RESUMO
Here, we characterize two novel GH5 endoglucanases (GH5CelA and GH5CelB) from an uncultured bacterium identified in termite gut microbiomes. Both genes were codon-optimized, synthetized, cloned, and expressed as recombinant proteins in Escherichia coli for subsequent purification. Both enzymes showed activity on the pNPC and barley ß-glucan substrates, whereas GH5CelB also showed low activity on carboxymethyl cellulose. The optimum conditions for both enzymes were an acid pH (5) and moderate temperature (35 to 50 °C). The enzymes differed in the kinetic profiles and patterns of the generated hydrolysis products. A structural-based modeling analysis indicated that both enzymes possess a typical (ß/α)8-barrel fold characteristic of GH5 family, with some differential features in the active site cleft. Also, GH5CelB presents a putative secondary binding site. Furthermore, adjacent to the active site of GH5CelA and GH5CelB, a whole subdomain rarely found in GH5 family may participate in substrate binding and thermal stability.Therefore, GH5CelA may be a good candidate for the production of cello-oligosaccharides of different degrees of polymerization applicable for feed and food industries, including prebiotics. On the other hand, GH5CelB could be useful in an enzymatic cocktail for the production of lignocellulosic bioethanol, because of the production of glucose as a hydrolysis product. Key Points ⢠Synthetic metagenomics is a powerful approach for discovering novel enzymes. ⢠Two novel GH5 endoglucanases from nonculturable microorganisms were characterized. ⢠Structural differences between them and other GH5 endoglucanases were observed. ⢠The enzymes may be good candidates for feed, food, and/or bioethanol industries.
Assuntos
Celulase , Isópteros , Microbiota , Animais , Celulase/genética , Celulase/metabolismo , Hidrólise , Metagenômica , Especificidade por SubstratoRESUMO
Programs for the eradication of bovine tuberculosis (bTB) focus on the tuberculin skin test (TST) and slaughter of reactor cattle. However, the disease remains an animal health concern in several countries and improving the efficiency of the TST has become a critical issue. The detection of Mycobacterium bovis antibodies in serum, within weeks after the TST, may be a rapid and inexpensive way to improve bTB control. This study reports the validation of an enzyme-linked immunosorbent assay (ELISA) to detect bovine tuberculosis as an ancillary test to TST in dairy farms in Argentina. The estimated validation parameters were within the established requirements of the World Organization for Animal Health (OIE). The test demonstrated high repeatability, with coefficients of variation <25%. High test reproducibility through interlaboratory testing was also found, with an estimated Pearson coefficient of 0.9648 (95% confidence intervals 0.9315-0.9820). The ELISA detected tuberculous cattle unidentified by the TST. Of 43 animals sent to slaughterhouses that were ELISA positive 15-17 days after a negative TST, 36 were confirmed as infected with M. bovis by histopathology and IS6110 PCR. According to ROC curve analysis of results of 145 cattle from M. bovis-free herds and the 36 M. bovis-infected cattle, at a corrected optical density cut-off point of 0.3853, specificity was 95.95% and the positive predictive value at this cut-off was 83.72%. The ELISA detection test validated in this study could be readily applied in dairy farms, to complement a prior TST and improve livestock health.
Assuntos
Ensaio de Imunoadsorção Enzimática/veterinária , Teste Tuberculínico/veterinária , Tuberculose Bovina/diagnóstico , Animais , Argentina , Bovinos , Indústria de Laticínios/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Reações Falso-Negativas , Feminino , Mycobacterium bovis/imunologia , Reprodutibilidade dos Testes , Teste Tuberculínico/métodosRESUMO
Mycobacterium avium sp. avium (MAA), M. avium sp. hominissuis (MAH), and M. avium sp. paratuberculosis (MAP) are the main members of the M. avium complex (MAC) causing diseases in several hosts. The aim of this study was to describe the genetic diversity of MAC isolated from different hosts. Twenty-six MAH and 61 MAP isolates were recovered from humans and cattle, respectively. GenoType CM® and IS1311-PCR were used to identify Mycobacterium species. The IS901-PCR was used to differentiate between MAH and MAA, while IS900-PCR was used to identify MAP. Genotyping was performed using a mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) scheme (loci: 292, X3, 25, 47, 3, 7, 10, 32) and patterns (INMV) were assigned according to the MAC-INMV database (http://mac-inmv.tours.inra.fr/). Twenty-two (22/26, 84·6%) MAH isolates were genotyped and 16 were grouped into the following, INMV 92, INMV 121, INMV 97, INMV 103, INMV 50, and INMV 40. The loci X3 and 25 showed the largest diversity (D: 0·5844), and the global discriminatory index (Hunter and Gaston discriminatory index, HGDI) was 0·9300. MAP (100%) isolates were grouped into INMV 1, INMV 2, INMV 11, INMV 8, and INMV 5. The HGDI was 0·6984 and loci 292 and 7 had the largest D (0·6980 and 0·5050). MAH presented a higher D when compared with MAP. The MIRU-VNTR was a useful tool to describe the genetic diversity of both MAH and MAP as well as to identify six new MAH patterns that were conveniently reported to the MAC-INMV database. It was also demonstrated that, in the geographical region studied, human MAC cases were produced by MAH as there was no MAA found among the human clinical samples.
