The IFNgamma-induced STAT1-CBP/P300 association, required for a normal response to the cytokine, is disrupted in Brucella-infected macrophages.

To develop intracellularly within phagocytes and cause chronic infection, Brucella must overcome different steps of the host immune responses. IFNgamma is a key mediator of the innate and adaptive responses produced during Brucella infection. Therefore, Brucella would control host defenses by impairing macrophage responses to IFNgamma. We first showed that in infected human macrophages (VD3-differentiated THP-1 cells) Brucella escaped the microbicidal environment generated by IFNgamma. We then analyzed the IFNgamma-mediated signaling in Brucella-infected cells. We observed no decrease in STAT1 tyrosine or serine phosphorylation, or in dimerization of phosphorylated STAT1 (P-STAT1) and P-STAT1 translocation to the nucleus or in P-STAT1 binding to GAS, a minimal IFNgamma-response DNA sequence. In contrast, immuno-precipitation experiments indicated that the IFNgamma-mediated association of P-STAT1 with CBP/P300 transactivators was markedly reduced in infected macrophages, demonstrating that P-STAT1 was unable to normally recruit these transactivators. The host cell cAMP pathway triggered by Brucella could be responsible for this defect, CBP/P300 mobilization by phosphorylated CREB (P-CREB) disrupting the IFNgamma-induced STAT1-CBP/P300 association, required for a normal response of macrophages to IFNgamma. In any case, the inhibition of an essential protein-protein interaction probably lead to a deteriorated response to IFNgamma and thus participated in the pathogen's establishment within its host.

VirB type IV secretory system does not contribute to Brucella suis' avoidance of human dendritic cell maturation.

Dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to infection with the intracellular bacteria Brucella. Infection with living Brucella prevents infected human DCs from engaging in maturation processes, thus impairing their capacity to present antigens to naïve T cells and to secrete IL-12. Recently, we have established that several attenuated mutants of Brucella (rough, omp25, bvrR) are unable to control DCs maturation and thus effectively stimulate naïve T cells, which could be the origin of the protective immunity elicited by these mutants in vivo. In this study, we investigate the interactions of a VirB-defective Brucella mutant with human DCs to determine whether its attenuation could be attributed to the induction of an adaptive immune response. We show here that in contrast to previously studied strains and similar to wild-type strains, this virB mutant was unable to trigger significant DC maturation. Together with recently published data describing infection with virB mutants in vivo, these results suggest that Brucella T4SS VirB is not involved in the control of DC maturation and does not interfere with the establishment of a T-helper type 1 adaptive immune response.

Interaction of Brucella suis and Brucella abortus rough strains with human dendritic cells.

Brucella is a facultative intracellular pathogen of various mammals and the etiological agent of brucellosis. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection. Furthermore, Brucella prevented the infected DCs from engaging in maturation processes and impaired their capacity to present antigen to naive T cells and to secrete interleukin-12 (IL-12). The lipopolysaccharide (LPS) phenotype is largely associated with the virulence of Brucella. Depending on whether they express the O-side chain of LPS or not, the bacteria display a smooth or rough phenotype. Rough Brucella mutants are attenuated and induce a potent protective T-cell-dependent immune response. Due to the essential role of DCs in the initiation of T-cell-dependent adaptive immune responses, it seemed pertinent to study the interaction between rough Brucella strains and human DCs. In the present paper, we report that, in contrast to smooth bacteria, infection of DCs with rough mutants of Brucella suis or Brucella abortus leads to both phenotypic and functional maturation of infected cells. Rough mutant-infected DCs then acquire the capacity to produce IL-12 and to stimulate naive CD4+ T lymphocytes. Experiments with rough and smooth purified LPS of Brucella supported the hypothesis of an indirect involvement of the O-side chain. These results provide new data concerning the role of LPS in Brucella virulence strategy and illuminate phenomena contributing to immune protection conferred by rough vaccine strains.

Brucella suis prevents human dendritic cell maturation and antigen presentation through regulation of tumor necrosis factor alpha secretion.

