Isolation and characterization of mini-Tn5Km2 insertion mutants of Brucella abortus deficient in internalization and intracellular growth in HeLa cells

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional and nonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. The mechanism and factors of virulence are not fully understood. To identify genes related to internalization and multiplication in host cells, Brucella abortus was mutagenized by mini-Tn5Km2 transposon that carryied the kanamycin resistance gene, 4,400 mutants were screened, and HeLa cells were infected with each mutant. Twenty-three intracellular-growth-defective mutants were screened and were characterized for internalization and intracellular growth. From these results, we divided the mutants into the following three groups: class 1, no internalization and intracellular growth within HeLa cells; class 11, an internalization similar to that of the wild type but with no intracellular growth; and class III, internalization twice as high as the wild type but with no intracellular growth. Sequence analysis of DNA flanking the site of transposon showed various insertion sites of bacterial genes that are virulence-associated genes, including virB genes, an ion transporter system, and biosynthesis- and metabolism-associated genes. These internalization and intracellular-growth-defective mutants in HeLa cells also showed defective intracellular growth in macrophages. These results suggest that the virulence-associated genes isolated here contributed to the intracellular growth of both nonprofessional and professional phagocytes.