Assuntos
Variação Genética , Genótipo , Complexo Mycobacterium avium/genética , Infecção por Mycobacterium avium-intracellulare/veterinária , Paratuberculose/epidemiologia , Tuberculose Bovina/epidemiologia , Animais , Argentina/epidemiologia , Bovinos , Humanos , Infecção por Mycobacterium avium-intracellulare/epidemiologia , Infecção por Mycobacterium avium-intracellulare/microbiologia , Paratuberculose/microbiologia , Filogenia , Reação em Cadeia da Polimerase/veterinária , Análise de Sequência de DNA/veterinária , Tuberculose Bovina/microbiologiaRESUMO
DNA damage induces apoptosis and many apoptotic genes are regulated via alternative splicing (AS), but little is known about the control mechanisms. Here we show that ultraviolet irradiation (UV) affects cotranscriptional AS in a p53-independent way, through the hyperphosphorylation of RNA polymerase II carboxy-terminal domain (CTD) and a subsequent inhibition of transcriptional elongation, estimated in vivo and in real time. Phosphomimetic CTD mutants not only display lower elongation but also duplicate the UV effect on AS. Consistently, nonphosphorylatable mutants prevent the UV effect. Apoptosis promoted by UV in cells lacking p53 is prevented when the change in AS of the apoptotic gene bcl-x is reverted, confirming the relevance of this mechanism. Splicing-sensitive microarrays revealed a significant overlap of the subsets of genes that have changed AS with UV and those that have reduced expression, suggesting that transcriptional coupling to AS is a key feature of the DNA-damage response.
Assuntos
Processamento Alternativo/efeitos da radiação , RNA Polimerase II/metabolismo , Raios Ultravioleta , Apoptose , Linhagem Celular Tumoral , Dano ao DNA , Diclororribofuranosilbenzimidazol/farmacologia , Fibronectinas/genética , Fibronectinas/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Humanos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , RNA Polimerase II/química , Transcrição GênicaRESUMO
Babesia bovis is the causative agent of babesiosis, a tick-borne disease that is a major cause of loss to livestock production in Latin America. Vaccination against Babesia species represents a major challenge against cattle morbidity and mortality in enzootic areas. The aim of this study was to evaluate the capacity of Bacille Calmette-Guerin (BCG) to deliver the rhoptry associated protein (RAP-1) antigen of B. bovis and to stimulate specific cellular and humoral immune responses in mice. Two of five mycobacterial expression vectors efficiently expressed the antigen. These constructs were subsequently studied in vivo following three immunization protocols. The construct with the greatest in vivo stability proved to be the one that induced the strongest immune responses. Our data support the hypothesis that specific T lymphocyte priming by rBCG can be employed as a component of a combined vaccine strategy to induce long-lasting humoral and cellular immune responsiveness towards B. bovis and encourage further work on the application of rBCG to the development of Babesia vaccines.
Assuntos
Babesia bovis/genética , Vacinas Bacterianas/genética , Técnicas de Transferência de Genes , Mycobacterium bovis/genética , Proteínas de Protozoários/biossíntese , Animais , Babesia bovis/imunologia , Vacinas Bacterianas/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium bovis/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Vacinas Combinadas/genética , Vacinas Combinadas/imunologiaRESUMO
A Mycobacterium avium subsp. paratuberculosis expression library in lambda ZAP was screened with immunized mice sera. One clone was selected, sequenced and further characterized. The sequence analysis of the hypothetical open-reading frame (ORF) predicts a protein of 20.8 kDa with a probable signal sequence compatible with Cys-acylation at Cys24, characteristic of lipoproteins. In consequence, the protein was termed Lpp34. Recombinant expression of Lpp34 was achieved by cloning the lpp34 gene into the histidine-tag expression vector pRSET-A. Western blot analysis showed a protein band with a molecular weight of 34 kDa. The native protein was localized in the membrane fraction of M. avium subsp. paratuberculosis and extracted in the detergent phase of Triton X-114. Southern blot and polymerase chain reaction showed that the gene is absent from all the non-M. avium complex mycobacterial genomes tested. Humoral reactivity using bovine sera demonstrated that this protein is widely recognized by both the infected and non-infected animals. This could partly be due to the conserved sequence in close-related environmental bacteria such as M. avium subsp. avium and to the presence of a conserved epitope in other bacteria such as Escherichia coli. In conclusion, these findings show that Lpp34 is a membrane protein and a putative lipoprotein present in M. avium complex mycobacteria and absent in the M. tuberculosis complex.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Lipoproteínas/isolamento & purificação , Mycobacterium avium subsp. paratuberculosis/imunologia , Mycobacterium avium subsp. paratuberculosis/isolamento & purificação , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/imunologia , Sequência de Bases , Western Blotting/veterinária , Bovinos , Clonagem Molecular , DNA Bacteriano/análise , Lipoproteínas/classificação , Lipoproteínas/imunologia , Dados de Sequência Molecular , Peso Molecular , Mycobacterium avium subsp. paratuberculosis/classificação , Fases de Leitura Aberta , Reação em Cadeia da Polimerase/veterinária , Proteínas Recombinantes/classificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNA , Análise de Sequência de ProteínaRESUMO
P36 is a member of a family of secreted proteins distributed throughout the genus Mycobacterium. The central domain of these proteins contains several amino acid PGLTS repeats, which differ considerably between species. P36, also called exported repetitive protein (Erp) in M. tuberculosis, has been shown to be associated with virulence since the disruption of its gene impaired multiplication of both virulent M. tuberculosis and M. bovis BCG in cultured macrophages and immunocompetent mice. In order to demonstrate that P36 is a putative virulence factor of wild-type Mycobacterium bovis we generated a P36 mutant by gene disruption and we evaluated its replication in spleen and lungs of infected mice. In this study, the mutant strain displays low levels of multiplication in mice, indicating that the P36 gene is important for in vivo growth of M. bovis.