 2005, p. 972-980, Vol. 73, No. 2
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.2.972-980.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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