RESUMO
Caseous lymphadenitis (CLA) is a chronic disease responsible for significant economic losses in sheep and goat breeding worldwide. The treatment for this disease is not effective, and an intense vaccination schedule would be the best control strategy. In this study, we evaluated the associations of rCP09720 or rCP01850 proteins from Corynebacterium pseudotuberculosis with recombinant exotoxin phospholipase D (rPLD) as subunit vaccines in mice. Four experimental groups (10 animals each) were immunized with a sterile 0.9% saline solution (G1), rPLD (G2), rPLDâ¯+â¯rCP09720 (G3), and rPLDâ¯+â¯rCP01850 (G4). The mice received two doses of each vaccine at a 21-day interval and were challenged 21â¯days after the last immunization. The animals were evaluated daily for 40â¯days after the challenge, and mortality rate was recorded. The total IgG production level increased significantly in the experimental groups on day 42 after the first vaccination. Similarly, higher levels of specific IgG2a were observed in experimental groups G2, G3, and G4 compared to the IgG1 levels on day 42. G4 showed a significant (pâ¯<â¯.05) humoral response against both antigens of the antigenic formulations. The cellular immune response induced by immunization was characterized by a significant (pâ¯<â¯.05) production of interferon-γ compared to that in the control, while the concentrations of interleukin (IL)-4 and IL-12 were not significant in any group. A significant increase of tumor necrosis factor was observed only in G4. The survival rates after the challenge were 30% (rPLD), 40% (rPLDâ¯+â¯rCP09720), and 50% (rPLDâ¯+â¯rCP01850). Thus, the association of rCP01850 with rPLD resulted in the best protection against the challenge with C. pseudotuberculosis and induced a more intense type 1 T-helper cell immune response.
Assuntos
Vacinas Bacterianas/imunologia , Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/veterinária , Fosfolipase D/imunologia , Proteínas Recombinantes/imunologia , Fosfatase Ácida/administração & dosagem , Fosfatase Ácida/genética , Fosfatase Ácida/imunologia , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/química , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/genética , Esterases/administração & dosagem , Esterases/genética , Esterases/imunologia , Cabras/microbiologia , Imunidade Celular , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interferon gama/imunologia , Linfadenite/imunologia , Linfadenite/microbiologia , Linfadenite/prevenção & controle , Camundongos , Fosfolipase D/administração & dosagem , Fosfolipase D/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Ovinos/microbiologia , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Doenças dos Ovinos/prevenção & controle , Células Th1/imunologia , Vacinação/veterinária , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
PURPOSE: We tested the efficacy of the esterase encoded by cp1002_RS09720 from Corynebacteriumpseudotuberculosis in recombinant subunit and DNA caseous lymphadenitis (CLA) vaccines. This target was predicted as one of the best CLA vaccine candidates by mature epitope density analysis. METHODOLOGY: Gene cp1002_RS09720 was cloned into two different vectors (pAE for subunit vaccine and pTARGET for DNA vaccine). Four groups of 15 mice each were immunized with the recombinant esterase rCP09720 associated with aluminium hydroxide adjuvant (G1), pTARGET/cp09720 DNA vaccine (G2), a naked pTARGET (G3) or PBS as a negative control (G4). Immunization occurred in two doses intercalated by a 21 day interval. Twenty-one days after the last dose administration, animals were challenged with a virulent C. pseudotuberculosis MIC-6 strain. RESULTS: G1 showed high levels of IgG1 and IgG2a on days 21 and 42 post-immunization and a significant level of IFN-γ (P<0.05), suggesting a Th1 response. The protection levels obtained were 58.3 and 16.6â% for G1 and G2, respectively. CONCLUSION: The subunit vaccine composed of the recombinant esterase rCP09720 and Al(OH)3 is a promising antigenic formulation for use against CLA.
