RESUMO
The adhesion of bacterial pathogens to host cells is an event that determines infection, and ultimately invasion and intracellular multiplication. Several evidences have recently shown that this rule is also truth for the intracellular pathogen Brucella. Brucella suis displays the unipolar BmaC and BtaE adhesins, which belong to the monomeric and trimeric autotransporter (TA) families, respectively. It was previously shown that these adhesins are involved in bacterial adhesion to host cells and components of the extracellular matrix (ECM). In this work we describe the role of a new member of the TA family of B. suis (named BtaF) in the adhesive properties of the bacterial surface. BtaF conferred the bacteria that carried it a promiscuous adhesiveness to various ECM components and the ability to attach to an abiotic surface. Furthermore, BtaF was found to participate in bacterial adhesion to epithelial cells and was required for full virulence in mice. Similar to BmaC and BtaE, the BtaF adhesin was expressed in a small subpopulation of bacteria, and in all cases, it was detected at the new pole generated after cell division. Interestingly, BtaF was also implicated in the resistance of B. suis to porcine serum. Our findings emphasize the impact of TAs in the Brucella lifecycle.
Assuntos
Adesinas Bacterianas/metabolismo , Aderência Bacteriana/fisiologia , Brucella suis/fisiologia , Brucella suis/patogenicidade , Adesinas Bacterianas/química , Adesinas Bacterianas/imunologia , Animais , Brucelose/imunologia , Brucelose/metabolismo , Linhagem Celular , Matriz Extracelular/metabolismo , Humanos , Masculino , Camundongos , Família Multigênica , Multimerização Proteica , Transporte Proteico , Suínos , VirulênciaRESUMO
Brucella is responsible for brucellosis, one of the most common zoonoses worldwide that causes important economic losses in several countries. Increasing evidence indicates that adhesion of Brucella spp. to host cells is an important step to establish infection. We have previously shown that the BmaC unipolar monomeric autotransporter mediates the binding of Brucella suis to host cells through cell-associated fibronectin. Our genome analysis shows that the B. suis genome encodes several additional potential adhesins. In this work, we characterized a predicted trimeric autotransporter that we named BtaE. By expressing btaE in a nonadherent Escherichia coli strain and by phenotypic characterization of a B. suis ΔbtaE mutant, we showed that BtaE is involved in the binding of B. suis to hyaluronic acid. The B. suis ΔbtaE mutant exhibited a reduction in the adhesion to HeLa and A549 epithelial cells compared with the wild-type strain, and it was outcompeted by the wild-type strain in the binding to HeLa cells. The knockout btaE mutant showed an attenuated phenotype in the mouse model, indicating that BtaE is required for full virulence. BtaE was immunodetected on the bacterial surface at one cell pole. Using old and new pole markers, we observed that both the BmaC and BtaE adhesins are consistently associated with the new cell pole, suggesting that, in Brucella, the new pole is functionally differentiated for adhesion. This is consistent with the inherent polarization of this bacterium, and its role in the invasion process.
Assuntos
Adesinas Bacterianas/metabolismo , Brucella suis/metabolismo , Brucella suis/patogenicidade , Brucelose/microbiologia , Proteínas de Transporte/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Adesinas Bacterianas/genética , Animais , Anticorpos Antibacterianos , Aderência Bacteriana/fisiologia , Brucella suis/genética , Proteínas de Transporte/genética , Polaridade Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Família Multigênica , VirulênciaRESUMO
Brucella suis biovar 1 is the causative agent of brucellosis in several domestic and wild animals and it is a common agent of human brucellosis. European hares (Lepus europaeus) have been shown to be infected by B. suis biovar 1 and the transmission to other animals has been suggested. In this work, experimental rabbits (Cuniculus orictolagus) were infected with B. suis biovar 1 isolated from wild hares. Infected rabbits showed high serological response in 2 weeks after discharge and typical granulomatous lesions (2mm diameter) were found in liver, spleen and kidneys after 50 days. B. suis biovar 1 was cultured from the lesion of the organs mentioned above as well as from urine, placenta and fetuses. These data suggest that hares are a potential source for horizontal transmission of B. suis biovar 1 to other mammalians.
Assuntos
Brucella suis/patogenicidade , Brucelose/veterinária , Lebres/microbiologia , Coelhos/microbiologia , Animais , Animais Selvagens/microbiologia , Brucelose/patologia , Feminino , Rim/microbiologia , Rim/patologia , Fígado/microbiologia , Fígado/patologia , Masculino , Gravidez , Complicações Infecciosas na Gravidez/microbiologia , Complicações Infecciosas na Gravidez/veterinária , Baço/microbiologia , Baço/patologiaRESUMO
Although vascular pathologies such as vasculitis, endocarditis and mycotic aneurysms have been described in brucellosis patients, the interaction of Brucella with the endothelium has not been characterized. In this study we show that Brucella abortus and Brucella suis can infect and replicate in primary human umbilical vein endothelial cells (HUVEC) and in the microvascular endothelial cell line HMEC-1. Infection led to an increased production of IL-8, MCP-1 and IL-6 in HUVEC and HMEC-1 cells, and an increased expression of adhesion molecules (CD54 in both cells, CD106 and CD62E in HUVEC). Experiments with purified antigens from the bacterial outer membrane revealed that lipoproteins (Omp19) but not lipopolysaccharide mediate these proinflammatory responses. Infection of polarized HMEC-1 cells resulted in an increased capacity of these cells to promote the transmigration of neutrophils from the apical to the basolateral side of the monolayer, and the same phenomenon was observed when the cells were stimulated with live bacteria from the basolateral side. Overall, these results suggest that Brucella spp. can infect and survive within endothelial cells, and can induce a proinflammatory response that might be involved in the vascular manifestations of brucellosis.