Brucella is a facultative intracellular pathogen and the etiological agent of brucellosis. In some cases, human brucellosis results in a persistent infection that may reactivate years after the initial exposure. The mechanisms by which the parasite evades clearance by the immune response to chronically infect its host are unknown. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection and are a preferential niche for the development of the bacteria. Here, we report that in contrast to several intracellular bacteria, Brucella prevented the infected DCs from engaging in their maturation process and impaired their capacities to present antigen to naïve T cells and to secrete interleukin-12. Moreover, Brucella-infected DCs failed to release tumor necrosis factor alpha (TNF-alpha), a defect involving the bacterial protein Omp25. Exogenous TNF-alpha addition to Brucella-infected DCs restored cell maturation and allowed them to present antigens. Two avirulent mutants of B. suis, B. suis bvrR and B. suis omp25 mutants, which do not express the Omp25 protein, triggered TNF-alpha production upon DC invasion. Cells infected with these mutants subsequently matured and acquired the ability to present antigens, two properties which were dramatically impaired by addition of anti-TNF-alpha antibodies. In light of these data, we propose a model in which virulent Brucella alters the maturation and functions of DCs through Omp25-dependent control of TNF-alpha production. This model defines a specific evasion strategy of the bacteria by which they can escape the immune response to chronically infect their host.

Characterization of SP41, a surface protein of Brucella associated with adherence and invasion of host epithelial cells.

Brucella is an invasive organism that multiplies and survives within eukaryotic cells. The brucellae are able to adhere to the surface of cultured epithelial cells, a mechanism that may facilitate penetration and dissemination to other host tissues. However, no adhesins that allow the bacteria to interact with the surface of epithelial cells before migration within polymorphonuclear leukocytes, monocytes and macrophages have been described. Here, we show that Brucella surface proteins (SPs) with apparent molecular masses of 14, 18 and 41 kDa bound selectively to HeLa cells. However, only antibodies directed against the 41 kDa surface protein (SP41) inhibited in dose-response manner, bacterial adherence and invasion of HeLa cells. HeLa cells treated with neuraminidase did not bind SP41, suggesting the involvement of cellular sialic acid residues in this interaction. Biochemical analysis of SP41 revealed that this protein is the predicted product of the ugpB locus, which showed significant homology to the glycerol-3-phosphate-binding ATP-binding cassette (ABC) transporter protein found in several bacterial species. SP41 appears to be exposed on the bacterial surface as determined by immunofluorescence and immunogold labelling with anti-SP41 antibody. An isogenic DeltaugpB mutant showed a significant inhibitory effect on Brucella adherence and invasion of human cultured epithelial cells and this effect could be reversed by restoration of the ugpB on a plasmid. Lastly, we also show that most of the sera from individuals with acute brucellosis, but not sera obtained from healthy donors or patients with chronic brucellosis, mount antibody reactivity against SP41, suggesting that this protein is produced in vivo and that it elicits an antibody immune response. These data are novel findings that offer new insights into understanding the interplay between this bacterium and host target cells, and identify a new target for vaccine development and prevention of brucellosis.

Requirement of norD for Brucella suis virulence in a murine model of in vitro and in vivo infection.

A mutant of Brucella suis bearing a Tn5 insertion in norD, the last gene of the operon norEFCBQD, encoding nitric oxide reductase, was unable to survive under anaerobic denitrifying conditions. The norD strain exhibited attenuated multiplication within nitric oxide-producing murine macrophages and rapid elimination in mice, hence demonstrating that norD is essential for Brucella virulence.

NnrA is required for full virulence and regulates several Brucella melitensis denitrification genes.

We identified two regulators of denitrification genes in Brucella melitensis 16M: NarR, which regulates the nitrate reductase (nar) operon, and NnrA, which is involved in the expression of the last three reductases of the denitrification pathway (nirK, norB, and nosZ). NnrA is required for virulence in mice and for intracellular resistance to nitric oxide.

High susceptibility of human dendritic cells to invasion by the intracellular pathogens Brucella suis, B. abortus, and B. melitensis.

Bacteria from the Brucella genus are able to survive and proliferate within macrophages. Because they are phylogenetically closely related to macrophages, myeloid dendritic cells (DCs) constitute potential targets for Brucella bacteria. Here we report that DCs display a great susceptibility to Brucella infection. Therefore, DCs might serve as a reservoir and be important for the development of Brucella bacteria within their host.

