Identificação e sequenciação da acetilase mediada pelo plasmídeo recombinante pMdT1 em Escherichia coli
Ficheiros
Data
2016-06-03
Título da revista
ISSN da revista
Título do Volume
Editora
Resumo
A resistência a antibióticos é um problema gobal na medicina humana e veterinária. Escherichia coli é um microrganismo comensal do trato gastrointestinal de animais e humanos. Esta pode ser utilizada em diversos estudos biológicos e genéticos sobre a resistência aos antibióticos, um problema emergente de saúde pública. A presença do plasmídeo pMdT1 é de grande relevância no estudo da resistência aos antibióticos, visto que contém um gene que codifica uma variante da proteína AAC(6’)-lb-cr e que esta, quando ativa, confere resistência à tobramicina e à canamicina, e diminui a susceptibilidade à ciprofloxacina e à norfloxacina.
Foram utilizadas duas amostras, Escherichia coli Electromax DH10B, recetora do processo de transformação e Escherichia coli TF-Se20 sendo esta transformada, pois contém o plasmídeo pMdT1 que expressa o gene da acetilase. Após a extração proteica, a separação e quantificação das proteínas foi realizada mediante a eletroforese monodimensional e posteriormente bidimensional, segundo os princípios de O’Farrel, contudo usando a tecnologia de IPG. As amostras foram sujeitas a uma focalização isoelétrica, seguida de SDS-PAGE. A identificação das proteínas foi realizada através da técnica MALDI-TOF/MS. Os picos de massa obtidos pelo motor de busca Mascot foram comparados com a base de dados Uniprot e NCBI. Para determinar a sequência da proteína de interesse, utilizou-se a técnica LC-MS/MS.
Da amostra Escherichia coli TF-Se20 foram excisados 260 spots. Destes, foram identificados 111, e foi possível identificar 76 proteínas distintas, 71 das quais com função conhecida. Foram excisados 225 spots da amostra de Escherichia coli Electromax DH10B, tendo sido identificados 119, dos quais foi possível identificar 72 proteínas diferentes (71 têm processo biológico). As proteínas identificadas participam em diversos processos biológicos como no processo da glicólise, no processo de oxidação-redução e biossíntese de proteínas. A proteína de interesse, aminoglicosidase N(6')-acetiltransferase tipo 1, foi identificada e posteriormente sequenciada. Com um sinal correto, os resultados foram analisados através do Mascot e do Peaks, permitindo a identificação da proteína AAC com seis péptidos. Foi possível a identificação do péptido YSIVTNS(T) DSVTLR, que apresenta uma mutação de substituição, Asn (Aspargina) por uma Thr (Treonina). Para o péptido SHIVEWWGGEEARPTLAD VQE, apenas se obteve o match no Mascot. Como não se confirmou o match no Peaks, a classificação desta identificação foi “possível mas incerta”. Por sua vez, o péptido IA MLNGEPIGYA QSYVALGSGDGR
foi confirmado, devido a ter sido validado várias vezes, quer na sua totalidade, quer de uma só parte. Os péptidos GLGTKLVR, MSNAKTKLGITK e QAFERTRSDA não foram detetados ou sequenciados corretamente. O péptido CYEKAGFE (G) QGTVTTPYG PAVYMVQTR foi validado pelo Mascot, apresentando uma mutação na qual uma Gly (Glicina) substitui uma Arg (Arginina).
Concluiu-se desta forma que a proteómica pode ser usada para obter mais informação sobre os mecanismos de resistência e diferentes processos biológicos.
