Etude structurale et fonctionnelle de la spécificité de substrat des carbapénèmases 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-48∆P217, 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 b5–b6 loop determines the specificity of the enzyme.Les b-lactamines, grâce à leur efficacité clinique, sont parmi les antibiotiques les plus prescrits pour traiter des infections bactériennes. Cependant, leur utilité est compromise par la prolifération des b-lactamases (BLs) avec des profils d’hydrolyse de substrats très larges. La résistance induite par les BLs compromet également les b-lactamines les plus puissantes (c-à-d les carbapénèmes). OXA-48, une carbapénèmase de classe D (CHDL), a été initialement identifiée dans une souche de K. pneumoniae de Turquie en 2001. OXA-48 hydrolysent fortement les pénicillines, faiblement les carbapénèmes, et quasiment pas les céphalosporines de troisième génération (C3G). Cependant certains variants comme OXA-163 ou OXA-405 hydrolysent les C3G et pas les carbapénèmes. La comparaison de la structure tri-dimensionnelle d’OXA-48 avec d’autres CHDLs a révélé de petites différences principalement localisées dans les boucles qui relient des éléments de structure. Notamment, la boucle localisée entre les feuillets b5 et b6 semble jouer un rôle majeur dans l’hydrolyse des carbapénèmes.Afin de mieux comprendre la contribution de la boucle b5-b6 dans l’hydrolyse des carbapénèmes, nous avons étudié, grâce à des outils biochimiques et structuraux, des variants naturels ou synthétiques d’OXA-48 présentant des modifications dans la boucle: le rôle du remplacement de chaque AA de la boucle par une alanine, des délétions croissantes ou de l’augmentation de la taille de la boucle. Nous avons également réalisé l’échange de boucle entre OXA-48 et OXA-18, une oxacillinase inhibée par l’acide clavulanique et hydrolysant fortement les C3G mais pas les carbapénèmes. La protéine recombinante OXA-48loop18 hydrolysait les C3G, et conservait une activité significative d’hydrolyse des carbapénèmes. L’échange de boucle a permis l’élargissement du site actif, permettant l’accès à des b-lactamines possédant un radical volumineux (e.g. ceftazidime). De plus, le remplacement de chaque AA de la boucle par une alanine a relevé de faibles changements hydrolytiques. En réalisant des délétions croissantes d’AA 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 montré que l’activité d’hydrolyse des carbapénèmes diminuait avec la taille des délétions, alors que celle des C3G augmentait. Les délétions de 4 AA présentent les plus fortes activités hydrolytiques des C3G et une perte totale de l’activité carbapénèmase, excepté pour le simple mutant, OXA-48∆P217, qui présentait un profile d’hydrolyse avec une forte activité carbapénèmase et C3G. La cristallographie et la modélisation moléculaire ont montré une grande flexibilité de la boucle, permettant l’entrée de b-lactamines de tailles variables. De plus, l’étude de nouveaux variants d’OXA-48 a permis d’identifier des déterminants structuraux importants dans le profil d’hydrolyse observé. Ainsi, la délétion I215-E216 associée à la substitution R214K dans la boucle b5-b6 de OXA-517 permet une forte hydrolyse des carbapénèmes et des C3G. De même, dans OXA-519, une substitution V120L située à proximité de la boucle b5-b6, a pour conséquence une diminution de l’affinité pour tous les substrats. La chaine latérale plus encombrante de la L120 empêche l’insertion des b-lactamines, diminuant l’affinité de l’enzyme. Finalement, nous avons caractérisé OXA-535, la b-lactamase naturelle et chromosomique de Shewanella bicestrii JAB-1 qui, n’ayant uniquement 91,5% d’identité en AA avec OXA-48, présente le même profil d’hydrolyse. OXA-535 présentait 98.9% d’identité en AA avec OXA-436, codé par un gène plasmidique, suggérant ainsi que S. bicestrii portant le gène blaOXA-535 pourrait être le progéniteur du gène plasmidique blaOXA-436.Nos travaux ont montré le formidable pouvoir d’adaptation de OXA-48 à évoluer par mutation afin d’accommoder différents substrats, et comment la nature et la longueur de la boucle b5-b6 pouvait influencer sur la spécificité de substrat

The Tequila Banking Crisis in Argentina

tequila, banks, crisis, Argentina

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 NCBI’s “nr” and “nr_env” 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 reprezintă o problemă majoră pentru sănătate, având învedere detectarea lor dificilă, comportamentul epidemic și tendința lor de a-și modifica spectrul de hidrolizăprin mutații punctiforme.Obiectiv: Pentru a obține o imagine amplă asupra variabilității actuale a enzimelor OXA-48-like, amrecuperat toate secvențele disponibile de la NCBI (National Center for Biotechnology Information).Metodă: Am efectuat mai multe căutări BLAST (Basic Local Alignment Search Tool) în bazele de date„nr” și „nr_env” ale NCBI (descărcate pe 20 decembrie 2016), utilizând ca interogare membrii cunoscuți aisub-familiei OXA-48.Rezultate: În timp ce unui număr de 23 de variante li s-au atribuit numere OXA, au fost identificate 62 dealele noi. Acestea corespund noilor enzime cu mutații localizate în unele cazuri în cadrul motivelor conservateale site-ului activ. Numărul important de variante noi identificate în acest studiu este de mare interes, deoareceoferă o evaluare mai realistă a variantelor de tip OXA-48-like.Concluzie: O mare varietate de enzime OXA-48-like a fost descoperită în urma căutării bioinformaticea variantelor. Detectarea enzimelor OXA-48-like în izolate din mediu poate reflecta contaminarea cuEnterobactericeae producătoare de enzime OXA-48-like și/sau prezența de izolate Shewanella spp.Cuvinte-cheie: OXA-48, variabilitate, variante

Beta-lactamase database (BLDB) – 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 β-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