RESUMO
Developing an effective Staphylococcus aureus (S. aureus) vaccine has been a challenging endeavor, as demonstrated by numerous failed clinical trials over the years. In this study, we formulated a vaccine containing a highly conserved moonlighting protein, the pyruvate dehydrogenase complex E2 subunit (PDHC), and showed that it induced strong protective immunity against epidemiologically relevant staphylococcal strains in various murine disease models. While antibody responses contributed to bacterial control, they were not essential for protective immunity in the bloodstream infection model. Conversely, vaccine-induced systemic immunity relied on γδ T cells. It has been suggested that prior S. aureus exposure may contribute to the reduction of vaccine efficacy. However, PDHC-induced protective immunity still facilitated bacterial clearance in mice previously exposed to S. aureus. Collectively, our findings indicate that PDHC is a promising serotype-independent vaccine candidate effective against both methicillin-sensitive and methicillin-resistant S. aureus isolates.
Assuntos
Infecções Estafilocócicas , Vacinas Antiestafilocócicas , Staphylococcus aureus , Animais , Infecções Estafilocócicas/prevenção & controle , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Camundongos , Staphylococcus aureus/imunologia , Staphylococcus aureus/enzimologia , Vacinas Antiestafilocócicas/imunologia , Complexo Piruvato Desidrogenase/metabolismo , Complexo Piruvato Desidrogenase/imunologia , Feminino , Anticorpos Antibacterianos/imunologia , Modelos Animais de Doenças , Humanos , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Camundongos Endogâmicos C57BL , Staphylococcus aureus Resistente à Meticilina/imunologia , Piruvato Desidrogenase (Lipoamida)/imunologia , Piruvato Desidrogenase (Lipoamida)/metabolismo , Piruvato Desidrogenase (Lipoamida)/genéticaRESUMO
Autoantibodies have been implicated in schizophrenia aetiology. Here, novel autoantibodies were isolated from patients with schizophrenia. Autoantibody candidates were searched using two-dimensional gel electrophoresis and western blotting with rat brain proteins as antigens and two sera pools (25 schizophrenia patients versus 25 controls) as antibodies. Immunoreactive antigens were identified by mass spectrometry. Antibody prevalence were evaluated by western blotting using human recombinant proteins. Furthermore, brain magnetic resonance imaging data (regional brain volumes and diffusion tensor imaging measures) were compared. Two proteins of the mitochondrial respiration pathway were identified as candidate antigens. Three patients with schizophrenia, but no controls, expressed antibodies targeting one of the candidate antigens, i.e., pyruvate dehydrogenase E1 component subunit alpha, somatic form, mitochondrial (PDHA1, EC 1.2.4.1), which is related to mitochondrial energy production. Anti-PDHA1 antibody-positive patients (n = 3) had increased volumes in the left occipital fusiform gyrus compared to both controls (n = 23, p = 0.017) and antibody-negative patients (n = 16, p = 0.009), as well as in the left cuneus compared to antibody-negative patients (n = 16, p = 0.018). This is the first report of an anti-PDHA1 antibody in patients with schizophrenia. Compatible with recent findings of mitochondrial dysfunction in schizophrenia, this antibody may be involved in the pathogenesis of a specific subgroup of schizophrenia.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Suscetibilidade a Doenças , Piruvato Desidrogenase (Lipoamida)/imunologia , Esquizofrenia/etiologia , Adulto , Animais , Autoanticorpos/sangue , Biomarcadores , Encéfalo/diagnóstico por imagem , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Estudos de Casos e Controles , Imagem de Tensor de Difusão , Suscetibilidade a Doenças/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteômica/métodos , Piruvato Desidrogenase (Lipoamida)/antagonistas & inibidores , Ratos , Esquizofrenia/diagnóstico por imagem , Esquizofrenia/metabolismo , Adulto JovemRESUMO
Streptococcus iniae is a major Gram-positive pathogen that causes invasive disease in fish worldwide. In this study, in order to identify immunogenic proteins for developing highly effective vaccine against S. iniae, whole-cell lysate proteins of S. iniae were analyzed by western blotting using flounder anti-S. iniae antibodies, and two positive protein bands of molecular weight 37 kDa and 40 kDa were screened, which were identified as pyruvate dehydrogenase E1 subunit alpha (PDHA1), BMP family ABC transporter substrate-binding protein (BMP) and L-lactate dehydrogenase (LDH), as well as ornithine carbamoyltransferase (OCT), lactate oxidas (LOx) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by mass spectrometry. Subsequently, the six recombinant proteins were produced and used to immunize healthy flounder, and the relative percent survival (RPS) value was 72.73%, 27.27%, 36.36%, 9.09%, 36.36% and 63.64% respectively after intraperitoneal challenge with live S. iniae, revealing that rPDHA1 and rGAPDH produced higher relative percent survival than formalin-killed S. iniae (36.36%). To further investigate the protective efficacy of rPDHA1 and rGAPDH, the proliferation of surface membrane immunoglobulin-positive (sIg+) lymphocytes in peripheral blood leucocytes, the total serum IgM, specific IgM against S. iniae and RPS were detected. The results showed that rPDHA1, rGAPDH and formalin-killed S. iniae significantly induced the proliferation of sIg+ lymphocytes, the production of total serum IgM and specific IgM as compared with the control group, and rGAPDH and rPDHA1 provide higher RPS (62.5% and 75%, respectively) again. These results demonstrated that rPDHA1 and rGAPDH are promising vaccine candidates against S. iniae infection in flounder.
