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Infection and Immunity, November 2007, p. 5167-5174, Vol. 75, No. 11
0019-9567/07/$08.00+0     doi:10.1128/IAI.00690-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification and Isolation of Brucella suis Virulence Genes Involved in Resistance to the Human Innate Immune System

CNRS-UMR 5236 and Université Montpellier 2, place Eugène Bataillon, 34000 Montpellier, France

Received 22 May 2007/ Returned for modification 21 June 2007/ Accepted 9 August 2007

Brucella strains are facultative intracellular pathogens that induce chronic diseases in humans and animals. This observation implies that Brucella subverts innate and specific immune responses of the host to develop its full virulence. Deciphering the genes involved in the subversion of the immune system is of primary importance for understanding the virulence of the bacteria, for understanding the pathogenic consequences of infection, and for designing an efficient vaccine. We have developed an in vitro system involving human macrophages infected by Brucella suis and activated syngeneic 92 T lymphocytes. Under these conditions, multiplication of B. suis inside macrophages is only slightly reduced. To identify the genes responsible for this reduced sensitivity, we screened a library of 2,000 clones of transposon-mutated B. suis. For rapid and quantitative analysis of the multiplication of the bacteria, we describe a simple method based on Alamar blue reduction, which is compatible with screening a large library. By comparing multiplication inside macrophages alone and multiplication inside macrophages with activated 92 T cells, we identified four genes of B. suis that were necessary to resist to the action of the 92 T cells. The putative functions of these genes are discussed in order to propose possible explanations for understanding their exact role in the subversion of innate immunity.

Published ahead of print on 20 August 2007.

Editor: D. L. Burns