Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The gene encoding Mhp1, a 124 kDa protein from Mycoplasma hyopneumoniae, has been cloned, sequenced, and its product characterized. No significant homology to the gene or encoded polypeptide was found in the Genbank, NBRF, or PIR databases, though this protein appears similar to p97, a putative adhesin of M. hyopneumoniae described by Zhang et al. (Infect. Immun. 63, 1013-1019, 1995). Two repeated motifs were identified within the 3' end of the gene and encoded polypeptide. The mhp1 gene was fused to the glutathione S-transferase (GST) gene from Schistosoma japonicum, enabling high-level expression and purification of the protein. Both the authentic and recombinant proteins were recognized by sera from pigs infected with M. hyopneumoniae. In an induced-disease model in pigs, coughing was reduced in animals vaccinated with recombinant GST-Mhp1, although differences were not significant. Only minimal protection against lung lesion formation was provided, and again differences between the Mhpl-vaccinated and nonvaccinated groups were not significant.
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PMID:Characterization of the gene encoding Mhp1 from Mycoplasma hyopneumoniae and examination of Mhp1's vaccine potential. 904 63

A hypothetical ORF of Mycoplasma gallisepticum with a putative 99-amino-acid product (ORF99) was noted previously in the upstream region from the type II topoisomerase gene. The amino acid sequence shows weak homology with the Escherichia coli histone-like protein HU. To identify and characterize the protein product of ORF99, we prepared mouse antiserum against recombinant GST-ORF99 fusion protein. The antiserum reacted with an 11-kDa peptide in the crude cell extract of M. gallisepticum, indicating that this protein is an ORF99 product. ORF99 protein binds to DNA, although its binding affinity is weaker than that of E. coli HU. When ORF99 was cloned in a plasmid and expressed in E. coli cells depleted of HU, Mu phage growth was strongly promoted in the cells, showing the presence of HU activity. The effect of IHF mutation was suppressed when a high level of ORF99 protein was expressed in an E. coli mutant deficient in IHF.
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PMID:Identification and characterization of HU protein from Mycoplasma gallisepticum. 970 29

The 36kDa L-lactate dehydrogenase (LDH) and a 29kDa partial fragment of an ABC transporter ATP-binding protein analogue/multidrug resistance protein homologue (PR2) of Mycoplasma hyopneumoniae were tested for their potential as diagnostic antigens. Recombinant LDH was genetically engineered to contain six histidine residues at its C-terminal end, expressed in Escherichia coli and purified to a high degree using Ni(2+)-chelate affinity chromatography. A partial 262 amino acid segment representing the C-terminal end of the PR2 protein was cloned as a glutathione S-transferase (GST) fusion protein, expressed in E. coli and purified by urea extraction. Purified recombinant LDH-6xHis and PR2-GST were then reacted with pig sera in immunoblot assays. Our immunoblots showed that both proteins detected anti-M. hyopneumoniae antibodies in field and experimentally infected pig sera but not in any of the SPF control sera. The two proteins were specific for M. hyopneumoniae as they did not react with sera of pigs infected with the closely related Mycoplasma flocculare and Mycoplasma hyorhinis which are frequently isolated in pigs but are not of particular concern.
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PMID:Immunoblot assays using recombinant antigens for the detection of Mycoplasma hyopneumoniae antibodies. 1086 56

The p36 protein of Mycoplasma hyopneumoniae is a cytosolic protein carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the p36-encoding gene (948 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species and pathogenic bacteria that commonly colonize the porcine respiratory tract. The amplified p36 gene was subcloned into the pGEX-4T-1 vector to be expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The GST-p36 recombinant fusion protein was purified by affinity chromatography and cut by thrombin, and the enriched p36 protein was used to immunize female BALB/c mice for the production of anti-p36 monoclonal antibodies (MAbs). The polypeptide specificity of the nine MAbs obtained was confirmed by Western immunoblotting with cell lysates prepared from the homologous strain. Cross-reactivity studies of the anti-p36 MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture suggested that these anti-p36 MAbs were directed against a highly conserved epitope, or closely located epitopes, of the p36 protein. No reactivity was demonstrated against other mycoplasma species tested. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs infected experimentally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. The bacteria could be recovered from lung homogenates of pigs that were killed after the 3-week observation period by both PCR and cultivation procedures. Furthermore, the anti-p36 MAbs permitted effective detection by indirect immunofluorescence of M. hyopneumoniae in frozen lung sections from experimentally infected pigs. However, attempts to use the recombinant p36 protein as an antigen in an indirect enzyme-linked immunosorbent assay for the detection of antibodies in sera from convalescent pigs showed no correlation with clinical and pathological findings.
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PMID:Species-specific monoclonal antibodies to Escherichia coli-expressed p36 cytosolic protein of Mycoplasma hyopneumoniae. 1088 46