Assuntos
Doenças dos Peixes/imunologia , Linguado/imunologia , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Piruvato Desidrogenase (Lipoamida)/imunologia , Proteínas Recombinantes/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus iniae/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Doenças dos Peixes/prevenção & controle , Linguado/microbiologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/administração & dosagem , Vacinas Estreptocócicas/imunologia , VacinaçãoRESUMO
The identification of environmental factors that lead to loss of tolerance has been coined the holy grail of autoimmunity. Our work has focused on the reactivity of antimitochondrial autoantibodies (AMA) to chemical xenobiotics and has hypothesized that a modified peptide within PDC-E2, the major mitochondrial autoantigen, will have been immunologically recognized at the time of loss of tolerance. Herein, we successfully applied intein technology to construct a PDC-E2 protein fragment containing amino acid residues 177-314 of PDC-E2 by joining a recombinant peptide spanning residues 177-252 (PDC-228) with a 62-residue synthetic peptide from 253 to 314 (PP), which encompasses PDC-E2 inner lipoyl domain (ILD). We named this intein-constructed fragment PPL. Importantly, PPL, as well as lipoic acid conjugated PPL (LA-PPL) and xenobiotic 2-octynoic acid conjugated PPL (2OA-PPL), are recognized by AMA. Of great importance, AMA has specificity for the 2OA-modified PDC-E2 ILD peptide backbone distinct from antibodies that react with native lipoylated PDC-E2 peptide. Interestingly, this unique AMA subfraction is of the immunoglobulin M isotype and more dominant in early-stage primary biliary cholangitis (PBC), suggesting that exposure to 2OA-PPL-like compounds occurs early in the generation of AMA. To understand the structural basis of this differential recognition, we analyzed PPL, LA-PPL, and 2OA-PPL using electron paramagnetic resonance spectroscopy, with confirmations by enzyme-linked immunosorbent assay, immunoblotting, and affinity antibody analysis. We demonstrate that the conformation of PDC-E2 ILD is altered when conjugated with 2OA, compared to conjugation with lipoic acid. CONCLUSION: A molecular understanding of the conformation of xenobiotic-modified PDC-E2 is critical for understanding xenobiotic modification and loss of tolerance in PBC with widespread implications for a role of environmental chemicals in the induction of autoimmunity. (Hepatology 2017;65:1670-1682).
Assuntos
Autoanticorpos/sangue , Colangite/induzido quimicamente , Mitocôndrias/imunologia , Piruvato Desidrogenase (Lipoamida)/efeitos dos fármacos , Xenobióticos/toxicidade , Afinidade de Anticorpos , Estudos de Casos e Controles , Colangite/sangue , Colangite/imunologia , Espectroscopia de Ressonância de Spin Eletrônica , Ensaio de Imunoadsorção Enzimática , Humanos , Inteínas , Piruvato Desidrogenase (Lipoamida)/química , Piruvato Desidrogenase (Lipoamida)/imunologiaRESUMO
Mycoplasma hyorhinis is commonly found in the respiratory tract of pigs and is the etiological agent of polyserositis. The metabolic enzymes of M. hyorhinis may play important roles in host-pathogen interactions. We immunized BALB/c mice with sodium deoxycholate-extracted antigens (DOC-Ags) and screened 10 hybridomas that secreted antibodies against various M. hyorhinis proteins. Pyruvate dehydrogenase E1 complex subunit alpha (PDHA) was identified as the protein that reacted with five of the 10 monoclonal antibodies (mAbs). Sequence analysis indicated that PDHA was highly conserved among M. hyorhinis strains, but not among other mycoplasmas. We predicted the three-dimensional structure of PDHA and identified three epitopes ((277)RTEEEEK(283), (299)KDKKYITDE(307), and (350)LKEQKQHAKDY(360)). The mAb 1H12 we generated was used to detect M. hyorhinis PDHA in vitro and in piglets infected with M. hyorhinis. We observed that PDHA was mainly located in the epithelial cells of the lungs. Our results indicate that the mAbs we generated could be used to further investigate the structure and function of M. hyorhinis PDHA. In addition, they could be used in the differential diagnosis of M. hyorhinis and other mycoplasmas.
