Professor Claire Janoir

Professor Neil Wo

Professor, Gabriel Gutkind

PhD Pierre Bogaerts

Wladimir Mcu

MCU Thierry Naas

Bogdan Iorga

Remy Bonnin

Thèse Prés

Maria Laura

Dabos Président

Examinateur Rapporteur Rapporteur

Composition du Jury

Paper I. Role of the loop β5-β6 in the substrate specificity of OXA-48. Paper VII. Beta-lactamase database (BLDB) -structure and function. C. Analysis of the dimerization of OXA-48. Paper XI. Escherichia coli clinical isolates harbouring mcr-1 and mcr-1.5

genes recovered from a University Hospital in Buenos Aires, Argentina.

Tables

Introduction Table 1. Bush and Jacoby β-lactamases classification. Table 2. Kinetics parameters of OXA-48 with representative β-lactams substrates.

Paper I. Role of the loop β5-β6 in the substrate specificity of OXA-48. Moreover, the characterization of three novel natural OXA variants revealed structural features important in the observed hydrolysis profile. Thus, the I215 E216 deletion and R214K substitution in the loop of OXA carbapenems and C3G at high level. In OXA the V120L substitution is located at the bottom of the binding site, in the close vicinity of the active Ser70 and the higher 48. The bulkier side chain of L120 in OXA hampers the approach of resulting in a decrease of the substrate affinity. Finally, we have characterized the chromosomally encoded OXA O hydrolysis profiles. Interestingly, OXA presented 98.9% of AA identity with the plasmid mediated OXA gene might be the progenitor of the plasmid encoded propensity of OXA to accommodate different substrates in its active site and how the specificity of the enzyme.

Aubin, France

Structural and functional insights into the substrate specificity of OXA to β-lactams with bulky sidechains e.g. ceftazidime. Additionally, by performing alanine replacements in the β5-β6 loop we could show reduced hydrolysis of carbapenems, mostly reflected by changes in k cat loop, starting from Tyr Pro-217 to Tyr carbapenems decreased with the size of the deletion whereas the activity against ceftazidime increased. 4 AA deletions revea 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 enter the active site.

Moreover, the characterization of three novel natural OXA variants revealed structural features important in the observed hydrolysis profile. Thus, the I215 E216 deletion and R214K substitution in the loop of OXA carbapenems and C3G at high level. In OXA the V120L substitution is located at the bottom of the binding site, in the close vicinity of the active Ser70 and the higher Km values were observed c 48. The bulkier side chain of L120 in OXA hampers the approach of resulting in a decrease of the substrate affinity. Finally, we have characterized the chromosomally encoded OXA-OXA-48 (91.5% AA identity), despite similar hydrolysis profiles. Interestingly, OXA presented 98.9% of AA identity with the plasmid mediated OXA gene might be the progenitor of the plasmid encoded bla OXA Taken together, our work illustrates the propensity of OXA to accommodate different substrates in its active site and how the specificity of the enzyme.

Structural and functional insights into the substrate specificity of OXA-48 lactams with bulky sidechains e.g. ceftazidime. Additionally, by performing alanine replacements 6 loop we could show reduced hydrolysis of carbapenems, mostly reflected by cat . By increasing deletions in the loop, starting from Tyr-211 to Pro 217 to Tyr-211, the activity against carbapenems decreased with the size of the deletion whereas the activity against ceftazidime increased. 4 AA deletions revea activity, except for one single AA mutant, OXA ∆P217, with high level carbapenem and ceftazidime hydrolysis. Crystallography along with molecular modelling showed an increased flexibility of this loop allowing different sized enter the active site. Moreover, the characterization of three novel natural OXA variants revealed structural features important in the observed hydrolysis profile. Thus, the I215 E216 deletion and R214K substitution in the loop of OXA-517 induced the hydrolysis of carbapenems and C3G at high level. In OXA the V120L substitution is located at the bottom of the binding site, in the close vicinity of the active Ser70 and the β5-β6 loop, and therefore overall m values were observed c 48. The bulkier side chain of L120 in OXA hampers the approach of resulting in a decrease of the substrate affinity. Finally, we have characterized the chromosomally -535 that is more distantly related to 48 (91.5% AA identity), despite similar hydrolysis profiles. Interestingly, OXA presented 98.9% of AA identity with the plasmid mediated OXA-436 suggesting that the gene might be the progenitor of the plasmid OXA-436 gene. en together, our work illustrates the propensity of OXA-48 to evolve through mutations to accommodate different substrates in its active site and how the β5-β6 loop determines the specificity of the enzyme.