Assuntos
Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/imunologia , Esterases/genética , Linfadenite/prevenção & controle , Vacinas de DNA/imunologia , Adjuvantes Imunológicos , Animais , Infecções por Corynebacterium/imunologia , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/patogenicidade , Citocinas/metabolismo , Esterases/administração & dosagem , Esterases/imunologia , Imunoglobulina G/sangue , Interferon gama/imunologia , Linfadenite/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/imunologia , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificaçãoRESUMO
Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from Loxosceles intermedia and Corynebacterium pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. J. Cell. Biochem. 118:2053-2063, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas de Artrópodes/toxicidade , Proteínas de Bactérias/toxicidade , Permeabilidade Capilar/efeitos dos fármacos , Corynebacterium pseudotuberculosis/química , Diester Fosfórico Hidrolases/toxicidade , Aranhas/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/patogenicidade , Eritrócitos/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hemólise/efeitos dos fármacos , Cavalos , Humanos , Camundongos , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Carneiro Doméstico , Pele/efeitos dos fármacos , Pele/patologia , Aranhas/enzimologia , Aranhas/patogenicidadeRESUMO
The gram-positive bacteria Corynebacterium pseudotuberculosis, the causative agent of caseous lymphadenitis in livestock significantly reduces productivity and often causes death. The adenine/guanine-specific DNA glycosylase (MutY) prevents mutations in the DNA of the pathogen and a unique feature of the MutY protein family is the [4Fe-4S]2+ cluster that interlinks two protein subdomains. MutY from C. pseudotuberculosis was expressed in E. coli and purified, the CD experiments indicate a high content of α-helices and random coiled secondary structure and a typical near-UV CD fingerprint for the [4Fe-4S]2+ cluster. EDTA and copper sulfate possess a strong destabilizing effect on the [4Fe-4S]2+ cluster. UV-vis and fluorescence spectroscopy results demonstrate that between pH3.0 and 4.0 the integrity of the [4Fe-4S]2+ cluster is destroyed. To investigate the thermal stability of the protein differential scanning calorimetry and fluorescence spectroscopy were used and the Tm was determined to be 45°C. The analysis presented provides information concerning the protein stability under different physio-chemical conditions.
Assuntos
Proteínas de Bactérias/química , Corynebacterium pseudotuberculosis/enzimologia , DNA Glicosilases/química , Proteínas Ferro-Enxofre/química , Proteínas de Bactérias/genética , Dicroísmo Circular , Sulfato de Cobre/química , Corynebacterium pseudotuberculosis/genética , DNA Glicosilases/genética , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Proteínas Ferro-Enxofre/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Despite the economic importance of caseous lymphadenitis (CLA), a chronic disease caused by Corynebacterium pseudotuberculosis, few genes related to the virulence of its etiologic agent have been characterized. The oligopeptide permease (Opp) transporters are located in the plasma membrane and have functions generally related to the uptake of peptides from the extracellular environment. These peptide transporters, in addition to having an important role in cell nutrition, also participate in the regulation of various processes involving intercellular signaling, including the control of the expression of virulence genes in pathogenic bacteria. To study the role of Opp in C. pseudotuberculosis, an OppD deficient strain was constructed via simple crossover with a nonreplicative plasmid carrying part of the oppD gene sequence. As occurred to the wild-type, the ΔoppD strain showed impaired growth when exposed to the toxic glutathione peptide (GSH), indicating two possible scenarios: (i) that this component can be internalized by the bacterium through an Opp-independent pathway or (ii) that there is toxicity while the peptide is extracellular. Additionally, the ΔoppD mutant presented a reduced ability to adhere to and infect macrophages compared to the wild-type, although both strains exhibit the same potential to colonize spleens and cause injury and death to infected mice.
Assuntos
Proteínas de Bactérias/genética , Transporte Biológico/genética , Corynebacterium pseudotuberculosis/genética , Linfadenite/genética , Proteínas de Membrana Transportadoras/genética , Animais , Proteínas de Bactérias/metabolismo , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/patogenicidade , Humanos , Linfadenite/microbiologia , Linfadenite/patologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Mutação , Óperon/genéticaRESUMO
Sphingomyelinases D (SMases D) or dermonecrotic toxins are well characterized in Loxosceles spider venoms and have been described in some strains of pathogenic microorganisms, such as Corynebacterium sp. After spider bites, the SMase D molecules cause skin necrosis and occasional severe systemic manifestations, such as acute renal failure. In this paper, we identified new SMase D amino acid sequences from various organisms belonging to 24 distinct genera, of which, 19 are new. These SMases D share a conserved active site and a C-terminal motif. We suggest that the C-terminal tail is responsible for stabilizing the entire internal structure of the SMase D Tim barrel and that it can be considered an SMase D hallmark in combination with the amino acid residues from the active site. Most of these enzyme sequences were discovered from fungi and the SMase D activity was experimentally confirmed in the fungus Aspergillus flavus. Because most of these novel SMases D are from organisms that are endowed with pathogenic properties similar to those evoked by these enzymes alone, they might be associated with their pathogenic mechanisms.