Assuntos
Anticorpos Monoclonais/análise , Proteínas de Bactérias/imunologia , Infecções por Mycoplasma/veterinária , Mycoplasma hyorhinis/isolamento & purificação , Piruvato Desidrogenase (Lipoamida)/imunologia , Doenças dos Suínos/microbiologia , Animais , Anticorpos Monoclonais/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Mapeamento de Epitopos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Mycoplasma/diagnóstico , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/microbiologia , Mycoplasma hyorhinis/enzimologia , Mycoplasma hyorhinis/genética , Mycoplasma hyorhinis/imunologia , Piruvato Desidrogenase (Lipoamida)/química , Piruvato Desidrogenase (Lipoamida)/genética , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/imunologiaRESUMO
Primary biliary cirrhosis is an immune-mediated chronic liver disease whose diagnosis relies on the detection of serum antimitochondrial antibodies directed against a complex set of proteins, among which pyruvate dehydrogenase complex is considered the main autoantigen. We studied the immunological role of the lipoyl domain of this protein using synthetic lipoylated peptides, showing that the lipoyl chain chirality does not affect autoantibody recognition and, most importantly, confirming that both lipoylated and unlipoylated peptides are able to recognize specific autoantibodies in patients sera. In fact, 74% of patients sera recognize at least one of the tested peptides but very few positive sera recognized exclusively the lipoylated peptide, suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope. These results are supported by a conformational analysis showing that the lipoyl moiety of pyruvate dehydrogenase complex appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex antigenic epitope. A preliminary analysis of the specificity of the two most active peptides indicates that they could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex positivity pattern detected in immunofluorescence.
Assuntos
Epitopos Imunodominantes/imunologia , Cirrose Hepática Biliar/diagnóstico , Peptídeos , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/imunologia , Antígenos/química , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/enzimologia , Conformação Molecular , Peptídeos/síntese química , Peptídeos/química , Piruvato Desidrogenase (Lipoamida)/sangue , Piruvato Desidrogenase (Lipoamida)/química , Piruvato Desidrogenase (Lipoamida)/imunologia , Complexo Piruvato Desidrogenase/sangue , Relação Estrutura-AtividadeRESUMO
The pathogen Mycoplasma bovis (M. bovis) is a major cause of respiratory disease, mastitis, and arthritis in cattle. Screening the key immunogenic proteins and updating rapid diagnostic techniques are necessary to the prevention and control of M. bovis infection. In this study, 19 highly immunogenic proteins from M. bovis strain PD were identified using 2-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF MS. Of these 19 proteins, pyruvate dehydrogenase E1 component beta subunit (PDHB) showed excellent immune reactivity and repeatability. PDHB was found to be conserved in different M. bovis isolates, as indicated by Western blot analysis. On the basis of these results, a rPDHB-based indirect ELISA (iELISA) was established for the detection of serum antibodies using prokaryotically expressed recombinant PDHB protein as the coating antigen. The specificity analysis result showed that rPDHB-based iELISA did not react with other pathogens assessed in our study except M. agalactiae (which infects sheep and goats). Moreover, 358 serum samples from several disease-affected cattle feedlots were tested using this iELISA system and a commercial kit, which gave positive rates of 50.8% and 39.9%, respectively. The estimated Kappa agreement coefficient between the two methods was 0.783. Notably, 39 positive serum samples that had been missed by the commercial kit were all found to be positive by Western blot analysis. The detection rate of rPDHB-based iELISA was significantly higher than that of the commercial kit at a serum dilution ratio of 1â¶5120 to 1â¶10,240 (P<0.05). Taken together, these results provide important information regarding the novel immunogenic proteins of M. bovis. The established rPDHB-based iELISA may be suitable for use as a new method of antibody detection in M. bovis.