48-like

lactams with bulky sidechains e.g. ceftazidime. Additionally, by performing alanine replacements 6 loop we could show reduced hydrolysis of carbapenems, mostly reflected by

. By increasing deletions in the 211 to Pro-217 and from the 211, 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 ∆P217, with high level carbapenem and ceftazidime hydrolysis. Crystallography along with molecular modelling showed an increased flexibility of this loop allowing different sized βenter the active site.

Moreover, the characterization of three novel natural OXA variants revealed structural features important in the observed hydrolysis profile. Thus, the I215 E216 deletion and R214K substitution in the duced the hydrolysis of carbapenems and C3G at high level. In OXA the V120L substitution is located at the bottom of the binding site, in the close vicinity of the active 6 loop, and therefore overall m values were observed compared to OXA 48. The bulkier side chain of L120 in OXA hampers the approach of β-lactam substrate, resulting in a decrease of the substrate affinity. Finally, we have characterized the chromosomally 535 that is more distantly related to 48 (91.5% AA identity), despite similar hydrolysis profiles. Interestingly, OXA presented 98.9% of AA identity with the plasmid 436 suggesting that the gene might be the progenitor of the plasmid en together, our work illustrates the 48 to evolve through mutations to accommodate different substrates in its active 6 loop determines the lactams with bulky sidechains e.g. ceftazidime. Additionally, by performing alanine replacements 6 loop we could show reduced hydrolysis of carbapenems, mostly reflected by

. By increasing deletions in the β5-β6 217 and from the 211, the activity against carbapenems decreased with the size of the deletion whereas the activity against ceftazidime led the highest 3GC activity, except for one single AA mutant, OXA-∆P217, with high level carbapenem and ceftazidime hydrolysis. Crystallography along with molecular modelling showed an increased flexibility -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 β5-β6 duced 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 6 loop, and therefore overall ompared to OXA-48. The bulkier side chain of L120 in OXA-519 lactam substrate, resulting in a decrease of the substrate affinity.

Figure 1 .

 1 Figures

Figure 2 .

 2 Figure 2. Representation of the peptidoglycan structure.

Figure 3 .

 3 Figure 3. Representation of the cross-link formation.

Figure 4 .

 4 Figure 4. Structure of the principal β-lactams.

Figure 5 .

 5 Figure 5. Mechanism of action of β-lactams.

Figure 6 .

 6 Figure 6. Mechanisms of β-lactam resistance in Enterobacteriaceae.

Figure 7 .

 7 Figure 7. Hydrolytic mechanism of serine β-lactamases.

Figure 8 .

 8 Figure 8. Worldwide distribution of carbapenemases.

Figure 9 .

 9 Figure 9. Global distribution of IMP, VIM and NDM MBLs.

Figure 10 .

 10 Figure 10. Antibiogram of E. coli Top10 expressing OXA-48.

Figure 11 .

 11 Figure 11. Alignment of OXA-48 variants recovered from Enterobacteriacea isolates.

Figure 12 .

 12 Figure 12. Phylogenetic tree of all OXA-48 variants.

Figure 13 .

 13 Figure 13. Representation of the diversity among some allelic variants of the OXA-48 linage.

Figure 14 .

 14 Figure 14. Worldwide distribution of OXA-48-like producer in Enterobacteriaceae.

Figure 15 .

 15 Figure 15. Distribution of Carbapenemase Producing Enterobacteriaceae (CPE) in France.

Figure 16 .

 16 Figure 16. Major structural features of the plasmids IncL bla OXA-48 plasmid (pOXA-48a).

Figure 17 .

 17 Figure 17. Genetic environments of the bla OXA-48 -bearing Tn1999-like transposon structures in Enterobacteriaceae isolates.

Figure 18 .

 18 Figure 18. Schematic representation of the genetic environment of bla OXA-48 -like βlactamases.

Figure 19 .

 19 Figure 19. Tertiary Structure of OXA-48.

Figure 20 .

 20 Figure 20. Superposition of OXA-10, OXA-24 and OXA-48.

Figure 1 .

 1 Figure 1. Amino acids sequences comparison of the β5-β6 loop region in different mutants.

Figure 2 .

 2 Figure 2. Docking of imipenem on the OXA-48 structure.

Figure 3 .

