RESUMO
Carbapenems are "last resort" ß-lactam antibiotics used to treat serious and life-threatening health care-associated infections caused by multidrug-resistant Gram-negative bacteria. Unfortunately, the worldwide spread of genes coding for carbapenemases among these bacteria is threatening these life-saving drugs. Metallo-ß-lactamases (MßLs) are the largest family of carbapenemases. These are Zn(II)-dependent hydrolases that are active against almost all ß-lactam antibiotics. Their catalytic mechanism and the features driving substrate specificity have been matter of intense debate. The active sites of MßLs are flanked by two loops, one of which, loop L3, was shown to adopt different conformations upon substrate or inhibitor binding, and thus are expected to play a role in substrate recognition. However, the sequence heterogeneity observed in this loop in different MßLs has limited the generalizations about its role. Here, we report the engineering of different loops within the scaffold of the clinically relevant carbapenemase NDM-1. We found that the loop sequence dictates its conformation in the unbound form of the enzyme, eliciting different degrees of active-site exposure. However, these structural changes have a minor impact on the substrate profile. Instead, we report that the loop conformation determines the protonation rate of key reaction intermediates accumulated during the hydrolysis of different ß-lactams in all MßLs. This study demonstrates the existence of a direct link between the conformation of this loop and the mechanistic features of the enzyme, bringing to light an unexplored function of active-site loops on MßLs.
Assuntos
Antibacterianos/química , Ceftazidima/química , Imipenem/química , Meropeném/química , Zinco/química , beta-Lactamases/química , Sequência de Aminoácidos , Antibacterianos/metabolismo , Domínio Catalítico , Cefepima/química , Cefepima/metabolismo , Cefotaxima/química , Cefotaxima/metabolismo , Ceftazidima/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Imipenem/metabolismo , Cinética , Meropeném/metabolismo , Modelos Moleculares , Piperacilina/química , Piperacilina/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Engenharia de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Zinco/metabolismo , Resistência beta-Lactâmica , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-ß-lactamases (MßLs) are the main mechanism of resistance against carbapenems. Clinically approved inhibitors of MBLs are currently unavailable as design has been limited by the incomplete knowledge of their mechanism. Here, we report a biochemical and biophysical study of carbapenem hydrolysis by the B1 enzymes NDM-1 and BcII in the bi-Zn(II) form, the mono-Zn(II) B2 Sfh-I and the mono-Zn(II) B3 GOB-18. These MßLs hydrolyse carbapenems via a similar mechanism, with accumulation of the same anionic intermediates. We characterize the Michaelis complex formed by mono-Zn(II) enzymes, and we identify all intermediate species, enabling us to propose a chemical mechanism for mono and binuclear MßLs. This common mechanism open avenues for rationally designed inhibitors of all MßLs, notwithstanding the profound differences between these enzymes' active site structure, ß-lactam specificity and metal content.Carbapenem-resistant bacteria pose a major health threat by expressing metallo-ß-lactamases (MßLs), enzymes able to hydrolyse these life-saving drugs. Here the authors use biophysical and computational methods and show that different MßLs share the same reaction mechanism, suggesting new strategies for drug design.
Assuntos
Carbapenêmicos/metabolismo , Zinco/metabolismo , beta-Lactamases/química , beta-Lactamases/metabolismo , Carbapenêmicos/química , Hidrólise , Imipenem/química , Imipenem/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Espectroscopia por Absorção de Raios XRESUMO
The number and type of outer membrane (OM) channels responsible for carbapenem uptake in Acinetobacter are still not well defined. Here, we addressed these questions by using Acinetobacter baylyi as a model species and a combination of methodologies aimed to characterize OM channels in their original membrane environment. Kinetic and competition analyses of imipenem (IPM) uptake by A. baylyi whole cells allowed us to identify different carbapenem-specific OM uptake sites. Comparative analyses of IPM uptake by A. baylyi wild-type (WT) cells and ΔcarO mutants lacking CarO indicated that this OM protein provided a carbapenem uptake site displaying saturable kinetics and common binding sites for basic amino acids compatible with a specific channel. The kinetic analysis uncovered another carbapenem-specific channel displaying a somewhat lower affinity for IPM than that of CarO and, in addition, common binding sites for basic amino acids as determined by competition studies. The use of A. baylyi gene deletion mutants lacking OM proteins proposed to function in carbapenem uptake in Acinetobacter baumannii indicated that CarO and OprD/OccAB1 mutants displayed low but consistent reductions in susceptibility to different carbapenems, including IPM, meropenem, and ertapenem. These two mutants also showed impaired growth on l-Arg but not on other carbon sources, further supporting a role of CarO and OprD/OccAB1 in basic amino acid and carbapenem uptake. A multiple-carbapenem-channel scenario may provide clues to our understanding of the contribution of OM channel loss or mutation to the carbapenem-resistant phenotype evolved by pathogenic members of the Acinetobacter genus.