Regulation of the mitogen-activated protein kinases by Brucella spp. expressing a smooth and rough phenotype: relationship to pathogen invasiveness.

By comparing smooth wild-type Brucella spp. to their rough mutants, we show that the LPS O chain restricted the activation of the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) pathways, thus preventing the synthesis of immune mediators that regulate host defense. We conclude that the MAPKs are a target for immune intervention by virulent smooth Brucella.

Cellular bioterrorism: how Brucella corrupts macrophage physiology to promote invasion and proliferation.

Brucellosis is a worldwide human zoonosis caused by intracellular bacteria of the genus Brucella. Virulence factors play an important role in allowing Brucella infection and proliferation within macrophages. Brucella enters macrophages through lipid raft microdomains, avoids phagolysosome fusion, and inhibits TNF-alpha secretion and apoptosis. Furthermore, Brucella can perturb bactericidal activity in macrophages by influencing the host cell response to its advantage through its LPS or by activating the cAMP/PKA pathway. To date, small steps have been taken in defining and understanding the virulence factors of Brucella used in macrophage subversion, but further investigation is required to fully explain virulence and persistence.

Adherence of Brucella to human epithelial cells and macrophages is mediated by sialic acid residues.

The basis for the interaction of Brucella species with the surface of epithelial cells before migration in the host within polymorphonuclear leucocytes is largely unknown. Here, we studied the ability of Brucella abortus and Brucella melitensis to adhere to cultured epithelial (HeLa and HEp-2) cells and THP-1-derived macrophages, and to bind extracellular matrix proteins (ECM). The brucellae adhered to epithelial cells forming localized bacterial microcolonies on the cell surface, and this process was inhibited significantly by pretreatment of epithelial cells with neuraminidase and sodium periodate and by preincubation of the bacteria with heparan sulphate and N-acetylneuraminic acid. Trypsinization of epithelial cells yielded increased adherence, suggesting unmasking of target sites on host cells. Notably, the brucellae also adhered to cultured THP-1 cells, and this event was greatly reduced upon removal of sialic acid residues from these cells with neuraminidase. B. abortus bound in a dose-dependent manner to immobilized fibronectin and vitronectin and, to a lesser extent, to chondroitin sulphate, collagen and laminin. In sum, our data strongly suggest that the adherence mechanism of brucellae to epithelial cells and macrophages is mediated by cellular receptors containing sialic acid and sulphated residues. The recognition of ECM (fibronectin and vitronectin) by the brucellae may represent a mechanism for spread within the host tissues. These are novel findings that offer new insights into understanding the interplay between Brucella and host cells.

Impairment of intramacrophagic Brucella suis multiplication by human natural killer cells through a contact-dependent mechanism.

Brucella spp. are facultative intracellular bacteria that can establish themselves and cause chronic disease in humans and animals. NK cells play a key role in host defense. They are implicated in an early immune response to a variety of pathogens. However, it was shown that they do not control Brucella infection in mice. On the other hand, NK cell activity is impaired in patients with acute brucellosis, and recently it was demonstrated that human NK cells mediate the killing of intramacrophagic Mycobacterium tuberculosis in in vitro infection. Therefore, we have analyzed the behavior of Brucella suis infecting isolated human macrophages in the presence of syngeneic NK cells. We show that (i) NK cells impair the intramacrophagic development of B. suis, a phenomenon enhanced by NK cell activators, such as interleukin-2; (ii) NK cells cultured in the presence of infected macrophages are highly activated and secrete gamma interferon and tumor necrosis factor alpha; (iii) impairment of bacterial multiplication inside infected cells is marginally associated with the cytokines produced during the early phase of macrophage-NK cell cocultures; (iv) direct cell-to-cell contact is required for NK cells to mediate the inhibition of B. suis development; and (v) inhibition of B. suis development results from an induction of NK cell cytotoxicity against infected macrophages. Altogether, these findings show that NK cells could participate early in controlling the intramacrophagic development of B. suis in humans. It seems thus reasonable to hypothesize a role for NK cells in the control of human brucellosis. However, by impairing the activity of these cells in the acute phase of the illness, the pathogen should avoid this control.

