RESUMO
OBJECTIVE: Electronic cigarette (e-cig) use has recently been implicated in promoting atherosclerosis. In this study, we aimed to investigate the mechanism of e-cig exposure accelerated atherosclerotic lesion development. Approach and Results: Eight-week-old ApoE-/- mice fed normal laboratory diet were exposed to e-cig vapor (ECV) for 2 hours/day, 5 days/week for 16 weeks. We found that ECV exposure significantly induced atherosclerotic lesions as examined by Oil Red O staining and greatly upregulated TLR9 (toll-like receptor 9) expression in classical monocytes and in the atherosclerotic plaques, which the latter was corroborated by enhanced TLR9 expression in human femoral artery atherosclerotic plaques from e-cig smokers. Intriguingly, we found a significant increase of oxidative mitochondria DNA lesion in the plasma of ECV-exposed mice. Administration of TLR9 antagonist before ECV exposure not only alleviated atherosclerosis and the upregulation of TLR9 in plaques but also attenuated the increase of plasma levels of inflammatory cytokines, reduced the plaque accumulation of lipid and macrophages, and decreased the frequency of blood CCR2+ (C-C chemokine receptor type 2) classical monocytes. Surprisingly, we found that cytoplasmic mitochondrial DNA isolated from ECV extract-treated macrophages can enhance TLR9 activation in reporter cells and the induction of inflammatory cytokine could be suppressed by TLR9 inhibitor in macrophages. CONCLUSIONS: E-cig increases level of damaged mitochondrial DNA in circulating blood and induces the expression of TLR9, which elevate the expression of proinflammatory cytokines in monocyte/macrophage and consequently lead to atherosclerosis. Our results raise the possibility that intervention of TLR9 activation is a potential pharmacological target of ECV-related inflammation and cardiovascular diseases.
Assuntos
Aorta/metabolismo , Aterosclerose/etiologia , Dano ao DNA , DNA Mitocondrial/metabolismo , Vapor do Cigarro Eletrônico/efeitos adversos , Inflamação/etiologia , Macrófagos/metabolismo , Mitocôndrias/metabolismo , Receptor Toll-Like 9/metabolismo , Animais , Aorta/patologia , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , DNA Mitocondrial/genética , Modelos Animais de Doenças , Feminino , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/patologia , Células RAW 264.7 , Transdução de Sinais , Fumantes , VapingRESUMO
The alveolus participates in gas exchange, which can be impaired by environmental factors and toxins. There is an increase in using electronic cigarettes (e-cigarettes); however, their effect on human primary alveolar epithelial cells is unknown. Human lungs were obtained from nonsmoker organ donors to isolate alveolar type II (ATII) cells. ATII cells produce and secrete pulmonary surfactant and restore the epithelium after damage, and mitochondrial function is important for their metabolism. Our data indicate that human ATII cell exposure to e-cigarette aerosol increased IL-8 levels and induced DNA damage and apoptosis. We also studied the cytoprotective effect of DJ-1 against ATII cell injury. DJ-1 knockdown in human primary ATII cells sensitized cells to mitochondrial dysfunction as detected by high mitochondrial superoxide production, decreased mitochondrial membrane potential, and calcium elevation. DJ-1 knockout (KO) mice were more susceptible to ATII cell apoptosis and lung injury induced by e-cigarette aerosol compared with wild-type mice. Regulation of the oxidative phosphorylation (OXPHOS) is important for mitochondrial function and protection against oxidative stress. Major subunits of the OXPHOS system are encoded by both nuclear and mitochondrial DNA. We found dysregulation of OXPHOS complexes in DJ-1 KO mice after exposure to e-cigarette aerosol, which could disrupt the nuclear/mitochondrial stoichiometry, resulting in mitochondrial dysfunction. Together, our results indicate that DJ-1 deficiency sensitizes ATII cells to damage induced by e-cigarette aerosol leading to lung injury.
Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Sistemas Eletrônicos de Liberação de Nicotina , Interleucina-8/genética , Nicotina/farmacologia , Proteína Desglicase DJ-1/genética , Aerossóis , Células Epiteliais Alveolares/citologia , Células Epiteliais Alveolares/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Cálcio/metabolismo , Dano ao DNA , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Interleucina-8/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Cultura Primária de Células , Proteína Desglicase DJ-1/deficiência , Proteína Desglicase DJ-1/metabolismo , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/metabolismo , Superóxidos/metabolismoRESUMO
BACKGROUND: Brucella abortus is an intracellular pathogen which can infect and persist in host cells through multiple interactions. Above all, its interaction to host cell receptor is important to understand the pathogenic mechanisms of B. abortus. Accordingly, we demonstrated that platelet-activating factor receptor (PAFR) affects host cell response against B. abortus infection. RESULTS: First of all, B. abortus infection to macrophage induces secretion of platelet-activating factor (PAF), which is a PAFR agonist. The stimulation of PAFR by PAF remarkably increases B. abortus uptake into macrophages. It induces Janus kinase 2 (JAK2) and p38α phosphorylation, indicating that PAFR-mediated activation of JAK2 signaling leads to enhanced uptake of B. abortus. Moreover, the dynamics of F-actin polymerization revealed that PAFR-mediated B. abortus uptake is related with the reorganization of F-actin and JAK2. Upon B. abortus phagocytosis, reduced PAFR in the membrane and subsequently increased levels of PAFR colocalization with endosomes were observed which indicate that B. abortus uptake into macrophages allowed PAFR trafficking to endosomes. CONCLUSIONS: This study demonstrated that PAFR has a compelling involvement in B. abortus uptake as a promoter of phagocytosis, which is associated with JAK2 activation. Thus, our findings establish a novel insight into a receptor-related phagocytic mechanism of B. abortus.
Assuntos
Brucella abortus/patogenicidade , Macrófagos/imunologia , Macrófagos/microbiologia , Fagocitose , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Brucella abortus/metabolismo , Brucelose/metabolismo , Brucelose/microbiologia , Endossomos/metabolismo , Interações Hospedeiro-Patógeno , Janus Quinase 2/metabolismo , Macrófagos/enzimologia , Camundongos , Células RAW 264.7 , Transdução de SinaisRESUMO
Brucellosis is one of the major zoonoses worldwide that inflicts important health problems in animal and human. Here, we demonstrated that dextran sulfate sodium (DSS) significantly increased adhesion of Brucella (B.) abortus in murine macrophages compared to untreated cells. Even without infection, Brucella uptake into macrophages increased and F-actin reorganization was induced compared with untreated cells. Furthermore, DSS increased the phosphorylation of MAPKs (ERK1/2 and p38α) in Brucella-infected, DSS-treated cells compared with the control cells. Lastly, DSS markedly increased the intracellular survival of Brucella abortus in macrophages by up to 48 h. These results suggest that DSS enhanced the adhesion and phagocytosis of B. abortus into murine macrophages by stimulating the MAPK signaling proteins phospho-ERK1/2 and p38α and that DSS increased the intracellular survival of B. abortus by inhibiting colocalization of Brucella-containing vacuoles (BCVs) with the late endosome marker LAMP-1. This study emphasizes the enhancement of the phagocytic and intracellular modulatory effects of DSS, which may suppress the innate immune system and contribute to prolonged Brucella survival and chronic infection.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Brucella abortus/efeitos dos fármacos , Brucella abortus/crescimento & desenvolvimento , Brucelose/microbiologia , Sulfato de Dextrana/farmacologia , Macrófagos/microbiologia , Animais , Brucella abortus/fisiologia , Brucelose/genética , Brucelose/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Células RAW 264.7 , Regulação para Cima/efeitos dos fármacosRESUMO
