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A SacB Mutagenesis Strategy Reveals that the Bartonella quintana Variably Expressed Outer Membrane Proteins Are Required for Bloodstream Infection of the Host

Joanna K. MacKichan, Helen L. Gerns, Yu-Ting Chen, Peng Zhang, and Jane E. Koehler^*

Microbial Pathogenesis and Host Defense Program and Division of Infectious Diseases, Department of Medicine, University of California at San Francisco, San Francisco, California 94143-0654

Received 24 August 2007/ Returned for modification 11 October 2007/ Accepted 26 November 2007

Bartonella bacteria adhere to erythrocytes and persistently infect the mammalian bloodstream. We previously identified four highly conserved Bartonella quintana adhesin genes that undergo phase variation during prolonged bloodstream infection. The variably expressed outer membrane proteins (Vomp) encoded by these genes are members of the trimeric autotransporter adhesin family. Each B. quintana Vomp appears to contribute a different adhesion phenotype, likely mediated by the major variable region at the adhesive tip of each Vomp. Although studies document that the Vomp adhesins confer virulence phenotypes in vitro, little is known about in vivo virulence strategies of Bartonella. We sought to determine whether the B. quintana Vomp adhesins are necessary for infection in vivo by using a vomp null mutant. It first was necessary to develop a system to generate in-frame deletions of defined genes by allelic exchange in a wild-type Bartonella background, which had not been achieved previously. We utilized sacB negative selection to generate a targeted, in-frame, markerless deletion of the entire vomp locus in B. quintana. We also recently developed the first animal model for B. quintana infection, and using this model, we demonstrate here that the deletion of the entire vomp locus, but not the deletion of two vomp genes, results in a null mutant strain that is incapable of establishing bloodstream infection in vivo. The Vomp adhesins therefore represent critical virulence factors in vivo, warranting further study. Finally, our allelic exchange strategy provides an important advance in the genetic manipulation of all Bartonella species and, combined with the animal model that recapitulates human disease, will facilitate pathogenesis studies of B. quintana.

Published ahead of print on 10 December 2007.

Editor: W. A. Petri, Jr.

Present address: Institute of Environmental Science and Research, P.O. Box 50-348, Porirua 5240, New Zealand.