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

Abstract

Bacteria of the genus Brucella are facultative intracellular pathogens which have developed the capacity to survive and multiply in professional and nonprofessional phagocytes. The genetic basis of this aspect of Brucella virulence is still poorly understood. To identify new virulence factors, we have adapted signature-tagged transposon mutagenesis, which has been used essentially 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 pools, was 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 avirulent mutant, a genomic fragment containing the transposon was cloned. The genomic DNA sequence flanking the transposon allowed us to assign functions to all of the inactivated genes. Transposon integration had occurred in 14 different genes, some of which were known virulence genes involved in intracellular 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.

FIG. 1

Identification of attenuated mutants by differential hybridization. A dot blot of plasmids carrying the 96 tagged mini-Tn5 Km2 transposons was hybridized with probes prepared from the output and input pools from plate IX. In this pool clone H9 (arrows) was attenuated (lysR-like). The apparent attenuation of clones A4, G3, and E5 was not confirmed by individual testing. Clones C4 and E6 did not grow, and well H7 was the negative control (no innoculum).

FIG. 2

Growth curves of wild type and four mini-Tn5 Km2 mutants of B. suis in THP1 macrophages. Differentiated THP1 cells were infected as described in Materials and Methods. The four representative mini-Tn5 Km2 mutants shown here are IXH9 (lysR-like), IVA10 (virB4), IIIF11 (gpt-like), and IVH4 (pgi-like). The data points are the means of three wells with the standard deviations. This data set is from one of three independent experiments with similar results. The 48-h-postinfection points for all 18 mutants are detailed in Table 1.)