Currently, there are several serodiagnostic tools available for brucellosis, however, it is difficult to differentiate an active infection from vaccination. Hence, there is a great need to develop alternative means that can distinguish between these two conditions without utilizing lipopolysaccharide (LPS). This study was an attempt to determine the efficacy of combined recombinant Brucella (B.) abortus outer membrane proteins (rOmps) and individual rOmps in the serodiagnosis of brucellosis by enzyme linked immunosorbent assay (ELISA), utilizing both that standard tube agglutination test (TAT)-positive and -negative serum samples from Korean native cattle. The results are very interesting and promising because the combined rOmp antigens used in the study were highly reactive with the TAT-positive serum samples. The combined rOmps sensitivity, specificity and accuracy were 215/232 (92.67%), 294/298 (98.66%) and 509/530 (96.04%), respectively. While these results are preliminary, the tests performed have very high potential in the serodiagnosis of brucellosis and likewise, the combined rOmps can be used for future vaccine production.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Brucelose Bovina/diagnóstico , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Medicina Veterinária/métodos , Testes de Aglutinação , Animais , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Bovinos , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes/genética , República da Coreia , Sensibilidade e EspecificidadeRESUMO
Brucellosis is a vital zoonotic disease caused by Brucella, which infects a wide range of animals and humans. Accurate diagnosis and reliable vaccination can control brucellosis in domestic animals. This study examined novel immunogenic proteins that can be used to detect Brucella abortus infection or as an effective subcellular vaccine. In an immunoproteomic assay, 55 immunodominant proteins from B. abortus 544 were observed using two dimensional electrophoresis (2DE) and immunoblot profiles with antisera from B. abortus-infected cattle at the early (week 3), middle (week 7), and late (week 10) periods, after excluding protein spots reacting with antisera from Yersinia enterocolitica O:9-infected and non-infected cattle. Twenty-three selected immunodominant proteins whose spots were observed at all three infection periods were identified using MALDI-MS/MS. Most of these proteins identified by immunoblot and mass spectrometry were determined by their subcellular localization and predicted function. We suggest that the detection of prominent immunogenic proteins during the infection period can support the development of advanced diagnostic methods with high specificity and accuracy; subsidiarily, these proteins can provide supporting data to aid in developing novel vaccine candidates.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Brucella abortus/imunologia , Brucelose Bovina/imunologia , Soros Imunes/imunologia , Epitopos Imunodominantes/imunologia , Animais , Bovinos , Feminino , Immunoblotting/veterinária , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária , Espectrometria de Massas em Tandem/veterináriaRESUMO
Lipid raft-associated clathrin is essential for host-pathogen interactions during infection. Brucella abortus is an intracellular pathogen that circumvents host defenses, but little is known about the precise infection mechanisms that involve interaction with lipid raft-associated mediators. The aim of this study was to elucidate the clathrin-mediated phagocytic mechanisms of B. abortus. The clathrin dependence of B. abortus infection in HeLa cells was investigated using an infection assay and immunofluorescence microscopy. The redistribution of clathrin in the membrane and in phagosomes was investigated using sucrose gradient fractionation of lipid rafts and the isolation of B. abortus-containing vacuoles, respectively. Clathrin and dynamin were concentrated into lipid rafts during B. abortus infection, and the entry and intracellular survival of B. abortus within HeLa cells were abrogated by clathrin inhibition. Clathrin disruption decreased actin polymerization and the colocalization of B. abortus-containing vacuoles with clathrin and Rab5 but not lysosome-associated membrane protein 1 (LAMP-1). Thus, our data demonstrate that clathrin plays a fundamental role in the entry and intracellular survival of B. abortus via interaction with lipid rafts and actin rearrangement. This process facilitates the early intracellular trafficking of B. abortus to safe replicative vacuoles.
