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

ABSTRACTBrucellastrains are facultative intracellular pathogens that induce chronic diseases in humans and animals. This observation implies that Brucellasubverts 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 suisand activated syngeneic γ9δ2 T lymphocytes. Under these conditions, multiplication of B. suisinside 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 γ9δ2 T cells, we identified four genes of B. suisthat were necessary to resist to the action of the γ9δ2 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.