Assuntos
Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Mycoplasma bovis/imunologia , Piruvato Desidrogenase (Lipoamida)/imunologia , Proteínas Recombinantes/imunologia , Animais , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/química , Bovinos , Biologia Computacional , Eletroforese em Gel Bidimensional , Immunoblotting , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/microbiologia , Mycoplasma bovis/isolamento & purificação , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (AMA). Autoantibodies specific for the mitochondrial M4 antigen can be detected by a complement fixation test (CFT) but not by immunoblotting. The aim of this study was to elucidate the identity of the M4 antigen. PATIENTS AND METHODS: M4 proteins were purified by affinity chromatography using IgG fractions of PBC marker sera being CFT positive (n=5) or negative (n=5) and identified by Western blotting, silver staining and sequence analysis. Further, a cohort of 57 PBC patients was tested for the reactivity to M4 and pyruvate dehydrogenase complex (PDC). RESULTS: Two AMA patterns of the marker sera were visualized: CFT-positive sera were defined as PDC-E2(+)/E1(+) and the CFT-negative sera as PDC-E2(+)/E1(-). The major proteins in the M4 fraction could be related to the PDC-E1 subunits. A clear-cut association between anti-M4 reactivity in the CFT and the reactivity to both PDC subunits could also be documented in the cohort of 57 PBC patients showing anti-PDC-E1alpha and E1beta antibodies at a frequency of 74% and 67%. CONCLUSIONS: CFT reactivity against M4 antigens could be preferentially identified as a reaction against PDC-E1. As PDC-E1 subunits as compared with PDC-E2 lack lipoyl-binding sites, they probably have to be considered as an independent and important target.
Assuntos
Autoantígenos/imunologia , Cirrose Hepática Biliar/imunologia , Piruvato Desidrogenase (Lipoamida)/imunologia , Idoso , Idoso de 80 Anos ou mais , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Estudos de Coortes , Testes de Fixação de Complemento , Feminino , Humanos , Epitopos Imunodominantes/imunologia , Cirrose Hepática Biliar/sangue , Masculino , Pessoa de Meia-Idade , Mitocôndrias/imunologia , Complexo Piruvato Desidrogenase/imunologia , RatosRESUMO
Unravelling the molecular basis of capacitation is crucial to our understanding the basis of acquisition of fertilization competence by spermatozoa. In two recent studies, we have demonstrated that dihydrolipoamide dehydrogenase, which is a post-pyruvate metabolic enzyme and one of the components of pyruvate dehydrogenase complex, undergoes capacitation-dependent tyrosine phosphorylation, and that the activity of the enzyme correlates with capacitation events in the hamster spermatozoa. However, it is not clear as to whether other components of the pyruvate dehydrogenase complex are also crucial for sperm capacitation. In this report, we have identified pyruvate dehydrogenase A2 (PDHA2), a constituent of pyruvate dehydrogenase A (PDHA), which is a component of pyruvate dehydrogenase complex that exhibits tyrosine phosphorylation during hamster spermatozoal capacitation. This is the first report showing that hamster sperm PDHA2 is a testis-specific phosphotyrosine that is associated with the fibrous sheath of hamster spermatozoa. The localization of PDHA2 in spermatozoa was investigated using antibodies to PDHA, which is the active tetrameric protein that consists of a homodimer of PDHA2 and PDHB. Both immunofluorescence and confocal studies indicated a unique non-canonical, extramitochondrial localization for PDHA in the principal piece of hamster spermatozoa. It was also observed that PDHA colocalized with AKAP4 in the fibrous sheath of the spermatozoon. The enzymatic activity of PDHA was positively correlated with hyperactivation but not with the acrosome reaction. Given the localization of PDHA and the evidence that its activity correlates positively with hyperactivation and that its PDHA2 subunit exhibits capacitation-associated protein tyrosine phosphorylation, it appears that PDHA2 is associated with the process of capacitation.
