RESUMEN
Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolates recovered from cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a hallmark of chronic infections in CF patients. However, the within-patient genetic diversity of P. aeruginosa populations related to antibiotic resistance remains unexplored. Here, we show the evolution of the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years of chronic infection. Our results show the accumulation of thousands of mutations, with an overall evolutionary history characterized by purifying selection. However, mutations in antibiotic resistance genes appear to have been positively selected, driven by antibiotic treatment. Antibiotic resistance increased as infection progressed toward the establishment of a population constituted by genotypically diversified coexisting sublineages, all of which converged to multidrug resistance. These sublineages emerged by parallel evolution through distinct evolutionary pathways, which affected genes of the same functional categories. Interestingly, ampC and ftsI, encoding the ß-lactamase and penicillin-binding protein 3, respectively, were found to be among the most frequently mutated genes. In fact, both genes were targeted by multiple independent mutational events, which led to a wide diversity of coexisting alleles underlying ß-lactam resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. aeruginosa strategies for persistence.
Asunto(s)
Antibacterianos/farmacología , Fibrosis Quística/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Proteínas Bacterianas/genética , Fibrosis Quística/patología , Humanos , Mutación/genética , Proteínas de Unión a las Penicilinas/genética , Peptidoglicano Glicosiltransferasa/genética , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/aislamiento & purificación , Sistema Respiratorio/microbiología , Resistencia betalactámica/genética , beta-Lactamasas/genéticaRESUMEN
A sequence similar to prokaryotic transposable elements was identified in the long 5' untranslated region (5'UTR) of the butanediol dehydrogenase cDNA isolated from a bovine brain lambda gt11 library. Several observations suggested that this sequence could be related to bacterial IS elements: (a) 58% nucleotide sequence identity, (b) 56% amino acid sequence identity, and (c) the presence of inverted terminal repeats. However, nucleotide sequence analyses of the 5'UTR bovine cDNA showed the presence of chain-terminating nucleotide substitutions that would render it incapable of encoding a functional transposase. Finally, it was observed that different vertebrate genomes have sequences related to this putative transposable element.
Asunto(s)
Regiones no Traducidas 5'/genética , Oxidorreductasas de Alcohol/genética , Bacterias/genética , Elementos Transponibles de ADN , ADN Complementario/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/enzimología , Bovinos , Datos de Secuencia Molecular , Homología de Secuencia de Ácido NucleicoRESUMEN
Using a polyclonal antibody against a bovine brain 30-kDa protein (p30), we isolated from a lambda gt11 bovine brain expression library a cDNA that codifies a protein with an apparent molecular mass of 30 kDa. The cDNA nucleotide sequence contained a unique open reading frame encoding a 26.7 kDa polypeptide. The 257 amino acids deduced sequence showed a significant homology with several dehydrogenases, mainly with a bacterial acetoin reductase (62%). The cloned cDNA identity was confirmed by the determination of acetoin reductase activity in lysogens of lambda phage constructions containing the full length cDNA. The results described in this report are to our knowledge the first molecular characterization of a 2,3-butanediol dehydrogenase in mammals.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Química Encefálica , ADN Complementario/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Escherichia coli/genética , Hígado/química , Datos de Secuencia Molecular , ARN Mensajero/análisis , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
A preparation of tubulin carboxypeptidase partially purified from bovine brain was found to contain a protein of molecular mass 30 kDa (P30) as determined by SDS-PAGE, that is recognized by a polyclonal anti-bovine pancreatic carboxypeptidase A. However, this protein is different from pancreatic carboxypeptidase A as judged by the isoelectric point and the pattern of peptides produced by trypsin digestion. The isoelectric point of P30 was similar to that found for tubulin carboxypeptidase (9 +/- 0.2). When the tubulin carboxypeptidase preparation was subjected to gel filtration chromatography under low salt concentration, P30 behaved as a protein of molecular mass 38 kDa whereas tubulin carboxypeptidase eluted at a position of 75 kDa molecular mass. However, when the chromatography was performed at relatively high salt concentration they behaved as proteins of 49 and 56 kDa, respectively. We considered that P30 may be an inactive monomeric form of the dimeric tubulin carboxypeptidase. However we can not rule out the possibility that it represents another carboxypeptidase not yet described.
Asunto(s)
Encéfalo/enzimología , Carboxipeptidasas/química , Carboxipeptidasas/aislamiento & purificación , Animales , Anticuerpos , Western Blotting , Carboxipeptidasas/metabolismo , Carboxipeptidasas A , Bovinos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , ConejosRESUMEN
It was found that the detyrosination of tyrosinated tubulin by tubulin carboxypeptidase can occur when both the enzyme and the substrate are adsorbed on nitrocellulose. This, and the use of a specific antibody that recognizes detyrosinated tubulin allowed us to localize tubulin carboxypeptidase on a nitrocellulose membrane after agarose gel electrophoresis and blotting. The method was also extended to detect pancreatic carboxypeptidase A.