Assuntos
Corynebacterium pseudotuberculosis/enzimologia , Fungos/enzimologia , Ixodes/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Aranhas/enzimologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biocatálise , Domínio Catalítico , Corynebacterium pseudotuberculosis/classificação , Corynebacterium pseudotuberculosis/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/classificação , Fungos/genética , Ixodes/classificação , Ixodes/genética , Modelos Moleculares , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Esfingomielinas/química , Esfingomielinas/metabolismo , Aranhas/classificação , Aranhas/genéticaRESUMO
Corynebacterium pseudotuberculosis é de grande importância veterinária e econômica por causar linfadenite caseosa em diversos rebanhos, principalmente em ovelhas e cabras. Apesar de existir em todo o mundo, sua ocorr?ncia é maior em países como a Australia, Brasil e Canadá, o que provoca perdas significativas devido ao elevado custo no tratamento, bem como a inexistência de testes de diagnósticos específicos. Neste trabalho, nós apresentamos o genoma de Corynebacterium pseudotuberculosis linhagem 267 isolado de Lhama
Assuntos
Animais , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/genética , Corynebacterium pseudotuberculosis/químicaRESUMO
Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).
Assuntos
Animais , Bovinos , Coelhos , Infecções por Corynebacterium , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/isolamento & purificação , Fosfolipase D/genética , Fosfolipase D/isolamento & purificação , Regulação da Expressão Gênica , Técnicas In Vitro , Linfadenite , RNA , Búfalos , Eletroforese , Ativação Enzimática , Métodos , Coelhos , OvinosRESUMO
Metalloendopeptidases are zinc-dependent hydrolases enzymes with many different roles in biological systems, ranging from remodeling conjunctive tissue to removing signaling sequences from nascent proteins. Here, we describe the three-dimensional structure of the metalloendopeptidase from Corynebacterium pseudotuberculosis generated by homology modeling and molecular dynamics. Analysis of key distances shows that His-132, Asp-136, His-211, Leu-212 and one molecule of water play an important role in the protein-Zn(2+) ion interaction. The model obtained may provide structural insights into this enzyme and can be useful for the design of new caseous lymphadenitis vaccines based on genetic attenuation from key point mutation.
Assuntos
Corynebacterium pseudotuberculosis/enzimologia , Metaloendopeptidases/química , Sequência de Aminoácidos , Metaloendopeptidases/isolamento & purificação , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Caseous lymphadenitis, caused by Corynebacterium pseudotuberculosis, has a high prevalence in many regions of the world, including Argentina and Brazil. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the identification of this microorganism was designed based on the hypervariable region of the polymorphic RNA polymerase ß-subunit gene (rpoB). All available CorynebacteriumrpoB sequences were analyzed by computer-assisted restriction analysis. The rpoB PCR-RFLP pattern predicted by using endonucleases MseI and StuI clearly differentiated C. pseudotuberculosis from sixty-one other Corynebacterium species. This method was successfully applied to identify twelve wild C. pseudotuberculosis ovine isolates and one caprine isolate. It was also used to differentiate C. pseudotuberculosis from Arcanobacterium pyogenes, an ovine pathogen with similar clinical characteristics. These results indicate that this new molecular method can be used for the reliable identification of the pathogen, essential for the timely detection of infected animals and for epidemiological studies.
Assuntos
Infecções por Corynebacterium/veterinária , Corynebacterium pseudotuberculosis/genética , RNA Polimerases Dirigidas por DNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Doenças dos Ovinos/microbiologia , Animais , Infecções por Corynebacterium/diagnóstico , Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/enzimologia , Polimorfismo de Fragmento de Restrição/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência/veterinária , Ovinos/microbiologia , Doenças dos Ovinos/diagnósticoRESUMO
Bites by Loxosceles spiders can induce severe clinical symptoms, including dermonecrosis, thrombosis, vascular leakage, haemolysis and persistent inflammation. The causative toxin is a sphingomyelinase D (SMase D) that cleaves sphingomyelin into choline and ceramide-1-phosphate. A similar enzyme, showing comparable bioactivity, is secreted by certain pathogenic corynebacteria and acts as a potent virulence factor. We have previously found that SMase D toxins led to an increased susceptibility of human erythrocytes (E) to activation of complement (C) via the classical pathway (CP) in the absence of antibodies. In the present study we have investigated the CP initiating components involved in the haemolysis induced by SMases from Corynebacterium pseudotuberculosis (PLD) and from Loxosceles intermedia venom (P1). When P1 or PLD treated E were incubated with C8-depleted human serum, an increase in C1q, serum amyloid protein (SAP) and C-reactive protein (CRP) binding was observed. While purified C1q, SAP and CRP were found to bind to P1 or PLD treated E, depletion of SAP or CRP from human serum did not prevent C-mediated lysis, suggesting that pentraxins are not involved in the initiation of C-activation. However depletion of C1 lead to a greatly reduced haemolysis, demonstrating that the activation of the CP is caused by direct binding of C1q to the SMase treated cells. Binding of fluid phase C-regulators C4b-binding protein and factor H was also observed, however these C-regulators in conjunction with the membrane bound C-regulators were unable to prevent haemolysis, demonstrating the potency of SMase D facilitated binding of C1 and activation of C.