RESUMEN

The liver is frequently affected in patients with active brucellosis. The present study demonstrates that Brucella abortus infection induces the activation of the autophagic pathway in hepatic stellate cells to create a microenvironment that promotes a profibrogenic phenotype through the induction of transforming growth factor-ß1 (TGF-ß1), collagen deposition, and inhibition of matrix metalloproteinase-9 (MMP-9) secretion. Autophagy was revealed by upregulation of the LC3II/LC3I ratio and Beclin-1 expression as well as inhibition of p62 expression in infected cells. The above-described findings were dependent on the type IV secretion system (VirB) and the secreted BPE005 protein, which were partially corroborated using the pharmacological inhibitors wortmannin, a phosphatidyl inositol 3-kinase inhibitor, and leupeptin plus E64 (inhibitors of lysosomal proteases). Activation of the autophagic pathway in hepatic stellate cells during Brucella infection could have an important contribution to attenuating inflammatory hepatic injury by inducing fibrosis. However, with time, B. abortus infection induced Beclin-1 cleavage with concomitant cleavage of caspase-3, indicating the onset of apoptosis of LX-2 cells, as was confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay and Hoechst staining. These results demonstrate that the cross talk of LX-2 cells and B. abortus induces autophagy and fibrosis with concomitant apoptosis of LX-2 cells, which may explain some potential mechanisms of liver damage observed in human brucellosis.

Asunto(s)

Autofagia/fisiología , Brucella abortus/patogenicidad , Fibrosis/microbiología , Fibrosis/patología , Células Estrelladas Hepáticas/microbiología , Células Estrelladas Hepáticas/patología , Apoptosis/fisiología , Beclina-1/metabolismo , Brucelosis/metabolismo , Brucelosis/microbiología , Brucelosis/patología , Caspasa 3/metabolismo , Línea Celular , Colágeno/metabolismo , Fibrosis/metabolismo , Células Estrelladas Hepáticas/metabolismo , Humanos , Hígado/metabolismo , Hígado/microbiología , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/microbiología , Cirrosis Hepática/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Fenotipo , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta1/metabolismo , Sistemas de Secreción Tipo IV/metabolismo , Regulación hacia Arriba/fisiología

RESUMEN

The liver is frequently affected in patients with active brucellosis. In the present study, we identified a virulence factor involved in the modulation of hepatic stellate cell function and consequent fibrosis during Brucella abortus infection. This study assessed the role of BPE005 protein from B. abortus in the fibrotic phenotype induced on hepatic stellate cells during B. abortus infection in vitro and in vivo. We demonstrated that the fibrotic phenotype induced by B. abortus on hepatic stellate (LX-2) cells was dependent on BPE005, a protein associated with the type IV secretion system (T4SS) VirB from B. abortus. Our results indicated that B. abortus inhibits matrix metalloproteinase 9 (MMP-9) secretion through the activity of the BPE005-secreted protein and induces concomitant collagen deposition by LX-2 cells. BPE005 is a small protein containing a cyclic nucleotide monophosphate binding domain (cNMP) that modulates the LX-2 cell phenotype through a mechanism that is dependent on the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway. Altogether, these results indicate that B. abortus tilts LX-2 cells to a profibrogenic phenotype employing a functional T4SS and the secreted BPE005 protein through a mechanism that involves the cAMP and PKA signaling pathway.

Asunto(s)

Proteínas Bacterianas/química , Brucella abortus/metabolismo , Colágeno/metabolismo , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/microbiología , Hígado/patología , Metaloproteinasa 9 de la Matriz/genética , Factor de Crecimiento Transformador beta/metabolismo , Animales , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Brucella abortus/química , Brucella abortus/genética , Brucella abortus/patogenicidad , Brucelosis/microbiología , Brucelosis/patología , Línea Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación hacia Abajo , Femenino , Fibrosis , Regulación Enzimológica de la Expresión Génica , Células Estrelladas Hepáticas/patología , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones Endogámicos BALB C , Fenotipo , Transducción de Señal , Sistemas de Secreción Tipo IV , Factores de Virulencia

RESUMEN

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.

Asunto(s)

Vacunas Bacterianas/inmunología , Vacunas Bacterianas/aislamiento & purificación , Brucella abortus/inmunología , Brucelosis Bovina/prevención & control , Infecciones por Escherichia coli/prevención & control , Escherichia coli O157/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Brucella abortus/genética , Brucelosis Bovina/inmunología , Brucelosis Bovina/microbiología , Bovinos , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Expresión Génica , Inmunoglobulina A/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/aislamiento & purificación

RESUMEN

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.

Asunto(s)

Linfocitos B/inmunología , Proteínas Bacterianas/inmunología , Proliferación Celular , Macrófagos/inmunología , Factores de Virulencia/inmunología , Isomerasas de Aminoácido/genética , Isomerasas de Aminoácido/inmunología , Isomerasas de Aminoácido/metabolismo , Animales , Linfocitos B/citología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Western Blotting , Brucella abortus/inmunología , Brucella abortus/metabolismo , Brucella abortus/patogenicidad , Línea Celular , Células Cultivadas , Femenino , Macrófagos/parasitología , Macrófagos/virología , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Miosina Tipo IIA no Muscular/inmunología , Miosina Tipo IIA no Muscular/metabolismo , Unión Proteica , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Trypanosoma cruzi/inmunología , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/patogenicidad , Virulencia/inmunología , Factores de Virulencia/genética , Factores de Virulencia/metabolismo

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas/metabolismo , Brucella abortus/metabolismo , Colina/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Transporte Biológico Activo , Membrana Celular , Femenino , Regulación Bacteriana de la Expresión Génica/fisiología , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Fosfatidilcolinas/biosíntesis

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas/metabolismo , Brucella abortus/metabolismo , Brucella abortus/patogenicidad , Macrófagos/microbiología , Factores de Virulencia/metabolismo , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Fusión Artificial Génica , Proteínas Bacterianas/genética , Brucella abortus/genética , Brucelosis/microbiología , Línea Celular , Biología Computacional/métodos , Modelos Animales de Enfermedad , Genes Reporteros , Genómica/métodos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Confocal , Transporte de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Virulencia/genética