Different responses of macrophages to smooth and rough Brucella spp.: relationship to virulence.

By comparing smooth wild-type Brucella strains to their rough mutants, we show that the lipopolysaccharide (LPS) O side chain of pathogenic Brucella has a dramatic impact on macrophage activation. It favors the development of virulent Brucella by preventing the synthesis of immune mediators, important for host defense. We conclude that this O chain property is firmly linked to Brucella virulence.

Impairment of Brucella growth in human macrophagic cells that produce nitric oxide.

In mice, nitric oxide (NO) production by inducible NO synthase (iNOS), is a component of the control of Brucella infection. In humans, the involvement of iNOS in infection is still a matter of debate. Based on in vitro experiments, it was recently postulated that in humans, Brucella infection tends to become chronic because NO cannot exert its deleterious effect. In fact, conditions allowing NO production by human macrophages in culture are poorly defined, rendering the in vitro study of NO function difficult. Using DFGiNOS U937 macrophagic cells engineered to produce NO and U937 cells activated by ligation of IgE receptors, we showed that the intracellular development of Brucella was impaired in human macrophages, which produced NO. Although Brucella-infected human macrophagic phagocytes did not release NO in commonly used models of infection, the machinery required to produce NO was expressed in these cells and could be triggered by cell membrane receptors present on the infected cells. Therefore, the lack of NO production in isolated human macrophages infected by Brucella under in vitro conditions did not exclude a possible involvement of NO in the control of human brucellosis.

Smooth and rough lipopolysaccharide phenotypes of Brucella induce different intracellular trafficking and cytokine/chemokine release in human monocytes.

Virulence of the intracellular pathogen Brucella for 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 Brucella organisms 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-alpha, IL-8) and CC (MIP-1alpha, MIP-1beta, MCP-1, RANTES) chemokines, as well as pro- (IL-6, TNF-alpha) 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 Brucella and 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.

Subversion and utilization of the host cell cyclic adenosine 5'-monophosphate/protein kinase A pathway by Brucella during macrophage infection.

Brucella spp. are intramacrophage pathogens that induce chronic infections in a wide range of mammals, including domestic animals and humans. Therefore, the macrophage response to infection has important consequences for both the survival of phagocytosed bacteria and the further development of host immunity. However, very little is known about the macrophage cell signaling pathways initiated upon infection and the virulence strategy that Brucella use to counteract these responses and secure their survival. In a previous study, we have shown that macrophages activated by SR141716A, a ligand of the cannabinoid receptor CB1, acquired the capacity to control Brucella and observed that the CB1 receptor-triggering engages the microbicidal activity of phagocytes. To analyze the perturbation of cell signaling pathway during macrophage infection by Brucella, we hypothesized that SR141716A provides cell signaling that interferes with the bacterial message leading to inhibition of macrophage functions. As CB1 receptor belongs to the family of G protein-linked receptors, we explored the cAMP signaling pathway. In this study, we show that the CB1 ligand inhibited the bacteria-induced cell signaling. Taking advantage of this result, we then demonstrated that Brucella infection elicited a rapid activation of the cAMP/protein kinase A pathway. This activation resulted in a prolonged phosphorylation of the transcription factor CREB. We finally demonstrate that the activation of the cAMP/protein kinase A pathway is crucial for the survival and establishment of Brucella within macrophages. For the first time in phagocytes, we thus characterized a primordial virulence strategy of Brucella involving the host signaling pathway, a novel point of immune intervention of this virulent pathogen.

Nramp1 is not a major determinant in the control of Brucella melitensis infection in mice.