Assuntos
Acinetobacter/metabolismo , Aminoácidos Básicos/metabolismo , Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Imipenem/metabolismo , Porinas/deficiência , Acinetobacter/genética , Acinetobacter baumannii/genética , Acinetobacter baumannii/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico , Membrana Celular/química , Membrana Celular/metabolismo , Ertapenem , Evolução Molecular , Deleção de Genes , Expressão Gênica , Cinética , Meropeném , Porinas/genética , Tienamicinas/metabolismo , beta-Lactamas/metabolismoRESUMO
Acinetobacter baumannii is an emerging multidrug-resistant pathogen and very little information is available regarding its imipenem resistance in Latin American countries such as Bolivia. This study investigated the antimicrobial resistance profile of 46 clinical strains from different hospitals in Cochabamba, Bolivia, from March 2008 to July 2009, and the presence of carbapenemases as a mechanism of resistance to imipenem. Isolates were obtained from 46 patients (one isolate per patient; 30 males,16 females) with an age range of 1 day to 84 years, and were collected from different sample types, the majority from respiratory tract infections (17) and wounds (13). Resistance to imipenem was detected in 15 isolates collected from different hospitals of the city. These isolates grouped into the same genotype, named A, and were resistant to all antibiotics tested including imipenem, with susceptibility only to colistin. Experiments to detect carbapenemases revealed the presence of the OXA-58 carbapenemase. Further analysis revealed the location of the bla(OXA-58) gene on a 40 kb plasmid. To our knowledge, this is the first report of carbapenem resistance in A. baumannii isolates from Bolivia that is conferred by the OXA-58 carbapenemase. The presence of this gene in a multidrug-resistant clone and its location within a plasmid is of great concern with regard to the spread of carbapenem-resistant A. baumannii in the hospital environment in Bolivia.
Assuntos
Infecções por Acinetobacter/epidemiologia , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/metabolismo , Carbapenêmicos/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Hospitais/estatística & dados numéricos , beta-Lactamases/biossíntese , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/genética , Adolescente , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/farmacologia , Bolívia/epidemiologia , Carbapenêmicos/farmacologia , Criança , Pré-Escolar , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Feminino , Humanos , Imipenem/metabolismo , Imipenem/farmacologia , Lactente , Recém-Nascido , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Plasmídeos/genética , Adulto Jovem , beta-Lactamases/genéticaRESUMO
Objetivo. Identificar la proteína de membrana externa ausente en los aislamientos resistentes y determinar tanto las causas de su ausencia en la membrana, como la presencia de otros mecanismos de resistencia a carbapenemes en aislamientos clínicos de Pseudomonas aeruginosa. Métodos. Se estudió un brote de 20 aislamientos de P. aeruginosa previamente caracterizados como productores de la metalobetalactamasa IMP-13. Estos aislamientos presentaron igual expresión de la enzima IMP-13, pero solo cinco de ellos fueron resistentes acarbapenemes. En esos cinco aislamientos resistentes se confirmó la ausencia de una proteína de membrana externa. Se secuenciaron oprD y ampC; se identificaron las proteínas de membrana externa por desorción/ionización láser asistida por matriz/espectometría de masa tiempo de vuelo (MALDI-TOF); se determinó el nivel de expresión de OprD, de AmpC y de los sistemas de eflujo tipo Mex, por reacción en cadena de polimerasa en tiempo real, y por último, se determinó la contribución del déficit de OprD a la resistencia a carbapenemes. Resultados. La proteína de la membrana externa ausente en el grupo R (resistentes a ambos carbapenemes) fue identificada como OprD-TS, pero no se observaron variaciones en suexpresión. El gen oprD presentó mutaciones en los cinco aislamientos resistentes. Se observó la misma producción de la enzima tipo AmpC PDC-5 y del sistema de eflujo Mex AB-OprM entre los aislamientos sensibles y resistentes a carbapenemes. Se analizó cómo la presencia conjunta de IMP-13 y el déficit de OprD contribuyen al aumento de la resistencia.Conclusiones. Distintos mecanismos contribuyen a la resistencia de aislamientos productores de IMP-13 a carbapenemes. La posibilidad de no detectar estos aislamientos productores de IMP-13 representa un riesgo latente de selección de mutantes con mecanismos de resistencia que se suman para aumentar la resistencia a carbapenemes.