The P46 and P65 proteins of Mycoplasma hyopneumoniae are two membranous proteins carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the genes encoding entire P46 (1,260 bp) and P65 (1,803 bp) and N-terminally truncated P65(c) (1,200 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species that commonly colonize the porcine respiratory tract. Both amplified genes were then cloned into the pGEX-4T-1 vector to be expressed in Escherichia coli cells as recombinant fusion proteins with glutathione S-transferase (GST). Prior to generation of expression constructs, TGA nonsense codons, exceptionally used for tryptophan residues by M. hyopneumoniae, had been converted to TGG codons by PCR-directed mutagenesis. Following induction by IPTG (isopropyl-beta-D-thiogalactopyranoside), both GST-P46 and GST-P65(c) recombinant fusion proteins were recovered by disrupting transformed cells by sonication, purified by affinity chromatography, and then cut with thrombin to release the P46 and P65(c) moieties. The enriched E. coli-expressed P46 and P65c proteins were used to immunize female BALB/c mice for the generation of anti-P46 and anti-P65(c) monoclonal antibodies (MAbs). The polypeptide specificities of MAbs obtained was confirmed by Western blotting with cell lysates prepared from the homologous strain. Cross-reactivity study of the anti-P46 and anti-P65(c) MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture, suggested that the MAbs obtained against both membranous proteins were directed against highly conserved species-specific epitopes. No reactivity to other mycoplasma species tested was demonstrated. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs that had been inoculated intratracheally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. Both anti-P46 and anti-P65(c) MAbs permitted effective detection by indirect immunofluorescence and indirect immunoperoxidase assay of M. hyopneumoniae in, respectively, frozen and formalin-fixed, paraffin-embedded lung sections from pigs that were killed after the 6- to 7-week observation period.
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PMID:Monoclonal antibodies to Escherichia coli-expressed P46 and P65 membranous proteins for specific immunodetection of Mycoplasma hyopneumoniae in lungs of infected pigs. 1273 49

Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine. p65 is an immunodominant surface lipoprotein of M. hyopneumoniae that is specifically recognized during disease. Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes. To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons. After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein. The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP). Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39 degrees C. Calcium ions did not increase the activity of recombinant GST-p65. Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M. hyopneumoniae in vitro. Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC. The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases. This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma.
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PMID:Mycoplasma hyopneumoniae p65 surface lipoprotein is a lipolytic enzyme with a preference for shorter-chain fatty acids. 1531 84

Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized, species-specific M. hyopneumoniae antigens. As a first step to solve these problems membrane and membrane-associated proteins were enriched from M. hyopneumoniae cells by Triton X-114 fractionation and further analyzed by 2D gel electrophoresis and Western blot analyses using convalescent sera. Two previously unknown immunogenic proteins were identified by quadrupole time-of-flight mass spectrometry and database analyses as the conserved putative lipoproteins, Mhp378 and Mhp651. Both proteins were expressed as recombinant GST fusion proteins and reacted with sera from convalescent pigs. Coated as solid-phase antigen, Mhp651 showed a distinct cross-reaction only with Mycoplasma flocculare specific rabbit hyperimmune serum, whereas Mhp378 was only recognized by the positive control serum directed against M. hyopneumoniae, thereby indicating its species specificity.
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PMID:Identification and immunological characterization of conserved Mycoplasma hyopneumoniae lipoproteins Mhp378 and Mhp651. 1665 Sep 45