 3 Figure 3. Oxyanion hole disruption for the last 4 N-terminus deletion mutants.

Figure 4 .

 4 Figure 4. Hydrolytic water position inside the active site cavity.

Figure 5 .

 5 Figure 5. Water network displacement in the OXA-48 deletion mutants.

Figure 6 .

 6 Figure 6. β5-β6 loop flexibility for OXA-48 and deletion mutants.

Figure 7 .

 7 Figure 7. Ceftazidime overlaps with OXA-48 and OXA-48ΔP.

Figure 8 .

 8 Figure 8. L158 C-Cα-Cβ-Cγ dihedral angle distribution during the MD simulation for OXA-48 and deletion mutants.

Figures S1- S14 .

 S14 Figures S1-S14. Hydrophobic/charged surface representations, and stick representations of the most representative structure extracted from MD simulations of OXA-48 and deletion mutants.

Figure 1 .

 1 Figure 1. Sequence alignment of β5-β6 loops and their phenotypic profiles.

Figure 2 .

 2 Figure 2. Crystal structure of OXA-48loop18.

Figure 3 .

 3 Figure 3. Structure conformational differences of OXA-48loop18 and OXA-48.

Figure 4 .

 4 Figure 4. Docking of ceftazidime on OXA-48loop18.

Figure 5 .

 5 Figure 5. Flexibility of OXA-48loop18.

Figure 6 .

 6 Figure 6. Water networks around OXA-48 and OXA-48loop18.

Figure 7 .

 7 Figure 7. Displacement of active site water molecules by imipenem.

Figure 8 .

 8 Figure 8. Water molecules flow into the active site cavity.

Figure 9 .

 9 Figure 9. Conserved water site in the hydrolytic water pocket.

Figure 10 .

 10 Figure 10. β5-β6 loop conformation of OXAs with different hydrolytic profiles.

Figure 1 .

 1 Figure 1. Sequence alignment of β5-β6 loops.

Figure 2 .

 2 Figure 2. Genetic environments of OXA-48-like genes.

Figure 3 .

 3 Figure 3. Cartoon model of a single subunit of OXA-517.

Figure 4 .

 4 Figure 4. Dimer interface of the OXA-517 crystal structure.

Figure 5 .

 5 Figure 5. Active site of the OXA-517 crystal structure.

Figure 6 .

 6 Figure 6. Comparison of crystal structures of OXA-517, OXA-48 and OXA-163.

Figure 1 .

 1 Figure 1. Genetic environments of OXA-48-like genes.

Figure 1 .

 1 Figure 1. Amino acid sequence alignment of different OXA-48-like variants and molecular modeling.

Figure 2 .

 2 Figure 2. Genetic contexts of the blaOXA-535 gene.

Figure 1 .

 1 Figure 1. Phylogenetic analysis of representative Shewanella spp.

Figure 2 .

 2 Figure 2. Genome representation of Shewanella sp. JAB-1 and its plasmids.

Figure 3 .

 3 Figure 3. Schematic representation of the three ESBL-encoding plasmids.

Figure 4 .

 4 Figure 4. Genetic environment of the blaCTX-M-15 gene of K. pneumoniae JAB-1.

Figure 1 .

 1 Figure 1. Global architecture of the Beta-Lactamase Database.

Figure 1 .

 1 Figure 1. Phylogeny of the chromosomally-and plasmid-encoded OXA-48-like βlactamases.

Figure 1 .

 1 Figure 1. Dimer of OXA-48 carbapenemase.

Figure 2 .

 2 Figure 2. Interaction between amino acids that conformed the interface chain A chain B in the dimer of OXA-48.

Figure 3 .

 3 Figure 3. Size-exclusion chromatography of OXA-48, OXA-18 and the different mutants.

Figure 1 .

 1 Figure 1. Schematic representation of the genetic structure of SmarGI1-1 of S. marcescens N11-02820 (KF318367) and S. marcescens 163 and flanking regions.

Figure 2 .

 2 Figure 2. S. marcescens Maximum Likelihood tree obtained from core non-recombinant SNPs.

Figure 3 .

 3 Figure 3. Comparison of the SmarGI1-1 and its genetic environments among the different bla SME -encoding strains.

Figure 4 .

 4 Figure 4. Insertion site for SmarGI1-1 in the SME subclade and its basal strains.

Figure 1 .

