Brucella spp. can establish themselves and cause disease in humans and animals. The mechanisms by which Brucella spp. evade the antibacterial defenses of their host, however, remain largely unknown. We have previously reported that live brucellae failed to induce tumor necrosis factor alpha (TNF-␣) production upon human macrophage infection. This inhibition is associated with a nonidentified protein that is released into culture medium. Outer membrane proteins (OMPs) of gram-negative bacteria have been shown to modulate macrophage functions, including cytokine production. Thus, we have analyzed the effects of two major OMPs (Omp25 and Omp31) of Brucella suis 1330 (wild-type [WT] B. suis) on TNF-␣ production. For this purpose, omp25 and omp31 null mutants of B. suis (⌬omp25 B. suis and ⌬omp31 B. suis, respectively) were constructed and analyzed for the ability to activate human macrophages to secrete TNF-␣. We showed that, in contrast to WT B. suis or ⌬omp31 B. suis, ⌬omp25 B. suis induced TNF-␣ production when phagocytosed by human macrophages. The complementation of ⌬omp25 B. suis with WT omp25 (⌬omp25-omp25 B. suis mutant) significantly reversed this effect: ⌬omp25-omp25 B. suis-infected macrophages secreted significantly less TNF-␣ than did macrophages infected with the ⌬omp25 B. suis mutant. Furthermore, pretreatment of WT B. suis with an anti-Omp25 monoclonal antibody directed against an epitope exposed at the surface of the bacteria resulted in substancial TNF-␣ production during macrophage infection. These observations demonstrated that Omp25 of B. suis is involved in the negative regulation of TNF-␣ production upon infection of human macrophages.Members of the genus Brucella are gram-negative, facultatively intracellular bacteria that can induce chronic infections in humans. Following invasion of the reticuloendothelial system, the bacteria develop intracellularly within mononuclear phagocytes. Chronic infection generally results in the fixation of infected macrophages at specific locations within the body (spleen, brain, heart, bones), and the human disease is characterized by undulant fever, endocarditis, arthritis, and osteomyelitis (42). Brucellae are also pathogenic for animals, but the pathophysiology of the human infection differs in many respects from the illness induced in animals. In domestic ruminants, infection results mainly in abortion in females and orchitis in males (15) whereas in mice, infection resembles septicemia and does not become truly chronic (18). These observations therefore suggest a species-specific interaction of Brucella organisms with the immune systems of their different hosts. To survive and multiply within the host, one of the major strategies of pathogens is to affect the expression of cytokines, which is necessary for the normal protective function of the immune response (26).In previous papers (6, 7) we have reported that brucellae can adopt the following strategy. (i) In human monocytic phagocytes (but not in mouse macrophages), Brucella spp. impair the production o...

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Expression of the virB operon, encoding the type IV secretion system required for Brucella suis virulence, occurred in the acidic phagocytic vacuoles of macrophages and could be induced in minimal medium at acidic pH values. To analyze the production of VirB proteins, polyclonal antisera against B. suis VirB5 and VirB8 were generated. Western blot analysis revealed that VirB5 and VirB8 were detected after 3 h in acidic minimal medium and that the amounts increased after prolonged incubation. Unlike what occurs in the related organism Agrobacterium tumefaciens, the periplasmic sugar binding protein ChvE did not contribute to VirB protein production, and B. suis from which chvE was deleted was fully virulent in a mouse model. Comparative analyses of various Brucella species revealed that in all of them VirB protein production increased under acidic conditions. However, in rich medium at neutral pH, Brucella canis and B. suis, as well as the Brucella abortusand Brucella melitensis-derived vaccine strains S19, RB51, and Rev.1, produced no VirB proteins or only small amounts of VirB proteins, whereas the parental B. abortus and B. melitensis strains constitutively produced VirB5 and VirB8. Thus, the vaccine strains were still able to induce virB expression under acidic conditions, but the VirB protein production was markedly different from that in the wild-type strains at pH 7. Taken together, the data indicate that VirB protein production and probably expression of the virB operon are not uniformly regulated in different Brucella species. Since VirB proteins were shown to modulate Brucella phagocytosis and intracellular trafficking, the differential regulation of the production of these proteins reported here may provide a clue to explain their role(s) during the infection process.Bacteria belonging to the genus Brucella are gram-negative facultative intracellular pathogens of various wild and domestic mammals, and they also cause severe zoonotic infections in humans. Traditionally, three major species are distinguished by their preferences for certain animal hosts; Brucella abortus has a preference for cattle, Brucella melitensis has a preference for caprines, and Brucella suis has a preference for hogs. Whereas B. abortus is the livestock pathogen with the greatest economic impact, B. melitensis and B. suis account for most clinical cases in humans (15,42).In an attempt to unravel Brucella virulence factors by transposon mutagenesis, the crucial role of an operon similar to the virB operon of Agrobacterium tumefaciens encoding a type IV secretion system (T4SS) was revealed (35). The importance of the virB operon for Brucella virulence was further confirmed by signature-tagged mutagenesis both in vitro in a human macrophage infection model (24) and in vivo with mice (26). Further studies indicated that a complete Brucella virB operon was required for wild-type virulence in mice (47) or in macrophagelike cells (52,53). In nonphagocytic HeLa cells, the absence of some functional VirB proteins (B2, B4, and B9) did not...