6 research outputs found
Etude structurale et fonctionnelle de la spécificité de substrat des carbapénÚmases de type OXA-48
Antimicrobial resistance is the most alarming emerging problem in infectious diseases. b-Lactams, due to their safety, reliable killing properties and clinical efficacy, are among the most frequently prescribed antibiotics used to treat bacterial infections. However, their utility is being threatened by the worldwide proliferation of b-lactamases (BLs). BL-mediated resistance does not spare even most powerful b-lactams, carbapenems, whose activity is challenged by carbapenemases. OXA-48, a carbapenem-hydrolyzing class D b-lactamase (CHDL) initially identified from a Klebsiella pneumoniae isolate from Turkey in 2001, has since spread globally with the isolation of more than 30 variants. Most OXA-48-like enzymes hydrolyze penicillins at high level, carbapenems at low level and lack significant expanded-spectrum cephalosporin (3GC) hydrolysis, others such as OXA-163 hydrolyze expanded-spectrum cephalosporins and poorly carbapenems. Comparison of OXA-48 tertiary structure with those of other CHDLs revealed small differences located mainly in the loops connecting secondary structure elements, which may vary in length and orientation. The loop located between the b5 and b6 strands (Tyr211 to Pro217) has been suggested to play a major role in carbapenem hydrolysis.To better understand the contribution of the b5-b6 loop in the carbapenem hydrolysis of OXA-48-like carbapenemases, we investigated, using biochemistry and structural biology, natural OXA-48 variants with changes in different loops, replaced each AA of the loop b5-b6 by alanines, performed increasing deletions or increased the size of this loop by replacing it with that of OXA-18, a clavulanic acid inhibited class D b-lactamase that presents activity against expanded-spectrum cephalosporins and none against carbapenems. The resulting OXA-48loop18 was able to hydrolyze expanded-spectrum cephalosporins and conserved partial carbapenem hydrolysis. Structural analysis demonstrated that the loop swap produced an opening of the active site, being now accessible to b-lactams with bulky sidechains e.g. ceftazidime. Additionally, by performing alanine replacements in the b5-b6 loop we could show reduced hydrolysis of carbapenems, mostly reflected by changes in kcat. By increasing deletions in the b5-b6 loop, starting from Tyr211 to Pro217 and from the Pro217 to Tyr211, the activity against carbapenems decreased with the size of the deletion whereas the activity against ceftazidime increased. 4 AA deletions revealed the highest 3GC activity, except for one single AA mutant, OXA-48âP217, with high level carbapenem and ceftazidime hydrolysis. Crystallography along with molecular modelling showed an increased flexibility of this loop allowing different sized b-lactams to enter the active site. Moreover, the characterization of three novel natural OXA-48 variants revealed structural features important in the observed hydrolysis profile. Thus, the I215-E216 deletion and R214K substitution in the b5-b6 loop of OXA-517 induced the hydrolysis of carbapenems and C3G at high level. In OXA-519, the V120L substitution is located at the bottom of the binding site, in the close vicinity of the active Ser70 and the b5-b6 loop, and therefore overall higher Km values were observed compared to OXA-48. The bulkier side chain of L120 in OXA-519 hampers the approach of b-lactam substrate, resulting in a decrease of the substrate affinity. Finally, we have characterized the chromosomally-encoded OXA-535 that is more distantly related to OXA-48 (91.5% AA identity), despite similar hydrolysis profiles. Interestingly, OXA-535 presented 98.9% of AA identity with the plasmid-mediated OXA-436 suggesting that the blaOXA-535 gene might be the progenitor of the plasmid-encoded blaOXA-436 gene.Taken together, our work illustrates the propensity of OXA-48 to evolve through mutations to accommodate different substrates in its active site and how the