 1 Figure 1. Genetic environment of bla LMB-1 compared with the environment of the blaLMB-1-like sequence in Rheinheimera pacifica and pNRZ-10170-LMB-1.

Figure 2 .DiscussionFigure 1 .

 21 Figure 2. Schematic representation of the natural plasmid pCf164 from C. freundii 164.

Figure 1 .

 1 Figure 1. Plasmid extraction migration.Figure 2. Sequence alignment of IncI2-type mcr-carrying plasmids.

Figure 2 .

 2 Figure 1. Plasmid extraction migration.Figure 2. Sequence alignment of IncI2-type mcr-carrying plasmids.

Figure 1 .

 1 Figure 1. Lipid A modifications caused by chromosome-encoded determinants or MCR enzymes that can be detected by the MALDIxin test.

Figure 2 .

 2 Figure 2. Results of the MALDIxin test on E. coli.

Figure 3 .

 3 Figure 3. Comparison of research (purple outline), routine clinical (green outline) and MALDIxin (blue outline) workflows for the detection of chromosome-and plasmid (MCR)encoded resistance to polymyxin in Enterobacteriaceae.

Figure 1 .

 1 Figure 1. Schematic representation of the genetic organization of pCf587.

Figure 2 .

 2 Figure 2. Schematic representation of the pCf587 resistance island (RI-pCf).

Figure 3 .

 3 Figure 3. Local colinear block comparison between pCf587, pRA1, p34998, and pIncAC-KP4898 by Mauve software.

Figure 4 .

 4 Figure 4. Phylogenetic relationships between pCf587, p34998, and the different IncA/C STs identified to date, based on maximum likelihood and Bayesian methods.

