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...

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...

Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca 2؉ -and Mg 2؉ -free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.Bacteria of the genus Brucella are facultative intracellular parasites in various wildlife and domestic mammals, causing a debilitating zoonotic infection in humans. Traditionally, three major Brucella species are distinguished on the basis of their virulence for humans and their predilection for certain animal hosts: B. abortus for cattle, B. melitensis for caprines, and B. suis for hogs, with B. melitensis and B. suis accounting for the majority of clinical cases in humans (13,42). In its mammalian hosts, the pathogen typically persists in tissues with a prominent reticuloendothelial component as well as in the reproductive organs of both genders (18). The mechanisms underlying this tissue tropism and the way in which Brucella organisms are able to enter and survive within such different host cells as resident macrophages and epithelial cells are not yet clear.With regard to the uptake of brucellae by their host cells, studies on the phagocytosis-promoting host cell receptors and bacterial ligands are rare and gave contradictory results (reviewed in references 29 and 42). However, opsonized and nonopsonized brucellae were internalized at different rates, replicated at different rates, and induced differences in the killing mechanisms and the reactive cytokine pattern of the host cell (5,9,23,24,30). These findings sugges...

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus which is of major public health concern. ZIKV infection is recognized as the cause of congenital Zika disease and other neurological defects, with no specific prophylactic or therapeutic treatments. As the humoral immune response is an essential component of protective immunity, there is an urgent need for effective vaccines that confer protection against ZIKV infection. In the present study, we evaluate the immunogenicity of chimeric viral clone ZIKBeHMR-2, in which the region encoding the structural proteins of the African strain MR766 backbone was replaced with its counterpart from the epidemic strain BeH819015. Three amino-acid substitutions I152T, T156I, and H158Y were introduced in the glycan loop of the E protein (E-GL) making ZIKBeHMR-2 a non-glycosylated virus. Adult BALB/c mice inoculated intraperitoneally with ZIKBeHMR-2 developed anti-ZIKV antibodies directed against viral proteins E and NS1 and a booster dose increased antibody titers. Immunization with ZIKBeHMR-2 resulted in a rapid production of neutralizing anti-ZIKV antibodies. Antibody-mediated ZIKV neutralization was effective against viral strain MR766, whereas epidemic ZIKV strains were poorly sensitive to neutralization by anti-ZIKBeHMR-2 immune sera. From our data, we propose that the three E-GL residues at positions E-152, E-156, and E-158 greatly influence the accessibility of neutralizing antibody epitopes on ZIKV.

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 ...

No abstract

Annexin I belongs to a family of calcium-dependent phospholipid-binding and membrane-binding proteins. Although many of the biochemical properties and the three-dimensional structure of this protein are known, its true physiological roles have yet to be thoroughly defined. Its putative functions indude participation in the regulation of actin microfilaments dynamics, proposed after the discovery of an interaction with actin. In accordance with this hypothesis, we found that annexin I can also interact with profilin. We used different methods, overlay and surface plasmon resonance (BIAcore), to measure the parameters of the association equilibrium, i.e. k,,,, k,,,, and Kd. The affinity of annexin I for profilin was between lo7 M and lox M. High concentrations of KC1 did not prevent the interaction, although a slight decrease in affinity was observed. Calcium, a modulator of annexin I functions interfered only marginally with the association, in a manner comparable to magnesium. Proteins or compounds known to interact with annexin I or profilin were found to inhibit the annexin-I-profilin interaction when added in the reaction medium. Recombinant profilin exhibited a slightly lower affinity than natural platelet protein when measured with BIAcore. Due to the submembrane localisation of annexin I and the regulatory activity of profilin on the cytoskeleton, an interaction between annexin I and profilin may therefore be implicated in the regulation of some cellular functions, particularly those governing membrane-cytoskeleton dynamic organization.

No abstract