Microbial resistance to antibiotics is a worldwide problem in human and veterinary medicine. Escherichia coli is a commensal microorganism of the gastrointestinal tract of animals and humans. Already a mainstay of molecular biology, the E. coli model is increasingly used to study the biology and genetics of antibiotic resistance, an emerging public health problem. The presence of the pMdT1 plasmid in E. coli is important in the study of antibiotic resistance as it contains a gene that encodes a variant of the AAC (6')-lb-cr protein which confers resistance to kanamycin and tobramycin, and decreases susceptibility to ciprofloxacin and norfloxacin. Two E. coli samples were analyzed. Electromax DH10B is a transformation-ready strain and TF-SE20 is a strain that contains the pMdT1 plasmid expressing the acetylase gene. After extraction, fractionation and quantification, proteins were separated by one dimensional and after by two-dimensional electrophoresis, following the principles of O'Farrell but using the Immobiline pH gradient (IPG). The samples were subjected to isoelectric focusing, followed by SDS-PAGE. Proteins were identified through matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS). Using the Mascot search engine, data from the mass peaks obtained was searched against the UniProt database. To determine the sequence of the protein of interest, liquid chromatography - mass spectrometry/ mass spectrometry (LC-MS/MS) was performed. From gels containing the Escherichia coli TF-SE20 protein sample, 260 spots were excised. Of these, 111 were identified and it was possible to identify 76 distinct proteins, 71 of which had a known function. From the E. coli Electromax DH10B protein sample, 225 spots were excised, 119 of which were identified. Among this number it was possible identify 72 different proteins, 71 being associated with a biological process. Proteins identified were related to biological processes such as glycolysis, oxidation-reduction processes and bossinthesis of proteins. The protein of interest, aminoglycoside N(6’)-acetyltransferase type 1, was sequenced and identified. With a correct signal, the results were analyzed through the Mascot and Peaks search engines, allowing the identification of a protein AAC with six peptides. It was possible to identify the peptide YSIVTNS (T) DSVTLR, which showed a substitution mutation of asparagine with threonine. The peptide SHIVEWWGGEEARPTLAD VQE had a match with Mascot only. As the match was not confirmed in Peaks, this identification is classified as "possible but uncertain”. The peptide IA MLNGEPIGYA QSYVALGSGDGR was confirmed and validated several times, both as a whole and in one piece. The peptides GLGTKLVR, MSNAKTKLGITK and QAFERTRSDA were not detected or sequenced correctly. The peptide CYEKAGFE (G)QGTVTTPYG PAVYMVQTR was validated by Mascot, presenting a mutation in which glycine replaced arginine. Overall, about 75% of the sequence was covered. Using proteomics methods made it possible to detect the protein expressed by the pMdT1 plasmid in a strain of E. coli. Proteomics was used to obtain more information on resistance mechanisms and different biological processes and how they function.
Microbial resistance to antibiotics is a worldwide problem in human and veterinary medicine. Escherichia coli is a commensal microorganism of the gastrointestinal tract of animals and humans. Already a mainstay of molecular biology, the E. coli model is increasingly used to study the biology and genetics of antibiotic resistance, an emerging public health problem. The presence of the pMdT1 plasmid in E. coli is important in the study of antibiotic resistance as it contains a gene that encodes a variant of the AAC (6')-lb-cr protein which confers resistance to kanamycin and tobramycin, and decreases susceptibility to ciprofloxacin and norfloxacin. Two E. coli samples were analyzed. Electromax DH10B is a transformation-ready strain and TF-SE20 is a strain that contains the pMdT1 plasmid expressing the acetylase gene. After extraction, fractionation and quantification, proteins were separated by one dimensional and after by two-dimensional electrophoresis, following the principles of O'Farrell but using the Immobiline pH gradient (IPG). The samples were subjected to isoelectric focusing, followed by SDS-PAGE. Proteins were identified through matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS). Using the Mascot search engine, data from the mass peaks obtained was searched against the UniProt database. To determine the sequence of the protein of interest, liquid chromatography - mass spectrometry/ mass spectrometry (LC-MS/MS) was performed. From gels containing the Escherichia coli TF-SE20 protein sample, 260 spots were excised. Of these, 111 were identified and it was possible to identify 76 distinct proteins, 71 of which had a known function. From the E. coli Electromax DH10B protein sample, 225 spots were excised, 119 of which were identified. Among this number it was possible identify 72 different proteins, 71 being associated with a biological process. Proteins identified were related to biological processes such as glycolysis, oxidation-reduction processes and bossinthesis of proteins. The protein of interest, aminoglycoside N(6’)-acetyltransferase type 1, was sequenced and identified. With a correct signal, the results were analyzed through the Mascot and Peaks search engines, allowing the identification of a protein AAC with six peptides. It was possible to identify the peptide YSIVTNS (T) DSVTLR, which showed a substitution mutation of asparagine with threonine. The peptide SHIVEWWGGEEARPTLAD VQE had a match with Mascot only. As the match was not confirmed in Peaks, this identification is classified as "possible but uncertain”. The peptide IA MLNGEPIGYA QSYVALGSGDGR was confirmed and validated several times, both as a whole and in one piece. The peptides GLGTKLVR, MSNAKTKLGITK and QAFERTRSDA were not detected or sequenced correctly. The peptide CYEKAGFE (G)QGTVTTPYG PAVYMVQTR was validated by Mascot, presenting a mutation in which glycine replaced arginine. Overall, about 75% of the sequence was covered. Using proteomics methods made it possible to detect the protein expressed by the pMdT1 plasmid in a strain of E. coli. Proteomics was used to obtain more information on resistance mechanisms and different biological processes and how they function.
Descrição
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde
Palavras-chave
Escherichia coli , Resistência a antibióticos