Keywords

  Antimicrobial resistance is the most alarming emerging problem in infectious diseases. Lactams, due to their safety, reliable killing properties and clinical efficacy, are among the most frequently prescribed antibiotic bacterial infections. However, their utility is being threatened by the worldwide proliferation of lactamases (BLs). BL spare even most powerful whose activity is challenged by carbapene OXA lactamase (CHDL) initially identified from a Klebsiella pneumoniae has since spread globally with the isolation of more than 30 variants. Most OXA hydrolyze peni low level and lack significant expanded cephalosporin (3GC) hydrolysis, others such as OXA cephalosporins and poorly carbapenems. Comparison of OXA 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 β6 strands (Tyr to play a ma To better understand the contribution of the loop in the carbapenem hydrolysis of OXA carbapenemases, we investigated, using biochemistry and structural biology, natural OXA variants with changes in diff each AA of the loop increasing deletions or increased the size of this loop by replacing it with that of OXA clavulanic acid inhibited class D presents activity against expanded cephalosporins and none against carbapenems. The resulting OXA expanded partial carbapenem hydrolysis. Structural analysis demonstrated that the loop swap produced an opening Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint Title: Structural and functional insights into the substrate specificity of OXA carbapenemases ywords: β-lactams, Carbapenemase, Inhibitor.Antimicrobial resistance is the most alarming emerging problem in infectious diseases. Lactams, due to their safety, reliable killing properties and clinical efficacy, are among the most frequently prescribed antibiotic bacterial infections. However, their utility is being threatened by the worldwide proliferation of lactamases (BLs). BL spare even most powerful whose activity is challenged by carbapene OXA-48, a carbapenem lactamase (CHDL) initially identified from a Klebsiella pneumoniae has since spread globally with the isolation of more than 30 variants. Most OXA hydrolyze penicillins at high level, carbapenems at low level and lack significant expanded cephalosporin (3GC) hydrolysis, others such as OXA-163 hydrolyze expanded cephalosporins and poorly carbapenems. Comparison of OXA 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 6 strands (Tyr-211 to Pro to play a major role in carbapenem hydrolysis. To better understand the contribution of the loop in the carbapenem hydrolysis of OXA carbapenemases, we investigated, using biochemistry and structural biology, natural OXA variants with changes in diff each AA of the loop increasing deletions or increased the size of this loop by replacing it with that of OXA clavulanic acid inhibited class D presents activity against expanded 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 Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint Structural and functional insights into the substrate specificity of OXA carbapenemases. lactams, Carbapenemase, Inhibitor. Antimicrobial resistance is the most alarming emerging problem in infectious diseases. Lactams, due to their safety, reliable killing properties and clinical efficacy, are among the most frequently prescribed antibiotic bacterial infections. However, their utility is being threatened by the worldwide proliferation of lactamases (BLs). BL-mediated resistance does not spare even most powerful βwhose activity is challenged by carbapene 48, a carbapenem-hydrolyzing class D 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 cillins at high level, carbapenems at low level and lack significant expanded cephalosporin (3GC) hydrolysis, others such as 163 hydrolyze expanded cephalosporins and poorly carbapenems. Comparison of OXA-48 tertiary structure with th 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 211 to Pro-217) has been suggested jor role in carbapenem hydrolysis. To better understand the contribution of the loop in the carbapenem hydrolysis of OXA carbapenemases, we investigated, using biochemistry and structural biology, natural OXA variants with changes in diff each AA of the loop β5-β6 by alanines, performed increasing deletions or increased the size of this loop by replacing it with that of OXA clavulanic acid inhibited class D presents activity against expanded cephalosporins and none against carbapenems. The 48loop18 was able to hydrolyze spectrum cephalosporins and conserved partial carbapenem hydrolysis. Structural analysis demonstrated that the loop swap produced an e active site, being Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint Structural and functional insights into the substrate specificity of OXA lactams, Carbapenemase, Inhibitor.Antimicrobial resistance is the most alarming emerging problem in infectious diseases. 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 mediated resistance does not -lactams,carbapenems, whose activity is challenged by carbapene hydrolyzing class D lactamase (CHDL) initially identified from a isolate from Turkey in 2001, has since spread globally with the isolation of more than 30 variants. Most OXA-48-like enzymes cillins at high level, carbapenems at low level and lack significant expanded-spectrum cephalosporin (3GC) hydrolysis, others such as 163 hydrolyze expanded-spectrum cephalosporins and poorly carbapenems. 48 tertiary structure with th 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 217) has been suggested jor role in carbapenem hydrolysis. To better understand the contribution of the loop in the carbapenem hydrolysis of OXA carbapenemases, we investigated, using biochemistry and structural biology, natural OXA variants with changes in different loops, replaced 6 by alanines, performed increasing deletions or increased the size of this loop by replacing it with that of OXA clavulanic acid inhibited class D β-lactamase that presents activity against expanded-spec cephalosporins and none against carbapenems. The 48loop18 was able to hydrolyze spectrum cephalosporins and conserved partial carbapenem hydrolysis. Structural analysis demonstrated that the loop swap produced an being now accessibleEspace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, FranceStructural and functional insights into the substrate specificity of OXA lactams, Carbapenemase, Inhibitor.Antimicrobial resistance is the most alarming emerging problem in infectious diseases. β-Lactams, due to their safety, reliable killing properties and clinical efficacy, are among the most s used to treat bacterial infections. However, their utility is being threatened by the worldwide proliferation of βmediated resistance does not lactams,carbapenems, whose activity is challenged by carbapenemases.hydrolyzing class D βlactamase (CHDL) initially identified from a isolate from Turkey in 2001, has since spread globally with the isolation of more like enzymes cillins at high level, carbapenems at spectrum cephalosporin (3GC) hydrolysis, others such as spectrum cephalosporins and poorly carbapenems. 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 β5 and 217) has been suggested jor role in carbapenem hydrolysis. To better understand the contribution of the β5-β6 loop in the carbapenem hydrolysis of OXA-48-like carbapenemases, we investigated, using biochemistry and structural biology, natural OXA-48 erent loops, replaced 6 by alanines, performed increasing deletions or increased the size of this loop by replacing it with that of OXA-18, a lactamase that spectrum cephalosporins and none against carbapenems. The 48loop18 was able to hydrolyze spectrum cephalosporins and conserved partial carbapenem hydrolysis. Structural analysis demonstrated that the loop swap produced an accessible to Additionally, by performing alanine replacements in the hydrolysis of carbapenems, mostly reflected by changes in loop, starting from Tyr Pro carbapenems decreased with the size of the deletion whereas the activity against ceftazidime increased. 4 AA deletions revea activity, except for one single AA mutant, OXA 48 ceftazidime hydrolysis. Crystallography along with molecular modelling showed an increased flexibility of this loop allowing different sized enter the active site.