Objective. To identify the outer membrane protein absent in the resistant isolates and to determine both the causes of its absence in the membrane and the presence of othermechanisms of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa. Methods. Twenty isolates from an outbreak of P. aeruginosa previously characterized as metallo-beta-lactamase IMP-13 producers were studied. All the isolates exhibitedequal expression of the IMP-13 enzyme, but only five of them were carbapenemresistant. It was found that the five resistant isolates lacked a outer membrane protein. The oprD and ampC genes were sequenced; the outer membrane proteins were identifiedusing matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; the OprD and AmpC expressions, as well as the Mex efflux system, were assessed by real-time polymerase chain reaction; and finally, the contribution of reduced OprD to carbapenem resistance was determined. Results. The absent outer membrane protein in group R was identified as OprD-TS; however, no variations in its expression were observed. The oprD gene presentedmutations in the five resistant isolates. The production of AmpC PDC-5-type enzyme and the MexAB-OprM efflux system was the same in both carbapenem-sensitive and‑resistant isolates. The contribution of the combined presence of IMP-13 and reducedOprD to increased resistance was examined. Conclusions. Different mechanisms contribute to carbapenem resistance in IMP-13-producing isolates. The possibility that these IMP-13-producing isolates could go undetected poses a latent risk when selecting mutants with added resistancemechanisms in order to enhance carbapenem resistance.
Assuntos
Humanos , Proteínas de Bactérias/fisiologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla/fisiologia , Porinas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Resistência beta-Lactâmica/fisiologia , beta-Lactamases/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/genética , Análise Mutacional de DNA , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/genética , Eletroforese em Gel de Campo Pulsado , Genes Bacterianos , Imipenem/metabolismo , Imipenem/farmacologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/fisiologia , Mutação , Porinas/deficiência , Porinas/fisiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Estudos Retrospectivos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tienamicinas/metabolismo , Tienamicinas/farmacologia , Resistência beta-Lactâmica/genética , beta-Lactamases/genéticaRESUMO
OBJECTIVE: To identify the outer membrane protein absent in the resistant isolates and to determine both the causes of its absence in the membrane and the presence of other mechanisms of carbapenem resistance in clinical isolates of Pseudomonas aeruginosa. METHODS: Twenty isolates from an outbreak of P. aeruginosa previously characterized as metallo-beta-lactamase IMP-13 producers were studied. All the isolates exhibited equal expression of the IMP-13 enzyme, but only five of them were carbapenem-resistant. It was found that the five resistant isolates lacked a outer membrane protein. The oprD and ampC genes were sequenced; the outer membrane proteins were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry; the OprD and AmpC expressions, as well as the Mex efflux system, were assessed by real-time polymerase chain reaction; and finally, the contribution of reduced OprD to carbapenem resistance was determined. RESULTS: The absent outer membrane protein in group R was identified as OprD-TS; however, no variations in its expression were observed. The oprD gene presented mutations in the five resistant isolates. The production of AmpC PDC-5-type enzyme and the MexAB-OprM efflux system was the same in both carbapenem-sensitive and -resistant isolates. The contribution of the combined presence of IMP-13 and reduced OprD to increased resistance was examined. CONCLUSIONS: Different mechanisms contribute to carbapenem resistance in IMP-13-producing isolates. The possibility that these IMP-13-producing isolates could go undetected poses a latent risk when selecting mutants with added resistance mechanisms in order to enhance carbapenem resistance.
Assuntos
Proteínas de Bactérias/fisiologia , Carbapenêmicos/farmacologia , Farmacorresistência Bacteriana Múltipla/fisiologia , Porinas/genética , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Resistência beta-Lactâmica/fisiologia , beta-Lactamases/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/genética , Análise Mutacional de DNA , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla/genética , Eletroforese em Gel de Campo Pulsado , Genes Bacterianos , Humanos , Imipenem/metabolismo , Imipenem/farmacologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/fisiologia , Meropeném , Mutação , Porinas/deficiência , Porinas/fisiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética , Estudos Retrospectivos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tienamicinas/metabolismo , Tienamicinas/farmacologia , Resistência beta-Lactâmica/genética , beta-Lactamases/genéticaRESUMO
Three carbapenem-resistant Acinetobacter baumannii isolates were collected at a hospital in Buenos Aires, Argentina. Isoelectric focusing revealed multiple beta-lactamases, with two of the isolates showing identical profiles. A pI 6.9 carbapenemase with a molecular weight of 30 kDa was purified from one of these two isolates. The enzyme was predominantly a penicillinase, with its highest Vmax for oxacillin but highest Vmax/Km for benzylpenicillin. First-generation cephalosporins and imipenem were weaker substrates than penicillins, and oxyimino-aminothiazolyl cephalosporins were essentially stable. Meropenem-hydrolysing activity was not detected, despite resistance. The carbapenemase was inhibited by clavulanic acid and tazobactam, but not by EDTA. These kinetics place the enzyme into functional group 2; as an oxacillinase it could be placed in sub-group 2d or, as a zinc-independent carbapenemase, in sub-group 2f.