Assuntos
Brucella abortus/imunologia , Brucella abortus/patogenicidade , Brucella suis/imunologia , Brucella suis/patogenicidade , Citocinas/metabolismo , Células Endoteliais/imunologia , Células Endoteliais/microbiologia , Antígenos de Bactérias/imunologia , Antígenos CD/biossíntese , Proteínas da Membrana Bacteriana Externa/imunologia , Células Cultivadas , Humanos , Lipoproteínas/imunologia , Neutrófilos/imunologia , Migração Transendotelial e TransepitelialRESUMO
Inhalation is a common route for Brucella infection. We investigated whether Brucella species can invade and replicate within alveolar(A549) and bronchial (Calu-6 and 16HBE14o-) human epithelial cells. The number of adherent and intracellular bacteria was higher for rough strains (Brucella canis and Brucella abortus RB51) than for smooth strains (B. abortus 2308 and Brucella suis 1330). Only smooth strains exhibited efficient intracellular replication (1.5-3.5 log increase at 24 h p.i.). A B. abortus mutant with defective expression of the type IV secretion system did not replicate. B. abortus internalization was inhibited by specific inhibitors of microfilaments, microtubules and PI3-kinase activity. As assessed with fluorescent probes, B. abortus infection did not affect the viability of A549 and 16HBE14o- cells, but increased the percentage of injured cells (both strains) and dead cells (RB51) in Calu-6 cultures. LDH levels were increased in supernatants of Calu-6 and 16HBE14o- cells infected with B. abortus RB51, and to a lower extent in Calu-6 infected with B. abortus 2308. No apoptosis was detected by TUNEL upon infection with smooth or rough B. abortus. This study shows that smooth brucellae can infect and replicate in human respiratory epithelial cells inducing minimal or null cytotoxicity.
Assuntos
Brucella abortus/patogenicidade , Brucella canis/patogenicidade , Brucella suis/patogenicidade , Morte Celular , Células Epiteliais/microbiologia , Aderência Bacteriana , Linhagem Celular , Sobrevivência Celular , Citoplasma/microbiologia , HumanosRESUMO
The RND-type efflux pumps are responsible for the multidrug resistance phenotype observed in many clinically relevant species. Also, RND pumps have been implicated in physiological processes, with roles in the virulence mechanisms of several pathogenic bacteria. We have previously shown that the BepC outer membrane factor of Brucella suis is involved in the efflux of diverse drugs, probably as part of a tripartite complex with an inner membrane translocase. In the present work, we characterize two membrane fusion protein-RND translocases of B. suis encoded by the bepDE and bepFG loci. MIC assays showed that the B. suis DeltabepE mutant was more sensitive to deoxycholate (DOC), ethidium bromide, and crystal violet. Furthermore, multicopy bepDE increased resistance to DOC and crystal violet and also to other drugs, including ampicillin, norfloxacin, ciprofloxacin, tetracycline, and doxycycline. In contrast to the DeltabepE mutant, the resistance profile of B. suis remained unaltered when the other RND gene (bepG) was deleted. However, the DeltabepE DeltabepG double mutant showed a more severe phenotype than the DeltabepE mutant, indicating that BepFG also contributes to drug resistance. An open reading frame (bepR) coding for a putative regulatory protein of the TetR family was found upstream of the bepDE locus. BepR strongly repressed the activity of the bepDE promoter, but DOC released the repression mediated by BepR. A clear induction of the bepFG promoter activity was observed only in the BepDE-defective mutant, indicating a regulatory interplay between the two RND efflux pumps. Although only the BepFG-defective mutant showed a moderate attenuation in model cells, the activities of both bepDE and bepFG promoters were induced in the intracellular environment of HeLa cells. Our results show that B. suis harbors two functional RND efflux pumps that may contribute to virulence.
Assuntos
Proteínas de Bactérias/metabolismo , Brucella suis/efeitos dos fármacos , Farmacorresistência Bacteriana , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Brucella suis/patogenicidade , Brucella suis/fisiologia , Ácido Desoxicólico/farmacologia , Células Epiteliais/microbiologia , Etídio/farmacologia , Deleção de Genes , Dosagem de Genes , Regulação Bacteriana da Expressão Gênica , Violeta Genciana/farmacologia , Células HeLa , Humanos , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , VirulênciaRESUMO
Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and other alpha-2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host.