  Finally, we have characterized the chromosomally-535 that is more distantly related to 48 (91.5% AA identity), despite similar hydrolysis profiles. Interestingly, OXA-535 presented 98.9% of AA identity with the plasmid-436 suggesting that the bla OXA-535 gene might be the progenitor of the plasmiden together, our work illustrates the 48 to evolve through mutations to accommodate different substrates in its active 6 loop determines theUniversitéParis Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint Titre OXA Mots clés Les sont parmi les antibiotiques les plus prescrits pour traiter des infections bactérien utilité est compromise par la prolifération des lactamases (BLs) avec des profils d'hydrolyse de substrats très larges. La résistance induite par les BLs compromet également les puissantes (c carbapénèmase de classe D ( initialement identifiée dans une souche de pneumoniae hydrolysent fortement les pénicillines, faiblement les carbapénèmes, et quasiment pas les céphalosporines Cependant certains variants comme OXA OXA carbapénèmes. La comparaison de la structure tri dimensionnelle d'OXA révélé de petites différences principalement localisées dans les boucles qui relient des éléments de structure. Notamment, la boucle localisée entre les feuillets dans l'hydrolyse des carbapénèmes. Afin de mieux comprendre la contribution de la boucle nous avons étudié, grâce à des outils biochimiques et structuraux, des variants naturels ou synthétiques d'OXA modifications dans la boucle: le rôle du remplacement de chaque AA de la boucle par une alanine, d l'augmentation de la taille de la boucle. Nous avons également réalisé l'échange de boucle entre OXA 48 et OXA clavulanique et hydrolysant fortement les C3G mais pas les carbapénèmes. OXA une activité significative d'hydrolyse des carbapénèmes. L'échange de boucle a permis l'élargissement du site actif, permettant l'accès à des (e.g. ceftazidime). De AGER Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint Titre: Etude structurale et fonctionnell OXA-48. Mots clés: β-lactamines, C Les β-lactamines, grâce à leur efficacité clinique, sont parmi les antibiotiques les plus prescrits pour traiter des infections bactérien utilité est compromise par la prolifération des lactamases (BLs) avec des profils d'hydrolyse de substrats très larges. La résistance induite par les BLs compromet également les puissantes (c-à-d les carbapénèmase de classe D ( initialement identifiée dans une souche de pneumoniae de Turquie en 2001. OXA hydrolysent fortement les pénicillines, faiblement les carbapénèmes, et quasiment pas les céphalosporines Cependant certains variants comme OXA OXA-405 hydrolysent les C3G et pas les carbapénèmes. La comparaison de la structure tri dimensionnelle d'OXA révélé de petites différences principalement localisées dans les boucles qui relient des éléments de structure. Notamment, la boucle localisée entre les feuillets β5 et dans l'hydrolyse des carbapénèmes. Afin de mieux comprendre la contribution de la boucle β5-β6 dans l nous avons étudié, grâce à des outils biochimiques et structuraux, des variants naturels ou synthétiques d'OXA 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. 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 β-lactamines possédant un radical volumin (e.g. ceftazidime). De plus, le remplacement AGER Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint Etude structurale et fonctionnell lactamines, Carbapénèmase, Inhibiteur lactamines, grâce à leur efficacité clinique, sont parmi les antibiotiques les plus prescrits pour traiter des infections bactérien utilité est compromise par la prolifération des lactamases (BLs) avec des profils d'hydrolyse de substrats très larges. La résistance induite par les BLs compromet également les d les carbapénèmes). OXA carbapénèmase de classe D ( initialement identifiée dans une souche de de Turquie en 2001. OXA hydrolysent fortement les pénicillines, faiblement les carbapénèmes, et quasiment pas les céphalosporines de troisième génération (C3G). Cependant certains variants comme 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 5 et β6 semble jouer un rôle majeur dans l'hydrolyse des carbapénèmes. Afin de mieux comprendre la contribution de la 6 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 es délétions croissantes ou de l'augmentation de la taille de la boucle. Nous avons également réalisé l'échange de boucle entre OXA 18, une oxacillinase inhibée par l'acide clavulanique et hydrolysant fortement les C3G mais pas les carbapénèmes. La protéine recombinante 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 lactamines possédant un radical volumin (e.g. ceftazidime). remplacement de Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint Etude structurale et fonctionnelle de la spécificité de substrat des carbapénèmases de type arbapénèmase, Inhibiteur 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 lactamases (BLs) avec des profils d'hydrolyse de substrats très larges. La résistance induite par les BLs compromet également les β-lactamines les plus carbapénèmes). OXA carbapénèmase de classe D (CHDL), a été initialement identifiée dans une souche de de Turquie en 2001. OXA hydrolysent fortement les pénicillines, faiblement les carbapénèmes, et quasiment pas les de troisième génération (C3G). Cependant certains variants comme OXA 405 hydrolysent les C3G et pas les carbapénèmes. La comparaison de la structure tri 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 6 semble jouer un rôle majeur dans l'hydrolyse des carbapénèmes. Afin de mieux comprendre la contribution de la 'hydrolyse des carbapénèmes, nous avons étudié, grâce à des outils biochimiques et structuraux, des variants naturels ou 48 présentant des modifications dans la boucle: le rôle du remplacement de chaque AA de la boucle par une es délétions croissantes ou de l'augmentation de la taille de la boucle. Nous avons également réalisé l'échange de boucle entre OXA 18, une oxacillinase inhibée par l'acide clavulanique et hydrolysant fortement les C3G mais La protéine recombinante 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 lactamines possédant un radical volumin de chaque AA Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France e de la spécificité de substrat des carbapénèmases de type arbapénèmase, Inhibiteur lactamines, grâce à leur efficacité clinique, sont parmi les antibiotiques les plus prescrits pour nes. Cependant, leur utilité est compromise par la prolifération des βlactamases (BLs) avec des profils d'hydrolyse de substrats très larges. La résistance induite par les lactamines les plus carbapénèmes). OXA-48, une ), a été initialement identifiée dans une souche de K. de Turquie en 2001. OXA-48 hydrolysent fortement les pénicillines, faiblement les carbapénèmes, et quasiment pas les de troisième génération (C3G). Cependant certains variants comme OXA-163 ou 405 hydrolysent les C3G et pas les carbapénèmes. La comparaison de la structure tri-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 6 semble jouer un rôle majeur Afin de mieux comprendre la contribution de la 'hydrolyse des carbapénèmes, nous avons étudié, grâce à des outils biochimiques et structuraux, des variants naturels ou 48 présentant des modifications dans la boucle: le rôle du remplacement de chaque AA de la boucle par une es délétions croissantes ou de l'augmentation de la taille de la boucle. Nous avons également réalisé l'échange de boucle entre OXA-18, une oxacillinase inhibée par l'acide clavulanique et hydrolysant fortement les C3G mais La protéine recombinante 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 lactamines possédant un radical volumineux AA de la boucle par une alanine a relevé de faibles changements hydrolytiques.

