Identification of Brucella suis genes affecting intracellular survival in an in vitro human macrophage infection model by signature-tagged transposonmutagenesis

Bacteria of the genus Brucella are facultative intracellular pathogens whic h have developed the capacity to survive and multiply in professional and n onprofessional phagocytes, The genetic basis of this aspect of Brucella vir ulence is still poorly understood. To identify new virulence factors, we ha ve adapted signature-tagged transposon mutagenesis, which has been used ess entially in animal models, to an in vitro human macrophage infection model. A library of 1,152 Brucella suis 1330 tagged mini-Tn5 Km2 mutants, in 12 p ools, mas screened for intracellular survival and multiplication in vitamin D-3-differentiated THP1 cells. Eighteen mutants were identified, and their attenuation was confirmed in THP1 macrophages and HeLa cells, For each avi rulent mutant, a genomic fragment containing the transposon was cloned. The genomic DNA sequence flanking the transposon allowed us to assign function s to all of the inactivated genes, Transposon integration had occurred in 1 4 different genes, some of which were known virulence genes involved in int racellular survival or biosynthesis of smooth lipopolysaccharide (the virB operon and manB), thus validating the model. Other genes identified encoded factors involved in the regulation of gene expression and enzymes involved in biosynthetic or metabolic pathways. Possible roles in the virulence of Brucella for the different factors identified are discussed.