b5âb6 loop determines the specificity of the enzyme.Les b-lactamines, grĂące Ă leur efficacitĂ© clinique, sont parmi les antibiotiques les plus prescrits pour traiter des infections bactĂ©riennes. Cependant, leur utilitĂ© est compromise par la prolifĂ©ration des b-lactamases (BLs) avec des profils dâhydrolyse de substrats trĂšs larges. La rĂ©sistance induite par les BLs compromet Ă©galement les b-lactamines les plus puissantes (c-Ă -d les carbapĂ©nĂšmes). OXA-48, une carbapĂ©nĂšmase de classe D (CHDL), a Ă©tĂ© initialement identifiĂ©e dans une souche de K. pneumoniae de Turquie en 2001. OXA-48 hydrolysent fortement les pĂ©nicillines, faiblement les carbapĂ©nĂšmes, et quasiment pas les cĂ©phalosporines de troisiĂšme gĂ©nĂ©ration (C3G). Cependant certains variants comme OXA-163 ou OXA-405 hydrolysent les C3G et pas les carbapĂ©nĂšmes. La comparaison de la structure tri-dimensionnelle dâOXA-48 avec dâautres CHDLs a rĂ©vĂ©lĂ© de petites diffĂ©rences principalement localisĂ©es dans les boucles qui relient des Ă©lĂ©ments de structure. Notamment, la boucle localisĂ©e entre les feuillets b5 et b6 semble jouer un rĂŽle majeur dans lâhydrolyse des carbapĂ©nĂšmes.Afin de mieux comprendre la contribution de la boucle b5-b6 dans lâhydrolyse des carbapĂ©nĂšmes, nous avons Ă©tudiĂ©, grĂące Ă des outils biochimiques et structuraux, des variants naturels ou synthĂ©tiques dâOXA-48 prĂ©sentant des modifications dans la boucle: le rĂŽle du remplacement de chaque AA de la boucle par une alanine, des dĂ©lĂ©tions croissantes ou de lâaugmentation de la taille de la boucle. Nous avons Ă©galement rĂ©alisĂ© lâĂ©change de boucle entre OXA-48 et OXA-18, une oxacillinase inhibĂ©e par lâacide clavulanique et hydrolysant fortement les C3G mais pas les carbapĂ©nĂšmes. La protĂ©ine recombinante OXA-48loop18 hydrolysait les C3G, et conservait une activitĂ© significative dâhydrolyse des carbapĂ©nĂšmes. LâĂ©change de boucle a permis lâĂ©largissement du site actif, permettant lâaccĂšs Ă des b-lactamines possĂ©dant un radical volumineux (e.g. ceftazidime). De plus, le remplacement de chaque AA de la boucle par une alanine a relevĂ© de faibles changements hydrolytiques. En rĂ©alisant des dĂ©lĂ©tions croissantes dâAA soit en partant de la gauche de la boucle (Tyr-211 vers Pro-217) ou de la droite (Pro-217 vers Tyr-211), nous avons montrĂ© que lâactivitĂ© dâhydrolyse des carbapĂ©nĂšmes diminuait avec la taille des dĂ©lĂ©tions, alors que celle des C3G augmentait. Les dĂ©lĂ©tions de 4 AA prĂ©sentent les plus fortes activitĂ©s hydrolytiques des C3G et une perte totale de lâactivitĂ© carbapĂ©nĂšmase, exceptĂ© pour le simple mutant, OXA-48âP217, qui prĂ©sentait un profile dâhydrolyse avec une forte activitĂ© carbapĂ©nĂšmase et C3G. La cristallographie et la modĂ©lisation molĂ©culaire ont montrĂ© une grande flexibilitĂ© de la boucle, permettant lâentrĂ©e de b-lactamines de tailles variables. De plus, lâĂ©tude de nouveaux variants dâOXA-48 a permis dâidentifier des dĂ©terminants structuraux importants dans le profil dâhydrolyse observĂ©. Ainsi, la dĂ©lĂ©tion I215-E216 associĂ©e Ă la substitution R214K dans la boucle b5-b6 de OXA-517 permet une forte hydrolyse des carbapĂ©nĂšmes et des C3G. De mĂȘme, dans OXA-519, une substitution V120L situĂ©e Ă proximitĂ© de la boucle b5-b6, a pour consĂ©quence une diminution de lâaffinitĂ© pour tous les substrats. La chaine latĂ©rale plus encombrante de la L120 empĂȘche lâinsertion des b-lactamines, diminuant lâaffinitĂ© de lâenzyme. Finalement, nous avons caractĂ©risĂ© OXA-535, la b-lactamase naturelle et chromosomique de Shewanella bicestrii JAB-1 qui, nâayant uniquement 91,5% dâidentitĂ© en AA avec OXA-48, prĂ©sente le mĂȘme profil dâhydrolyse. OXA-535 prĂ©sentait 98.9% dâidentitĂ© en AA avec OXA-436, codĂ© par un gĂšne plasmidique, suggĂ©rant ainsi que S. bicestrii portant le gĂšne blaOXA-535 pourrait ĂȘtre le progĂ©niteur du gĂšne plasmidique blaOXA-436.Nos travaux ont montrĂ© le formidable pouvoir dâadaptation de OXA-48 Ă Ă©voluer par mutation afin dâaccommoder diffĂ©rents substrats, et comment la nature et la longueur de la boucle b5-b6 pouvait influencer sur la spĂ©cificitĂ© de substrat