e

  de la spécificité de substrat des carbapénèmases de type boucle par une alanine a relevé de faibles changements hydrolytiques. En réalisant des délétions croissantes d'AA gauche de la boucle (Tyr-211 vers 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 ∆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 β variables.De plus, l'étude de nouveaux variants 48 a permis d'identifier des déterminants structuraux importants dans le profil d'hydrolyse observé. Ainsi, la délétion I215 substitution R214K dans la boucle β5 517 permet une forte hydrolyse des carbapénèmes et des C3G. De même, dans OXA substitution V120L située à proximité de la boucle β6, a pour conséquence une diminution de pour tous les substrats. La chaine latérale plus encombrante de la L120 empêche l'ainsertion lactamines, diminuant l'affinité de l'enzyme. Finalement, nous avons caractérisé OXA lactamase naturelle et chromosomique de Shewanella bicestrii JAB-1 qui, n'ayant uniquement 91,5% d'identité en AA avec OXA même profil d'hydrolyse. OXA 98.9% d'identité en AA avec OXA gène plasmidique, suggérant ainsi que portant le gène bla OXA progéniteur du gène plasmidique Nos travaux ont montré le formidable pouvoir d'adaptation de OXA mutation afin d'accommoder différents substrats, et comment la nature et la longueur de la boucle β6 pouvait influencer sur la spécificité de e de la spécificité de substrat des carbapénèmases de type boucle par une alanine a relevé de faibles changements hydrolytiques. En réalisant des délétions croissantes d'AA soit en partant de la 211 vers Pro-217) ou de 211), nous avons montré que l'activité d'hydrolyse des carbapénèmes diminuait avec la taille des délétions, alors que celle 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 présentait un profile d'hydrolyse avec une forte activité carbapénèmase et C3G. La cristallographie et la modélisation moléculaire ont e flexibilité de la boucle, β-lactamines de tailles variables.De plus, l'étude de nouveaux variants 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 β5-β6 de OXA 517 permet une forte hydrolyse des carbapénèmes et des C3G. De même, dans OXA substitution V120L située à proximité de la boucle équence une diminution de pour tous les substrats. La chaine latérale plus encombrante de la L120 empêche l'ainsertion lactamines, diminuant l'affinité de l'enzyme. Finalement, nous avons caractérisé OXA lactamase naturelle et chromosomique de 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 OXA-535 pourrait être le progéniteur du gène plasmidique bla OXA-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 nfluencer sur la spécificité de e de la spécificité de substrat des carbapénèmases de type boucle par une alanine a relevé de faibles changements hydrolytiques. En réalisant des soit en partant de la 17) ou de la 211), nous avons montré que l'activité d'hydrolyse des carbapénèmes diminuait avec la taille des délétions, alors que celle 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, présentait un profile d'hydrolyse avec une forte activité carbapénèmase et C3G. La cristallographie et la modélisation moléculaire ont e flexibilité de la boucle, lactamines de tailles variables.De plus, l'étude de nouveaux variants 48 a permis d'identifier des déterminants structuraux importants dans le profil d'hydrolyse 16 associée à la β6 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 équence une diminution de pour tous les substrats. La chaine latérale plus encombrante de la L120 empêche l'ainsertion lactamines, diminuant l'affinité de l'enzyme. Finalement, nous avons caractérisé OXA-535, la βlactamase naturelle et chromosomique de 1 qui, n'ayant uniquement 48, présente le 535 présentait 436, codé par un gène plasmidique, suggérant ainsi que S. bicestrii pourrait être le 436 . Nos travaux ont montré le formidable 48 à évoluer par mutation afin d'accommoder différents substrats, et comment la nature et la longueur de la boucle nfluencer sur la spécificité de

