RESUMO
Brucella spp. and Trypanosoma cruzi are two intracellular pathogens that have no evolutionary common origins but share a similar lifestyle as they establish chronic infections for which they have to circumvent the host immune response. Both pathogens have a virulence factor (prpA in Brucella and tcPrac in T. cruzi) that induces B-cell proliferation and promotes the establishment of the chronic phase of the infectious process. We show here that, even though PrpA promotes B-cell proliferation, it targets macrophages in vitro and is translocated to the cytoplasm during the intracellular replication phase. We observed that PrpA-treated macrophages induce the secretion of a soluble factor responsible for B-cell proliferation and identified nonmuscular myosin IIA (NMM-IIA) as a receptor required for binding and function of this virulence factor. Finally, we show that the Trypanosoma cruzi homologue of PrpA also targets macrophages to induce B-cell proliferation through the same receptor, indicating that this virulence strategy is conserved between a bacterial and a protozoan pathogen.
Assuntos
Linfócitos B/imunologia , Proteínas de Bactérias/imunologia , Proliferação de Células , Macrófagos/imunologia , Fatores de Virulência/imunologia , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/imunologia , Isomerases de Aminoácido/metabolismo , Animais , Linfócitos B/citologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Brucella abortus/imunologia , Brucella abortus/metabolismo , Brucella abortus/patogenicidade , Linhagem Celular , Células Cultivadas , Feminino , Macrófagos/parasitologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Miosina não Muscular Tipo IIA/imunologia , Miosina não Muscular Tipo IIA/metabolismo , Ligação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Baço/citologia , Baço/imunologia , Baço/metabolismo , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidade , Virulência/imunologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/metabolismo , Colina/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Transporte Biológico Ativo , Membrana Celular , Feminino , Regulação Bacteriana da Expressão Gênica/fisiologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosfatidilcolinas/biossínteseRESUMO
Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella abortus/metabolismo , Brucella abortus/patogenicidade , Macrófagos/microbiologia , Fatores de Virulência/metabolismo , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Animais , Fusão Gênica Artificial , Proteínas de Bactérias/genética , Brucella abortus/genética , Brucelose/microbiologia , Linhagem Celular , Biologia Computacional/métodos , Modelos Animais de Doenças , Genes Reporter , Genômica/métodos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Confocal , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Virulência/genéticaRESUMO
Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH7) from STEC, was constructed and expressed in B. abortus Δpgm vaccine strain (BabΔpgm). Mice orally immunized with BabΔpgm(EITH7) elicited an immune response with the induction of anti-EITH7 antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t=4 days) than mice immunized with BabΔpgm carrier strain (t=12 days). As expected, mice immunized with BabΔpgm(EITH7) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles-the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC.
Assuntos
Vacinas Bacterianas/imunologia , Vacinas Bacterianas/isolamento & purificação , Brucella abortus/imunologia , Brucelose Bovina/prevenção & controle , Infecções por Escherichia coli/prevenção & controle , Escherichia coli O157/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Brucella abortus/genética , Brucelose Bovina/imunologia , Brucelose Bovina/microbiologia , Bovinos , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Expressão Gênica , Imunoglobulina A/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
Several bacterial pathogens have TIR domain-containing proteins that contribute to their pathogenesis. We identified a second TIR-containing protein in Brucella spp. that we have designated BtpB. We show it is a potent inhibitor of TLR signaling, probably via MyD88. BtpB is a novel Brucella effector that is translocated into host cells and interferes with activation of dendritic cells. In vivo mouse studies revealed that BtpB is contributing to virulence and control of local inflammatory responses with relevance in the establishment of chronic brucellosis. Together, our results show that BtpB is a novel Brucella effector that plays a major role in the modulation of host innate immune response during infection.