RESUMO
In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.
Assuntos
Isomerases de Aminoácido/metabolismo , Ácido D-Aspártico/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Ilhas Genômicas , Shigella flexneri/genética , Isomerases de Aminoácido/genética , Proteínas de Bactérias/metabolismo , Ácido D-Aspártico/análise , Genes Bacterianos , Manose/metabolismo , Fases de Leitura Aberta/genética , Fenótipo , Shigella flexneri/enzimologia , Shigella flexneri/crescimento & desenvolvimento , Shigella sonnei/genéticaRESUMO
Plasmid-encoded toxin (Pet) and protein involved in colonization (Pic), are serine protease autotransporters of Enterobacteriaceae (SPATEs) secreted by enteroaggregative Escherichia coli (EAEC), which display the GDSGSG sequence or the serine motif. Our research was directed to localize functional sites in both proteins using the phage display method. From a 12mer linear and a 7mer cysteine-constrained (C7C) libraries displayed on the M13 phage pIII protein we selected different mimotopes using IgG purified from sera of children naturally infected with EAEC producing Pet and Pic proteins, and anti-Pet and anti-Pic IgG purified from rabbits immunized with each one of these proteins. Children IgG selected a homologous group of sequences forming the consensus sequence, motif, PQPxK, and the motifs PGxI/LN and CxPDDSSxC were selected by the rabbit anti-Pet and anti-Pic IgGs, respectively. Analysis of the amino terminal region of a panel of SPATEs showed the presence in all of them of sequences matching the PGxI/LN or CxPDDSSxC motifs, and in a three-dimensional model (Modeller 9v2) designed for Pet, both these motifs were found in the globular portion of the protein, close to the protease active site GDSGSG. Antibodies induced in mice by mimotopes carrying the three aforementioned motifs were reactive with Pet, Pic, and with synthetic peptides carrying the immunogenic mimotope sequences TYPGYINHSKA and LLPQPPKLLLP, thus confirming that the peptide moiety of the selected phages induced the antibodies specific for the toxins. The antibodies induced in mice to the PGxI/LN and CxPDDSSxC mimotopes inhibited fodrin proteolysis and macrophage chemotaxis biological activities of Pet. Our results showed that we were able to generate, by a phage display procedure, mimotopes with sequence motifs PGxI/LN and CxPDDSSxC, and to identify them as functional motifs of the Pet, Pic and other SPATEs involved in their biological activities.
Assuntos
Toxinas Bacterianas/química , Enterotoxinas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Epitopos Imunodominantes/química , Fragmentos de Peptídeos/química , Serina Endopeptidases/química , Serina Proteases/química , Shigella flexneri/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Epitopos Imunodominantes/imunologia , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Biblioteca de Peptídeos , Estrutura Secundária de Proteína , Serina Endopeptidases/imunologia , Serina Proteases/imunologiaRESUMO
A 9-year nation-wide survey of the presence of extended-spectrum beta-lactamases (ESBLs) in Shigella flexneri is described. Ten of 9033 (0.1%) isolates produced ESBLs, which were characterized by isoelectric focusing, PCR and DNA sequencing. These were CTX-M-2 (five isolates), TOHO-1 (one isolate), SHV-2 (two isolates) and PER-2 (two isolates, the first report in S. flexneri world wide). The emergence of each ESBL type in S. flexneri was not restricted to a particular region of Argentina. TOHO-1 showed a more basic isoelectric point (8.4) than that previously found (7.8) and its encoding gene (bla(TOHO-1a)) harboured a silent change, G825A, relative to the reported bla(TOHO-1). All the ESBL-encoding genes were transferred to Escherichia coli by conjugation. PFGE analysis indicated that the 10 ESBL-producing S. flexneri isolates were subtypes of a unique clone.
Assuntos
Antibacterianos/farmacologia , Shigella flexneri/efeitos dos fármacos , Shigella flexneri/enzimologia , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamas/farmacologia , Substituição de Aminoácidos , Argentina , Técnicas de Tipagem Bacteriana , Conjugação Genética , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Disenteria Bacilar/microbiologia , Eletroforese em Gel de Campo Pulsado , Transferência Genética Horizontal , Humanos , Focalização Isoelétrica , Epidemiologia Molecular , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Shigella flexneri/isolamento & purificação , beta-Lactamases/isolamento & purificaçãoRESUMO
The peptide containing the catalytic serine of beta-lactamase from Shigella flexneri was determined as V-D-E-R-F-P-M-M-S*-T-F-K. It is a local pathogenic strain which produces intestinal problems, especially in children. The highly purified enzyme was prepared by affinity chromatography in phenylboronic acid-agarose gels. The peptide was obtained by tryptic hydrolysis, with further purification by Bio-Gel P-4, Sephadex QAE-25 and Sephadex SP-25. The relevance of the serine, lysine and arginine residues was mainly shown by the loss of enzymatic activity after specific chemical modifications. Finally, this enzyme was classified as A, according to the similarity of this peptide with that of class A beta-lactamases such as R-TEM 1 and 2.
Assuntos
Shigella flexneri/enzimologia , beta-Lactamases/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Criança , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Shigella flexneri/genética , Shigella flexneri/patogenicidade , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
A beta-lactamase (EC 3.5.2.6 penicillinase, penicillin amino beta-lactam-hydrolase) was purified from Shigella flexneri USCF-129 by an efficient two-stage procedure involving chromatography in Sephadex G-75 and HPLC on a C18-reverse phase column. The homogeneity of the purified enzyme was confirmed by capillary zone electrophoresis (CZE), HPLC electrospray mass spectrometry (LC-ESMS) and amino acid sequence analyses. The highly purified enzyme was a monomeric protein with a molecular mass of 28.903 +/- 2 Da, as determined by LC-ESMS. The amino acid sequence of the first 49 N-terminal residues of this beta-lactamase revealed 100% similarity with the mature forms of the plasmid coded Escherichia coli enzymes (plasmid pBR 322 and R6K) a TEM-type beta-lactamase.
