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ABSTRACTThe survival and replication of Brucellain macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by Brucelladuring this early acidification step, we analyzed Brucella suisconditioned 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. suisreleased 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 melitensiswas 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. melitensisorthologue. B. suisin acetate buffer depended on neither the virBtype IV secretory system nor the omp31gene product. However, the integrity of the omp25gene was required for release at acidic pH, while the absence of omp25bor omp25cdisplayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of Brucellain acidic medium.

Virulence of the intracellular pathogen Brucellafor humans is mainly associated with its lipopolysaccharide (LPS) phenotype, with smooth LPS phenotypes generally being virulent and rough ones not. The reason for this association is not quite understood. We now demonstrate by flow cytometry, electron microscopy, and ELISA that human peripheral blood monocytes interact both quantitatively and qualitatively different with smooth and rough Brucellaorganisms in vitro. We confirm that considerably higher numbers of rough than smooth brucellae attach to and enter the monocytes in nonopsonic conditions; but only smooth brucellae replicate in the host cells. We show for the first time that rough brucellae induce higher amounts than smooth brucellae of several CXC (GRO‐α, IL‐8) and CC (MIP‐1α, MIP‐1β, MCP‐1, RANTES) chemokines, as well as pro‐ (IL‐6, TNF‐α) and anti‐inflammatory (IL‐10) cytokines released by challenged monocytes. Upon uptake, phagosomes containing rough brucellae develop selective fusion competence to form spacious communal compartments, whereas phagosomes containing smooth brucellae are nonfusiogenic. Collectively, our data suggest that rough brucellae attract and infect monocytes more effectively than smooth brucellae, but only smooth LPS phenotypes establish a specific host cell compartment permitting successful parasitism. These novel findings link the LPS phenotype of Brucellaand its virulence for humans at the level of the infected host cells. Whether this is due to a direct effect of the LPS molecules or to upstream bacterial mechanisms remains to be established.

ABSTRACTExpression of the virBoperon, encoding the type IV secretion system required for Brucella suisvirulence, 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. suisVirB5 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. suisfrom which chvEwas deleted was fully virulent in a mouse model. Comparative analyses of various Brucellaspecies revealed that in all of them VirB protein production increased under acidic conditions. However, in rich medium at neutral pH, Brucella canisand B. suis, as well as the Brucella abortus-and 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. abortusand B. melitensisstrains constitutively produced VirB5 and VirB8. Thus, the vaccine strains were still able to induce virBexpression 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 virBoperon are not uniformly regulated in different Brucellaspecies. Since VirB proteins were shown to modulate Brucellaphagocytosis 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.

ABSTRACTBrucellaspp. can establish themselves and cause disease in humans and animals. The mechanisms by whichBrucellaspp. 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-α) 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 suis1330 (wild-type [WT] B. suis) on TNF-α production. For this purpose, omp25and omp31null mutants of B. suis(Δomp25 B. suisand Δomp31 B. suis, respectively) were constructed and analyzed for the ability to activate human macrophages to secrete TNF-α. We showed that, in contrast to WTB. suisor Δomp31 B. suis, Δomp25 B. suisinduced TNF-α production when phagocytosed by human macrophages. The complementation of Δomp25 B. suiswith WT omp25(Δomp25-omp25 B. suismutant) significantly reversed this effect: Δomp25-omp25 B. suis-infected macrophages secreted significantly less TNF-α than did macrophages infected with the Δomp25 B. suismutant. Furthermore, pretreatment of WT B. suiswith an anti-Omp25 monoclonal antibody directed against an epitope exposed at the surface of the bacteria resulted in substancial TNF-α production during macrophage infection. These observations demonstrated that Omp25 of B. suisis involved in the negative regulation of TNF-α production upon infection of human macrophages.

ABSTRACTBrucellaspp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated howB. suisand B. melitensisenter 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 ofBrucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca2+- and Mg2+-free medium reduced the uptake. Engulfment of all Brucellaorganisms (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 internalizeBrucellain a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of someBrucellaphagosomes 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 Brucelladepends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall.

