Isolation and Characterization of Mini-Tn 5 Km2 Insertion Mutants of Brucella abortus Deficient in Internalization and Intracellular Growth in HeLa Cells

Abstract

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 I, no internalization and intracellular growth within HeLa cells; class II, 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.Brucellosis is a major bacterial zoonosis that causes a serious debilitating disease in humans and abortion and sterility in domestic animals. The etiologic agents of brucellosis are Brucella spp., small gram-negative and facultative intracellular pathogens that can multiply within professional and nonprofessional phagocytes (9, 10). In contrast to other intracellular pathogens, Brucella species do not produce exotoxins, antiphagocytic capsules or thick cell walls, resistance forms, or fimbriae and do not show antigenic variation (16). A key aspect of the virulence of brucella is its ability to proliferate within professional and nonprofessional phagocytic host cells and thereby successfully bypasses the bactericidal effects of phagocytes, and their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells (30, 41). The molecular mechanisms and genetic basis for intracellular survival and replication, however, are not understood completely. Some studies with nonprofessional phagocytes have shown that Brucella invades host cells and is contained within early endosome-like vacuoles. These vacuoles rapidly fuse with early autophagosomes that acquire vacuolar [H ϩ ]ATPase and lysosome-associated membrane proteins (LAMP), mature into a late autophagosome, inhibit fusion with lysosomes, and finally become a replicating vacuole normally associated with the endoplasmic reticulum (5,11,31,32). The genetic basis of Brucella virul...

Zinc Uptake System (znuA Locus) of Brucella abortus is Essential for Intracellular Survival and Virulence in Mice

“…Isogenic mutants of B. abortus 544 znuA::Tn5Km2 and ∆virB4 have been described previously [10,20]. B. abortus strains were maintained as frozen glycerol stocks and were cultured in Brucella broth (Becton Dickinson, Sparks, MD) or Brucella broth containing 1.5% agar.…”

“…Bone marrow-derived macrophages from female BALB/c mice were prepared by the method described previously [10]. After culturing in L-cell conditioned medium, the macrophages were replated for use by lifting cells in phosphate-buffered saline (PBS) on ice for 5 to 10 min, harvesting by centrifugation and resuspending in RPMI 1640 (Sigma) containing 10% FBS.…”

“…In Eschrerichia coli, two systems, a high-affinity system and a low-affinity system, have been described for zinc uptake [2,17], and the high-affinity zinc uptake system of Salmonella enterica serovar Typhimurium is important for virulence in mouse model [3]. However, B. abortus does not contain low-affinity zinc uptake system [6] and high-affinity zinc uptake system is still not understood.A preliminary characterization of B. abortus znuA (highaffinity zinc uptake system protein) mutant showed that it had reduced intracellular growth within HeLa cells [10], thus suggesting that this zinc uptake system protein might be a virulence-associated factor. In this study, we examined the growth of znuA mutant in zinc chelated medium and their intracellular replication in HeLa cells and macrophages.…”

“…In contrast to other intracellular pathogens, Brucella species do not produce exotoxins, antiphagocytic capsules or thick cell walls, resistance forms or fimbriae and do not show antigenic variation [7]. Intracellular survival and replication are key virulence features of Brucella because mutants defective for these attributes are also avirulent [10,14]. A key aspect of the virulence of Brucella successfully bypasses the bactericidal effects of phagocytes, and their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells [5,20].…”

“…A preliminary characterization of B. abortus znuA (highaffinity zinc uptake system protein) mutant showed that it had reduced intracellular growth within HeLa cells [10], thus suggesting that this zinc uptake system protein might be a virulence-associated factor. In this study, we examined the growth of znuA mutant in zinc chelated medium and their intracellular replication in HeLa cells and macrophages.…”