Assuntos
Brucella abortus/fisiologia , Clatrina/metabolismo , Regulação Enzimológica da Expressão Gênica , Fagocitose , Proteínas rab5 de Ligação ao GTP/metabolismo , Actinas/química , Transporte Biológico , Células HeLa , Humanos , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Microdomínios da Membrana/química , Microscopia de Fluorescência , Fagossomos/metabolismo , Fagossomos/microbiologia , Polimerização , RNA Interferente Pequeno/metabolismoRESUMO
Alveolar type II (ATII) cells proliferate and restore the injured epithelium. It has been described that SARS-CoV-2 infection causes diffuse alveolar damage in the lungs. However, host factors facilitating virus infection in ATII cells are not well known. We determined the SARS-CoV-2-related genes and protein expression using RT-PCR and Western blotting, respectively, in ATII cells isolated from young and elderly non-smokers, smokers, and ex-smokers. Cells were also obtained from lung transplants of emphysema patients. ACE2 has been identified as the receptor for SARS-CoV-2, and we found significantly increased levels in young and elderly smokers and emphysema patients. The viral entry depends on TMPRSS2 protease activity, and a higher expression was detected in elderly smokers and ex-smokers and emphysema patients. Both ACE2 and TMPRSS2 mRNA levels were higher in this disease in comparison with non-smokers. CD209L serves as a receptor for SARS-CoV-2, and we found increased levels in ATII cells obtained from smokers and in emphysema patients. Also, our data suggest CD209L regulation by miR142. Endoplasmic reticulum stress was detected in ATII cells in this disease. Our results suggest that upregulation of SARS-CoV-2 entry factors in ATII cells in aging, smokers, and emphysema patients may facilitate infection.
RESUMO
The Brucella mdh gene was successfully cloned and expressed in E. coli. The purified recombinant malate dehydrogenase protein (rMDH) was reactive to Brucella-positive bovine serum in the early stage, but not reactive in the middle or late stage, and was reactive to Brucella-positive mouse serum in the late stage, but not in the early or middle stage of infection. In addition, rMDH did not react with Brucella-negative bovine or mouse sera. These results suggest that rMDH has the potential for use as a specific antigen in serological diagnosis for early detection of bovine brucellosis.
Assuntos
Antígenos de Bactérias/imunologia , Brucella abortus/enzimologia , Brucelose/veterinária , Doenças dos Bovinos/diagnóstico , Malato Desidrogenase/genética , Malato Desidrogenase/imunologia , Proteínas Recombinantes/imunologia , Animais , Brucella abortus/imunologia , Brucelose/diagnóstico , Bovinos , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Malato Desidrogenase/isolamento & purificação , Camundongos , Proteínas Recombinantes/genéticaRESUMO
To date, detection of antibodies against the lipopolysaccharide portion is the backbone of most serodiagnostic methods for brucellosis screening. However this pose a risk for false positive reactions related to other pathogens especially that of Yersinia enterocolitica O:9 which has the most prominent cross reactivity with Brucella spp. In this study, cloning and expression of Brucella abortus bacterioferritin (Bfr) was accomplished by PCR amplification into an expression vector system, and purification of a recombinant B. abortus Bfr (rBfr). The immunogenicity of rBfr was confirmed by Western blot with Brucella-positive bovine serum. To determine whether rBfr has a potential benefit for use in the serodiagnosis of bovine brucellosis, rBfr-based ELISA was performed. Interestingly, rBfr was able to detect anti-Brucella antibodies in positive sera in a dependent manner of TAT values but did not show an immunoreaction with negative samples. Particularly, average OD492 values at the lowest, medium and highest TAT titer levels were 1.4, 2.2 and 2.6-fold increase compared with the cutoff value, respectively. The accuracy, specificity and sensitivity of rBfr showed 89.09%, 93.6% and 85.33%, respectively. These findings suggest that rBfr might be a good candidate for serological diagnosis development of bovine brucellosis.