Assuntos
Shigella flexneri/enzimologia , beta-Lactamases/química , Sequência de Aminoácidos , Aminoácidos/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
A probe was constructed by radioactive labelling, and enzymatically a DNA fragment of plasmid pMAM-1, which codes for a beta-lactamase in Shigella flexneri UCSM 129, was obtained by amplification of a small part of the gene using the polymerase chain reaction technique (PCR). Since previous published work indicated that this beta-lactamase was of the TEM type, the primers used to amplify the gene were two highly conserved DNA regions in all TEM beta-lactamases. A 500 bp DNA probe was obtained which, by hybridization assays, facilitated the identification of restriction fragments of the plasmid containing the beta-lactamase gene. Two DNA fragments were sequenced by the Sanger method adapted to the PCR technique, and the sequence obtained showed a 100% homology with beta-lactamases TEM-1, TEM-2, TEM-13 and TEM-19. An intragenic restriction site, detected for Pst I, suggested that there is only one copy of the beta-lactamase gene per plasmid copy.
Assuntos
DNA Bacteriano/genética , Genes Bacterianos , Shigella flexneri/enzimologia , beta-Lactamases/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Sondas de DNA , Eletroforese em Gel de Poliacrilamida , Dosagem de Genes , Immunoblotting , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência , Homologia de Sequência de Aminoácidos , Shigella flexneri/genética , beta-Lactamases/químicaRESUMO
The role of a residue of arginine at the active site of beta-lactamase from Shigella flexneri UCSF-129 was studied. It is a local pathogenic strain which produces intestinal problems, especially in children. Purified enzymes were obtained by affinity chromatography on phenylboronic acid-agarose gels. The enzyme was serine dependent with a molecular weight of 23.6 kD. It was specifically modified with phenylglyoxal (1/830 molar ratio) and incubated for 20 min in the presence of 50 mM sodium phosphate buffer at pH 8.3. The chemical change was established by isoelectric focusing, since a loss of one positive charge was detected. Protection by cephradine, a substrate, indicated the presence of a residue of arginine at its active site. Controls were conducted by differential spectroscopy. Similar results were obtained with 2,3,butanedione. This vital arginine stabilizes the negative charge of the carboxylic group of C-3 or C-4 from the substrates.
Assuntos
Arginina/química , Shigella flexneri/enzimologia , beta-Lactamases/química , Cefradina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Fenilglioxal/farmacologia , Shigella flexneri/classificação , beta-Lactamases/efeitos dos fármacos , beta-Lactamases/isolamento & purificaçãoRESUMO
The effect of a powerful inhibitor, BRL 42715, on beta-lactamase from Shigella flexneri UCSF-129, to overcome the problem of shigellosis and its resistance to ampicillin, was studied. The I50 was determined for BRL 42715 [C6-(N1-methyl-1,2,3 triazolylmethylene)penem] as 0.0049 microgram/ml being 20-fold lower than the best inhibitor, 6-beta-iodopenicillanic acid, previously reported. The MIC fell from 2,048 to 2 micrograms/ml in the presence of 1 microgram/ml of BRL 42715. The synergism of the ampicillin plus this inhibitor lasted for 9 h. BRL 42715 is an irreversible inhibitor, according to the dialysis results, and a substrate analogue.
Assuntos
Antibacterianos/farmacologia , Lactamas , Shigella flexneri/enzimologia , Inibidores de beta-Lactamases , beta-Lactamas , Resistência a Ampicilina , Testes de Sensibilidade Microbiana , beta-Lactamases/metabolismoRESUMO
The resistance to beta-lactam antibiotics shown by a strain of Shigella flexneri was plasmid-coded. This plasmid, pMAM-1, when transferred to Escherichia coli K-12 by conjugation, presented the same molecular weight (100 kbp) and conferred the same high level of resistance to ampicillin in the transconjugant as in the wild type strains (MIC, 2048-4096). Restriction analysis of the plasmid in transconjugants revealed various restrictive sites to some endonucleases (i.e. Bam HI, Eco RI, Pst I, Nco I, Cla I, Sf I and Sau 3AI, Nhe and Hin dIII), and no restrictive sites at all for other endonucleases (such as Xho I, Dra I, Kpn I, and Sal I). Some restricted DNA fragments were appropriate for cloning and isolation of the beta-lactamase gene present in Shigella flexneri UCSF 129. This work provides the first step in this direction.
Assuntos
Resistência a Ampicilina/genética , Resistência ao Cloranfenicol/genética , Plasmídeos/genética , Shigella flexneri/enzimologia , beta-Lactamases/genética , Plasmídeos/química , beta-Lactamases/biossínteseRESUMO
The effect of five irreversible inhibitors on purified enzyme from Shigella flexneri USCF-129 and also in vitro, was investigated. The I50 was 0.091, 0.30, 0.40 and 0.87 microgram/ml for 6 beta-iodopenicillanic, clavulanic, olivanic and 6 beta-bromopenicillanic acids, respectively, and 255 micrograms/ml for sulbactam. The synergic responses of these inhibitors (10 micrograms/ml) were studied with ampicillin and cephradin on S. flexneri. The MIC of ampicillin was reduced by 128 times using 6 beta-iodopenicillanic, 6 beta-bromopenicillanic and clavulanic acids. This indicates that the beta-lactamase of S. flexneri is a penicillinase. Since these inhibitors have a strong action against the beta-lactamase and last for at least 6 h, further work is expected to reveal their medical value.