Brucella, the causative agent of brucellosis in animals and humans, can survive and proliferate within macrophages. Macrophages mediate mouse resistance to various pathogens through the expression of the Nramp1 gene. The role of this gene in the control of Brucella infection was investigated. When BALB/c mice (Nramp1(s)) and C.CB congenic mice (Nramp1(r)) were infected with Brucella melitensis, the number of Brucella organisms per spleen was significantly larger in the C.CB mice than in the BALB/c mice during the first week postinfection (p.i.). This Nramp1-linked susceptibility to Brucella was temporary, since similar numbers of Brucella were recovered from the two strains of mice 2 weeks p.i. The effect of Nramp1 expression occurred within splenocytes intracellularly infected by BRUCELLA: However, there was no difference between in vitro replication rates of Brucella in macrophages isolated from the two strains of mice infected in vivo or in Nramp1 RAW264 transfectants. In mice, infection with Brucella induced an inflammatory response, resulting in splenomegaly and recruitment of phagocytes in the spleen, which was amplified in C.CB mice. Reverse transcription-PCR (RT-PCR), performed 5 days p.i., showed that inducible nitric oxide synthase, tumor necrosis factor alpha (TNF-alpha), interleukin-12 p40 (IL-12p40), gamma interferon (IFN-gamma), and IL-10 mRNAs were similarly induced in spleens of the two strains. In contrast, the mRNA of KC, a C-X-C chemokine, was induced only in infected C.CB mice at this time. This pattern of mRNA expression was maintained at 14 days p.i., with IFN-gamma and IL-12p40 mRNAs being more intensively induced in the infected C.CB mice, but TNF-alpha mRNA was no longer induced. The higher recruitment of neutrophils observed in the spleens of infected C.CB mice could explain the temporary susceptibility of C.CB mice to B. melitensis infection. In contrast to infections with Salmonella, Leishmania, and Mycobacterium, the expression of the Nramp1 gene appears to be of limited importance for the natural resistance of mice to Brucella.

The innate immune response against Brucella in humans.

Pathogens have developed different strategies to survive and multiply within their host. Among them is the ability to control phagocyte apoptosis while another is to affect the expression of cytokines which is necessary for a normal protective function of the immune response. To establish themselves and cause chronic disease in humans and animals, Brucella spp. invade and proliferate within monocytic phagocytes. We have established that in humans, Brucella suis impairs the apoptosis of monocytes and macrophages, thus preventing its host cell elimination. In mice, which are not naturally colonized by the bacteria, Brucella infection results in Type1 (Th1) cellular immune response which promotes a clearance of the bacterial organism. The development of this response is under the control of major cytokines like TNF-alpha, IFN-gamma and IL-12 produced at the onset of infection. We have observed that in humans, B. suis-infected macrophages which produce IL-1, IL-6, IL-10 and several chemokines including IL-8, do not secrete TNF-alpha. By constructing null mutants, we demonstrated that this inhibition involves the outer membrane protein Omp25 of Brucella, however the mechanism regulating the inhibition has not yet been clearly defined. It is likely that the Omp25-induced effect on TNF-alpha production assists bacterial evasion of antimicrobial defences at different levels. Firstly, by preventing the autocrine activation of macrophages thus inhibiting innate immunity and secondly by impairing the production of IL-12 and the development of a Th1 type specific immunity. In addition to the central role of the macrophage in Brucella infection, others cells of the innate immune response are recruited and influenced by the interactions between bacteria and host. For instance, human Vgamma9Vdelta2 T-cells play an important role in the early response to infection with intracellular pathogens. Evidence has been presented that their number dramatically increased in the peripheral blood of patients with acute brucellosis. We have shown that human Vgamma9Vdelta2 T-cells can be specifically activated by non-peptidic low molecular weight compound(s) from B. suis lysate or by soluble factors produced by B. suis-infected macrophages. Under these conditions, they produce TNF-alpha and IFN-gamma and reduce the bacterial multiplication inside infected autologous macrophages. This impairment of B. suis multiplication is due to both soluble factors released from activated gammadeltaT-cells (including TNF-alpha and IFN-gamma) and to a contact-dependent cytotoxicity directed against the infected cells. The interactions between the bacteria and these cells can counteract the intramacrophagic development of the bacteria and finally influence the further development of the host defense. We hypothesize that the chronicity or the elimination of the infection will depend on the balance between contradictory effects induced by the bacteria which favor either the host or the pathogen. Moreover, the interrelationship between the different cells must be taken into account in the analysis of the virulence of the bacteria and in the development of in vitro models of human macrophage infection.