Assuntos
Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/imunologia , Brucella abortus/imunologia , Brucelose Bovina/diagnóstico , Grupo dos Citocromos b/imunologia , Ensaio de Imunoadsorção Enzimática/veterinária , Ferritinas/imunologia , Testes Sorológicos/veterinária , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Western Blotting , Brucella abortus/genética , Bovinos , Clonagem Molecular , Reações Cruzadas , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Reações Falso-Positivas , Ferritinas/genética , Ferritinas/isolamento & purificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Yersinia enterocolitica/imunologiaRESUMO
Brucellosis affects a wide range of host species, including humans and many livestock animals. Chronic infections of the disease make antibiotic treatment costly, and the current vaccine used in livestock has not been approved for human use. This study investigated the possible use of the Brucella abortus outer membrane protein A (OmpA) as a candidate subunit vaccine in an infected mouse model. The ompA gene was cloned and overexpressed, and the recombinant OmpA (rOmpA) protein fused to maltose binding protein (MBP) was purified in Escherichia coli. Immunogenicity was verified through western blotting, and mice were immunized and challenged to evaluate its protective effect. Mice treated with rOmpA exhibited induced humoral and host cell-mediated responses, with a significant increase in immunoglobulin G (IgG1 and IgG2a) and cytokine levels, especially TNF-α and IL-12, compared with the control groups treated with either MBP or PBS. In conclusion, rOmpA should be highly considered as a future subunit vaccine for brucellosis, and further studies regarding rOmpA and its protective ability are suggested.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Brucella abortus/imunologia , Brucelose/prevenção & controle , Animais , Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Brucella abortus/genética , Brucelose/imunologia , Brucelose/microbiologia , Feminino , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis.
Assuntos
Brucella abortus/efeitos dos fármacos , Brucelose/prevenção & controle , Panax/química , Polissacarídeos/farmacologia , Animais , Brucelose/microbiologia , Brucelose/veterinária , Macrófagos/efeitos dos fármacos , Camundongos , Fagocitose/efeitos dos fármacos , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacosRESUMO
In this study, the Brucella abortus ohr gene coding for an organic hydroperoxide resistance protein (Ohr) was cloned into a maltose fusion protein expression system (pMAL), inserted into Escherichia coli, and purified, and its immunogenicity was evaluated by western blot analysis using Brucella-positive mouse sera. The purified recombinant Ohr (rOhr) was treated with adjuvant and injected intraperitoneally into BALB/c mice. A protective immune response analysis revealed that rOhr induced a significant increase in both the IgG1 and IgG2a titers, and IgG2a reached a higher level than IgG1 after the second and third immunizations. Additionally, immunization with rOhr induced high production of IFN-γ as well as proinflammatory cytokines such as TNF, MCP-1, IL-12p70, and IL-6, but a lesser amount of IL-10, suggesting that rOhr predominantly elicited a cell-mediated immune response. In addition, immunization with rOhr caused a significantly higher degree of protection against a virulent B. abortus infection compared with a positive control group consisting of mice immunized with maltose-binding protein. These findings showed that B. abortus rOhr was able to induce both humoral and cell-mediated immunity in mice, which suggested that this recombinant protein could be a potential vaccine candidate for animal brucellosis.
Assuntos
Proteínas de Bactérias/imunologia , Brucella abortus/imunologia , Brucelose/prevenção & controle , Animais , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/genética , Brucella abortus/genética , Brucella abortus/patogenicidade , Brucelose/imunologia , Brucelose/microbiologia , Feminino , Humanos , Imunização , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , VirulênciaRESUMO
In this study, we particularly evaluated the protective effect of recombinant protein encoded by Brucella abortus 544 ndk (nucleoside diphosphate kinase) gene against B. abortus infection in the BALB/c mice. Cloning and expression of B. abortus Ndk was accomplished by PCR amplification into a pMAL expression system, and purification of a recombinant Ndk (rNdk). As for the determination of IgG responses, rNdk induced vigorous IgG production, especially higher in IgG2a compared to IgG1 with titers of 5.2 and 4.8, respectively, whereas titers of these in mice immunized with MBP were 2.4 of IgG2a and 2.6 of IgG1. The analysis of cytokine has revealed that rNdk can strongly induce production of IFN-γ as well as proinflammatory cytokines (TNF, MCP1 and IL-6) but not much IL-10, suggesting rNdk elicited predominantly cell-mediated immune responses. Furthermore, the spleen proliferation and bacterial burden in the spleen of rNdk immunized mice were significantly lower than those of MBP-immunized mice against virulent B. abortus challenge (P < 0.01). Conclusionly, rNdk immunization enables to elicit both of the humoral and cellular response, ultimately enhancing protection level in experimental mice, suggesting that rNdk of B. abortus might be a useful candidate for subunit vaccine for brucellosis in animals.