Assuntos
Proteínas Tirosina Quinases/metabolismo , Piruvato Desidrogenase (Lipoamida)/metabolismo , Capacitação Espermática/fisiologia , Espermatozoides/enzimologia , Animais , Anticorpos , Bovinos , Cricetinae , DNA Complementar , Expressão Gênica , Isoenzimas/química , Masculino , Mesocricetus , Microscopia Confocal , Fosforilação , Piruvato Desidrogenase (Lipoamida)/química , Piruvato Desidrogenase (Lipoamida)/imunologia , Coelhos , Análise de Sequência de DNA , Espermatozoides/metabolismo , Testículo/enzimologiaRESUMO
BACKGROUND: A major mitochondrial autoantigen recognized by sera of patients with primary biliary cirrhosis (PBC) is dihydrolipoamide acetyltransferase (E2) of the pyruvate dehydrogenase complex (PDH). The alpha subunit of pyruvate decarboxylase (E1alpha) of PDH is also recognized in some E2-reactive PBC sera, suggesting that the occurrence of autoimmunity against Elalpha is subsequent to that against E2. METHODS. To investigate the mechanism inducing autoimmunity against E1alpha, we surveyed immunoreactive sequences of E1alpha by ELISA with synthesized oligopeptides, and determined minimum amino acid residues for each determinant. RESULTS: The major determinants of E1alpha appeared to reside in its N-terminal region, apparently forming 'nested epitopes', and all E1alpha-reactive PBC sera tested recognized these regions. Minor epitopes were also found scattered throughout the entire sequence. The reactivities of these minor epitopes to individual PBC sera were proportional to those of the major epitopes. All the epitopes were located in hydrophilic regions of E1alpha, and many of them were out of the known functional domains (TPP-binding domain, subunit interaction site, and phosphorylation sites) whose structures are phylogenically well conserved. Furthermore, the sequences of many epitopes appeared to be specific to humans. CONCLUSION: These observations suggest that determinant spreading might underlie the autoimmunity against Elalpha.
Assuntos
Autoanticorpos/farmacologia , Cirrose Hepática Biliar/imunologia , Piruvato Desidrogenase (Lipoamida)/genética , Piruvato Desidrogenase (Lipoamida)/imunologia , Sequência de Aminoácidos , Sequência Conservada , Mapeamento de Epitopos , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Dados de Sequência MolecularRESUMO
Deficiency of the E1 alpha-subunit of the pyruvate dehydrogenase (PDH) complex is an X-linked inborn error of metabolism and one of the major causes of lactic acidosis in children. Although most heterozygous females manifest symptoms of the disease, it is often difficult to establish the diagnosis as results based on measurement of total PDH activity, and E1 alpha-immunoreactive protein in patient fibroblasts may be ambiguous because of the variability in the pattern of X chromosome inactivation. We report the development of a set of monoclonal antibodies (MAbs) specific to four subunits of the PDH complex that can be used for detection of PDH E1 alpha deficiency. We also show that anti-E1 alpha and anti-E2 MAbs, when used in immunocytochemical analysis, can detect mosaicism in cell cultures from female patients in which as few as 2-5% of cells express the deficiency. This immunocytochemical approach, which is fast, reliable, and quantitative, will be particularly useful in identifying females with PDH E1 alpha-subunit deficiency as a precursor to mutation analysis.
Assuntos
Piruvato Desidrogenase (Lipoamida)/metabolismo , Doença da Deficiência do Complexo de Piruvato Desidrogenase/enzimologia , Anticorpos Monoclonais , Especificidade de Anticorpos , Western Blotting , Linhagem Celular , Feminino , Fibroblastos/enzimologia , Humanos , Imuno-Histoquímica , Masculino , Mosaicismo , Subunidades Proteicas , Piruvato Desidrogenase (Lipoamida)/genética , Piruvato Desidrogenase (Lipoamida)/imunologia , Doença da Deficiência do Complexo de Piruvato Desidrogenase/diagnóstico , Reprodutibilidade dos TestesRESUMO
Genetic defects in pyruvate dehydrogenase complex (PDC) cause lactic acidosis, neurological deficits, and often early death. Most mutations of PDC are localized in the alpha subunit of the pyruvate dehydrogenase (E1) component. We have kinetically characterized a patient's missense mutation alphaH44R in E1alpha by creating and purifying three recombinant human E1s (alphaH44R, alphaH44Q, and alphaH44A). Substitutions at histidine-15 resulted in decreased V(max) values (6% alphaH44R; 30% alphaH44Q; 90% alphaH44A) while increasing K(m) values for thiamine pyrophosphate (TPP) compared to wild-type (alphaH44R, 3-fold; alphaH44Q, 7-fold; alphaH44A, 10-fold). This suggests that the volume of the residue at site 15 is important for TPP binding and substitution by a residue with a longer side chain disrupts the active site more than the TPP binding site. The rates of phosphorylation and dephosphorylation of alphaH44R E1 by E1-kinase and phospho-E1 phosphatase, respectively, were similar to that of the wild-type E1 protein. These results provide a biochemical basis for altered E1 function in the alphaH44R E1 patient.