A greater than expected variability among OXA-48-like carbapenemases
International audienceBackground: OXA-48-like carbapenemases represent a major health concern given their difficult detection, their epidemic behavior and their propensity to modify their spectrum of hydrolysis through point mutations. Objective: To get an extensive view on the current variability among OXA-48-like enzymes, we haveretrieved all the sequences available from NCBI (National Center for Biotechnology Information).Method: We carried out several BLAST (Basic Local Alignment Search Tool) searches in the NCBIâs ânrâ and ânr_envâ databases (downloaded on December 20th, 2016) using known members of OXA-48-likesubfamily as query.Results: While 23 variants have assigned OXA-numbers, 62 novel alleles have been identified. Theycorrespond to novel enzymes with mutations located in some cases within the conserved active site motives. The important number of novel variants identified by this study is of great interest, since it provides a more realistic assessment of OXA-48-like variants.Conclusion: A large variety of OXA-48-like enzymes has been unraveled through our bioinformatic search for variants. The finding of OXA-48-like enzymes in environmental isolates may reflect the contamination by Enterobactericeae producing OXA-48-like enzymes and/or the presence of Shewanella spp. isolates.Introducere: Carbapenemazele OXA-48-like reprezintÄ o problemÄ majorÄ pentru sÄnÄtate, avĂąnd Ăźnvedere detectarea lor dificilÄ, comportamentul epidemic Èi tendinÈa lor de a-Èi modifica spectrul de hidrolizÄprin mutaÈii punctiforme.Obiectiv: Pentru a obÈine o imagine amplÄ asupra variabilitÄÈii actuale a enzimelor OXA-48-like, amrecuperat toate secvenÈele disponibile de la NCBI (National Center for Biotechnology Information).MetodÄ: Am efectuat mai multe cÄutÄri BLAST (Basic Local Alignment Search Tool) Ăźn bazele de dateânrâ Èi ânr_envâ ale NCBI (descÄrcate pe 20 decembrie 2016), utilizĂąnd ca interogare membrii cunoscuÈi aisub-familiei OXA-48.Rezultate: Ăn timp ce unui numÄr de 23 de variante li s-au atribuit numere OXA, au fost identificate 62 dealele noi. Acestea corespund noilor enzime cu mutaÈii localizate Ăźn unele cazuri Ăźn cadrul motivelor conservateale site-ului activ. NumÄrul important de variante noi identificate Ăźn acest studiu este de mare interes, deoareceoferÄ o evaluare mai realistÄ a variantelor de tip OXA-48-like.Concluzie: O mare varietate de enzime OXA-48-like a fost descoperitÄ Ăźn urma cÄutÄrii bioinformaticea variantelor. Detectarea enzimelor OXA-48-like Ăźn izolate din mediu poate reflecta contaminarea cuEnterobactericeae producÄtoare de enzime OXA-48-like Èi/sau prezenÈa de izolate Shewanella spp.Cuvinte-cheie: OXA-48, variabilitate, variante
Beta-lactamase database (BLDB) â structure and function
<p>Beta-Lactamase Database (BLDB) is a comprehensive, manually curated public resource providing up-to-date structural and functional information focused on this superfamily of enzymes with a great impact on antibiotic resistance. All the enzymes reported and characterised in the literature are presented according to the class (A, B, C and D), family and subfamily to which they belong. All three-dimensional structures of ÎČ-lactamases present in the Protein Data Bank are also shown. The characterisation of representative mutants and hydrolytic profiles (kinetics) completes the picture and altogether these four elements constitute the essential foundation for a better understanding of the structure-function relationship within this enzymes family. BLDB can be queried using different protein- and nucleotide-based BLAST searches, which represents a key feature of particular importance in the context of the surveillance of the evolution of the antibiotic resistance. BLDB is available online at <a href="http://bldb.eu" target="_blank">http://bldb.eu</a> without any registration and supports all modern browsers.</p