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...

Impairment of the omp25 gene in Brucella spp. leads to attenuated strains and confers protection to the host. Omp25 and Omp31, whose functions remain unknown, were the first characterized members of group 3 outer membrane proteins (Omps) (25 to 34 kDa). Recently, genomic and proteomic approaches identified five new putative members of this family, some of which are produced in B. melitensis or B. abortus. In the present study, using protein microsequencing, we identified new members of group 3 Omps proteins produced in B. suis. Since several monoclonal antibodies (MAbs) against Omp25 cross-reacted with other members of group 3 Omps, we also performed Western immunoblotting to compare wild-type B. suis with mutants systematically having B. suis omp25-related genes knocked out. We demonstrate the production of three paralogs of Omp31 and/or Omp25 in B. suis, and the existence of a common site of signal peptide cleavage (AXAAD), which is very similar to that present in the five homologous Omps of Bartonella quintana. The seven group 3 Omps were classified in four-subgroups on the basis of percentage amino acid sequence identities: Omp25 alone, the Omp25b-Omp25c-Omp25d cluster, the Omp31/31b subgroup, and the less related Omp22 protein (also called Omp3b). Together with previous data, our results demonstrate that all new members of group 3 Omps are produced in B. suis or in other Brucella species and we propose a nomenclature that integrates all of these proteins to facilitate the understanding of future Brucella interspecies study results.Brucellae are small, nonmotile, gram-negative coccobacilli that are able to infect a broad range of wildlife and domestic mammals. They remain a major zoonotic disease source affecting humans worldwide and are also a focus of concern as potential biological warfare agents (20). Although BrucelIa spp. are not particularly host specific, three major (B. abortus, B. melitensis, and B. suis) and minor (B. canis, B. ovis, and B. neotomae) species each have distinct host preferences and pathogenicities for humans (29). Malta fever (also known as Mediterranean, Gibraltar, or undulant fever) and porcine brucellosis-caused by B. melitensis and B. suis infection of humans, respectively-are usually by far more clinically apparent than Bang's disease (B. abortus infection), whereas among the remaining species only B. canis causes anecdotal mild infections in humans (10).The Brucella outer membrane was investigated to seek immunogenic and protective antigens for potential diagnostic and vaccine applications. The major outer membrane proteins (Omps) of Brucella spp. were thoroughly studied in this regard.

The survival and replication of Brucella in macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by Brucella during this early acidification step, we analyzed Brucella suis conditioned medium at various pH levels. No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, B. suis released a substantial amount of soluble proteins. Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location. Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of Agrobacterium tumefaciens. The absence of this homologue in Brucella melitensis was due to the deletion of a 7.7-kb DNA fragment in its genome. We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats. In B. suis, this protein contained 9 repeats, while 12 were present in the B. melitensis orthologue. B. suis in acetate buffer depended on neither the virB type IV secretory system nor the omp31 gene product. However, the integrity of the omp25 gene was required for release at acidic pH, while the absence of omp25b or omp25c displayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of Brucella in acidic medium.Bacteria of the genus Brucella are gram-negative facultative intracellular pathogens of various wild and domestic mammals and are able to cause severe zoonotic infections in humans. Traditionally, three major species are distinguished by their predilections for certain animal hosts: Brucella abortus for cattle, Brucella melitensis for caprines, and Brucella suis 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 (1, 2, 11).To evade host defenses, Brucella can inhibit neutrophil degranulation and block tumor necrosis factor (TNF) production by macrophages. It has been shown that membrane integrity, in terms of both smooth lipopolysaccharide and outer membrane proteins, is required for such virulent behavior. Furthermore, studies using transposon or signature-tagged mutagenesis have unraveled, with respect to Brucella virulence, the crucial role of an operon homologous to the virB operon of Agrobacterium tumefaciens encoding a type IV secretion system (16, 21, 28). The virulence regulon of A. tumefaciens is triggered in response to chemical signals released at the plant wound site, such as acetosyringone and low pH. Type IV secretion system production is potentiated by monosaccharides (galactose and arabinose) through binding to the periplasmic multiple sugar binding protein ChvE, as well as by low pH (6). However, it was found that under neutral conditions, this secretory system is already produced in B. melitensis or B. abortus, while ...

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The acetohydroxyacid synthase (AHAS) of Brucella suis can be effectively targeted by the sulfonylureas chlorimuron ethyl and metsulfuron methyl. Growth in minimal medium was inhibited, and multiplication in human macrophages was totally abolished with 100 M of sulfonylureas. Metsulfuron methyl-resistant mutants showed reduced viability in macrophages and reduced AHAS activity.Bacterial pathogens are generally sensitive to antibiotics. However, a constantly increasing number of drug-resistant strains are isolated (17). The need to identify alternative bacterial targets for antibacterial drugs is therefore evident. In intracellular bacteria, pathogenicity is linked to the capacity to multiply within the host cell, and we reasoned that antibacterials specifically active at the intracellular state would block multiplication of the bacteria without affecting extracellular bacteria, decreasing pressure for the selection of resistant mutants and reducing the probability of affecting the commensal flora. In this study we demonstrate the usefulness of this approach with the example of Brucella spp. This intracellular pathogen infects animals and humans, and brucellosis is considered a major zoonosis (4). Human brucellosis may become chronic, eventually causing death. The genes required for intramacrophagic replication of Brucella are a subset of the virulence genes of the pathogen (5) and were called the intramacrophagic virulome (11). Among those genes, we identified ilv (BR1389 and BR1388 loci) (15), which encodes the acetohydroxyacid synthase (AHAS), as a potential antimicrobial target. It participates in the biosynthesis of isoleucine, leucine, and valine; and its importance in virulence, together with other amino acid biosynthesis enzymes, led us to conclude that the Brucella-containing vacuole is nutrient poor (11,12). AHAS has been studied in a wide range of organisms such as Escherichia coli, Saccharomyces cerevisiae, and Arabidopsis thaliana (3,10,13,16). Its activity is inhibited by sulfonylureas, which show very low toxicity for mammals (6). In this study, the effects of sulfonylureas on brucellae were investigated. Brucella suis 1330 (ATCC 23444), used throughout the study, was grown in complex tryptic soy (TS) broth or in minimal medium (8). An AHAS-specific colorimetric assay was performed with Brucella lysates according to established protocols (7, 10). Macrophage infection experiments were performed as described previously (2) by using human macrophage-like THP-1 cells. Spontaneously metsulfuron methyl (MSM)-resistant mutants of B. suis were isolated after 8 days from MSM-containing minimal medium (10 M), followed by plating on the same solid medium.Sulfonylureas inhibit AHAS activity in B. suis and growth in minimal medium. Chlorimuron ethyl (CE) and MSM were the sulfonylureas that were the most effective in blocking AHAS activity in B. suis, with CE being more active (Fig. 1). As expected, the ilvI::Tn5 mutant did not grow in a minimal medium that mimicked the presumably nutrient-poor Brucellacontaining...

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