The nature of the G-proteins present in the pre- and post-synaptic plasma membranes and in the synaptic vesicles of cholinergic nerve terminals purified from the Torpedoelectric organ was investigated. In pre- and post-synaptic plasma membranes, Bordetellapertussis toxin, known to catalyze the ADP-ribosylation of the α-subunit of several G-proteins, labels two substrates at 41 and 39 kDa. The 39 kDa subunit detected by ADP-ribosylation in the synaptic plasma membrane fractions was immunologically similar to the G oα-subunit purified from calf brain. In contrast to bovine chromaffin cell granules, no G-protein could be detected in Torpedosynaptic vesicles either by ADP-ribosylation or by immunoblotting.

Alpha-2 adrenergic receptors were solubilized from calf cerebral cortex using the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. The soluble extract retained the ability to bind the partial α2-adrenergic agonist [3H] p-aminoclonidine. This binding was of high affinity (KD= 1.5 ± 0.3 nM) saturable (Bmax = 150 ± 24 fmol/mg) and reversible. The orders of potencies of α-adrenergic drugs and nucleotides, for inhibition of binding, paralleled those previously observed on membrane bound α2-adrenoceptors. Furthermore the very rapid decrease of [3H] p-amino-clonidine binding induced by GTP suggest that the guanyl nucleotide effect is due to an allosteric mechanism. These results indicate that in the soluble extract the interaction between the α2-adrenoceptor and the GTP-binding regulator protein is preserved thus providing an interesting model to study the molecular structure of this system.

GTP-binding proteins have been proposed to be involved in some secretory processes. Bordetella pertussistoxin is known to catalyze ADP-ribosylation of several GTP-binding proteins. In this paper, the subcellular localization of B. pertussistoxin substrates has been explored in chromaffin cells of bovine adrenal medulla. With appropriate gel electrophoresis conditions, three ADP-ribosylated substrates of 39, 40 and 41 kDa were detectable in both plasma and granule membranes. The more intense labelling occurred on the 40 kDa component, while the 41 kDa species exhibited electrophoretic mobility similar to that of G iα. Significant immunoreactivity with anti-G oα antibodies was detected at the level of the 39 kDa faster component. The association of G-proteins with granule and plasma membranes suggests the involvement of these proteins in the exocytotic process or in its regulation.

GTP‐binding proteins have been proposed to be involved in some secretory processes. Bordetella pertussistoxin is known to catalyze ADP‐ribosylation of several GTP‐binding proteins. In this paper, the subcellular localization of B. pertussistoxin substrates has been explored in chromaffin cells of bovine adrenal medulla. With appropriate gel electrophoresis conditions, three ADP‐ribosylated substrates of 39, 40 and 41 kDa were detectable in both plasma and granule membranes. The more intense labelling occurred on the 40 kDa component, while the 41 kDa species exhibited electrophoretic mobility similar to that of Giα. Significant immunoreactivity with anti‐Goα antibodies was detected at the level of the 39 kDa faster component. The association of G‐proteins with granule and plasma membranes suggests the involvement of these proteins in the exocytotic process or in its regulation.

The enteropathogenic bacterium Yersinia enterocoliticacounteracts host defense mechanisms by interfering with eukaryotic signal transduction pathways. In this study, we investigated the mechanism by which Y. enterocoliticaprevents macrophage tumor necrosis factor-α (TNFα) production. Murine J774A.1 macrophages responded to Y. enterocoliticainfection by rapid activation of mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase (ERK), p38, and c-Jun NH2-terminal kinase (JNK). However, after initial activation, the virulent Y. enterocoliticastrain harboring the Y. enterocoliticavirulence plasmid caused a substantial decrease in ERK1/2 and p38 tyrosine phosphorylation. Simultaneously, the virulent Y. enterocoliticastrain gradually suppressed phosphorylation of the transcription factors Elk-1, activating transcription factor 2 (ATF2), and c-Jun, indicating time-dependent inhibition of ERK1/2, p38, and JNK kinase activities, respectively. Analysis of different Y. enterocoliticamutants revealed that (i) MAPK inactivation parallels the inhibition of TNFα release, (ii) the suppressor effect on TNFα production, which originates from the lack of TNFα mRNA, is distinct from the ability of Y. enterocoliticato resist phagocytosis and to prevent the oxidative burst, (iii) the tyrosine phosphatase YopH, encoded by the Y. enterocoliticavirulence plasmid, is not involved in the decrease of ERK1/2 and p38 tyrosine phosphorylation or in the cytokine suppressive effect. Altogether, these results indicate that Y. enterocoliticapossesses one or more virulence proteins that suppress TNFα production by inhibiting ERK1/2, p38, and JNK kinase activities.