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, a highly contagious and often fatal disease. We have previously reported the construction and characterization of a single gene apxIIC deletion mutant HB04C(-) based on A. pleuropneumoniae serovar 7 which produces ApxII toxin and ApxIV. A precisely defined DeltaapxIICDeltaapxIVA double-deletion mutant of A. pleuropneumoniae was constructed based on HB04C(-) by transconjugation and counterselection, and the levels of virulence of the DeltaapxIIC single mutant and DeltaapxIICDeltaapxIVA double mutant were compared in an experimental infection in mice and pigs. The results demonstrated that the DeltaapxIICDeltaapxIVA double mutant strain was less virulent than HB04C(-). Despite attenuation of virulence, the DeltaapxIICDeltaapxIVA double mutant remains immunogenic and conferred a similar level of protective immunity to pigs against challenge with a lethal dose of a heterologous fully virulent standard serovar 1 strain of A. pleuropneumoniae. The results of the virulence study suggest that ApxIV is a critical virulence factor of A. pleuropneumoniae serovar 7 and is able to induce clinical disease, but it not required for efficient vaccination of pigs against A. pleuropneumoniae infection. Two weeks after the booster immunization, animals vaccinated with HB04C(-) were positive in the ApxIVAM-ELISA based on a recombinant GST-fusion protein GST-ApxIVAM as the solid-phase antigen while animals vaccinated with the DeltaapxIICDeltaapxIVA double mutant were negative. These data demonstrate that the double mutant DeltaapxIICDeltaapxIVA can be used as an effective live marker vaccine allowing serological differentiation between vaccinated and infected animals.
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PMID:Potential use an Actinobacillus pleuropneumoniae double mutant strain DeltaapxIICDeltaapxIVA as live vaccine that allows serological differentiation between vaccinated and infected animals. 1776 80

Actinobacillus suis is an important opportunistic pathogen of swine that can cause disease in pigs of all ages, especially in high-health status herds. Although A. suis shares many virulence factors in common with Actinobacillus pleuropneumoniae and can cause a haemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae, A. suis most often causes septicaemia and diseases such as arthritis and meningitis that are sequelae to septicaemia. In a recent signature-tagged transposon mutagenesis study, 30 colonization-essential genes of A. suis were identified. In the current study, the attachment and invasion patterns of strains harboring Tn10 insertions in ompA, pfhaB1, lcbB, and cpxR were evaluated using porcine palatine tonsil organ cultures, the swine kidney epithelial cell line, SK6, and a porcine brain microvascular endothelial cell line, PBMEC/C1-2. All of these mutants attached in lower numbers than wild type to the tonsillar explants and to the SK6 cells. The ompA mutant attached in significantly lower numbers than wild type to the porcine tonsil cells (P=0.02) and to PBMEC (P=0.0008) at 60 min time point. As well, the ompA mutant showed significantly greater sensitivity than wild type to chemical stressors and to swine serum. Using fluorescent microscopy, a GST-OmpA fusion protein could be demonstrated to interact with the crypt epithelial cells of porcine palatine tonsil.
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PMID:Characterization of colonization-deficient mutants of Actinobacillus suis. 1966 89

Contagious Bovine Pleuropneumonia caused by Mycoplasma mycoides subsp. mycoides small colony type is a respiratory disease of considerable economic importance in sub-Saharan Africa; control of the disease in Africa is hampered by diagnostic tests which are suited for herd-level but not for individual animal diagnostics. In the work presented we identified 22 potential immunogenic antigens of the Kenyan outbreak strain B237 by using phage display technology. We determined the relative strength of immunogenicity, the discriminatory capacity between bovine positive and negative sera, and the cross-reactivity with rabbit hyperimmune sera directed against 15 different mycoplasmal species. The three best-performing antigens, a conserved hypothetical protein (MSC_0636), a glycosyl transferase (MSC_0108), and an acyl carrier protein phosphodiesterase (MSC_0029) were considered candidate diagnostic proteins. They were expressed as GST-fusion proteins in Escherichia coli, purified, and used in an ELISA as solid phase antigens. The diagnostic potential of the recombinant antigens was tested using the sera of ten experimentally infected animals and six control animals. This prototype test resulted in 100% diagnostic sensitivity and specificity. In comparison, the complement fixation test and the competitive ELISA performed with a diagnostic sensitivity of 70% and 60%, respectively.
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PMID:Phage display-based identification and potential diagnostic application of novel antigens from Mycoplasma mycoides subsp. mycoides small colony type. 1990 Jul 69


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