Assuntos
Brucella abortus/genética , Brucella abortus/imunologia , Brucelose/imunologia , Imunização , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/imunologia , Animais , Brucelose/prevenção & controle , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/isolamento & purificação , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Vacinas de Subunidades AntigênicasRESUMO
This study indicated that RGSF-A caused a marked reduction in the adherence, internalization and intracellular growth of Brucella abortus in RGSF-A-treated cells. Furthermore, a decline in the intensity of F-actin fluorescence was observed in RGSF-A-treated cells compared with untreated B. abortus-infected cells. In addition, an evaluation of phagocytic signaling proteins by Western blot analysis revealed an apparent reduction of ERK and p38α phosphorylation levels in B. abortus-infected RGSF-A-treated cells compared with the control. Upon intracellular trafficking of the pathogen, a higher number of B. abortus-containing phagosomes colocalized with LAMP-1 in RGSF-A-treated cells compared with control cells. These results strongly suggest that inhibition of B. abortus uptake could be mediated by suppression in the activation of MAPKs signaling proteins phospho-ERK 1/2, and p38 levels. On the other hand, inhibition of intracellular replication results from the enhancement of phagolysosome fusion in host macrophages. This study highlights the phagocytic and intracellular modulating effect of RGSF-A and its potential as an alternative remedy to control B. abortus infection.
Assuntos
Brucella abortus/efeitos dos fármacos , Brucella abortus/fisiologia , Macrófagos/microbiologia , Panax/química , Fagocitose/efeitos dos fármacos , Saponinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Actinas/fisiologia , Animais , Western Blotting , Brucella abortus/ultraestrutura , Proteínas de Membrana Lisossomal/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Fagocitose/fisiologia , Fagossomos/efeitos dos fármacos , Células RAW 264.7RESUMO
The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.
Assuntos
Betaína-Aldeído Desidrogenase/genética , Betaína-Aldeído Desidrogenase/metabolismo , Brucella abortus/enzimologia , Brucella abortus/patogenicidade , Brucelose/microbiologia , Fatores de Virulência/genética , Animais , Aderência Bacteriana/genética , Vacinas Bacterianas , Brucella abortus/genética , Brucella abortus/imunologia , Brucelose/imunologia , Brucelose/prevenção & controle , Células HeLa , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Pressão Osmótica , Fagócitos/microbiologia , Deleção de Sequência , Baço/microbiologiaRESUMO
In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host-pathogen interaction.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Brucella abortus/imunologia , Brucella abortus/metabolismo , Brucelose/prevenção & controle , Meios de Cultivo Condicionados/metabolismo , Proteoma , Animais , Anticorpos Antibacterianos/imunologia , Formação de Anticorpos/imunologia , Proteínas de Bactérias/química , Linhagem Celular , Meios de Cultivo Condicionados/química , Modelos Animais de Doenças , Feminino , Imunização , Camundongos , ProteômicaRESUMO
Brucellosis is a major zoonotic disease caused by pathogens of the genus Brucella. The eradication of brucellosis in domestic animals, associated with the prevention of human infection, can be attained through accurate diagnosis. However, the conventional serological diagnosis of brucellosis has limitations, particularly in detecting the infection period. Accordingly, the aim of this study was to determine reliable immunogenic proteins to detect Brucella abortus infection according to time course responses to aid in the appropriate management of this disease. Proteomic identification through two-dimensional electrophoresis (2DE), followed by immunoblotting, revealed 13, 24, and 55 immunodominant B. abortus 544 proteins that were reactive to sera from experimentally infected mice at early (10 days), middle (30 days), and late (60 days) infection periods, respectively. After excluding several spots reactive to sera from Yersinia enterocolitica O:9-infected and noninfected mice, 17 of the 67 immunodominant proteins were identified through MALDI-TOF MS. Consequently, the identified proteins showed time course-dependent immunogenicity against Brucella infection. Thus, the results of this study suggest that the production of immunogenic proteins during infection periods improves the diagnosis and discovery of vaccine candidates.