Table 1 .

 1 MICs

	of β-lactams for E. coli TOP 10(p-TOPO-OXA-48), its variants and E. coli TOP
	10.

Table 2 .

 2 Steady-state kinetic parameters of OXA-48 and its mutants towards representative β-lactam substrates.

Table 3 .

 3 IC 50 values for OXA-48 deletion mutants for ampicillin and temocillin.

	Paper II. Substrate specificity of OXA-48 modified by β5-β6 loop
	replacement.

Table 1 .

 1 MIC

	of β-lactams for E. coli TOP10 pTOPO-OXA-48, E. coli TOP10 pTOPO-OXA-
	48Loop18, E. coli TOP 10 pTOPO-OXA-18 and E. coli TOP10.
	Table 2. Steady-state kinetic parameters of β-lactamases OXA-48, OXA-48Loop18 and
	OXA-18.

Table 3 .

 3 Crystallography data collection and refinement statistics.

	Paper III. Genetic, biochemical and structural characterization of OXA-517,
	an OXA-48-like extended-spectrum cephalosporins and carbapenems-
	hydrolyzing β-lactamase.

Table 1 .

 1 MICs

	of β-lactam antibiotics for K. pneumoniae 1219, E. coli TOP 10 (pOXA-517),
	E. coli TOP 10 (pTOPO-OXA-48), E. coli TOP 10 (pTOPO-OXA-517).
	Table 2. Kinetic parameters of OXA-48, OXA-517 and OXA-163, with representative β-
	lactam substrates.

Table 3 .

 3 Fifty percent inhibitory concentration (IC 50 ) of clavulanic acid, tazobactam and NaCl for β-lactamases OXA-48 and OXA-517.

Table 4 .

 4 X-ray data collection and structure refinement statistics.

	Paper IV. Genetic and biochemical characterization of OXA-519, a novel
	OXA-48-Like β-lactamase.

Table 1 .

 1 MICs of β-lactams for K. pneumoniae and E. coli strains.

Table 2 .

 2 Steady-state kinetic parameters of β-lactamases OXA-48, OXA-519, and OXA-163.

	Paper V. Genetic and Biochemical Characterization of OXA-535, a distantly-
	related OXA-48-like β-lactamase.

Table 1 .

 1 MIC of β-lactams for Shewanella spp. JAB-1 and E. coli TOP10 pTOPO-OXA-535, TOP10 pTOPO-OXA-48, and TOP10.

Table 2 .

 2 Comparison of the steady-state kinetic parameters of OXA-48 and OXA-535 β-lactamases.

	Paper VI. CTX-M-15-producing Shewanella species clinical isolate expressing
	OXA-535, a chromosome-encoded OXA-48 variant, putative progenitor of the
	plasmid-encoded OXA-436.

Table 1 .

 1 MIC values of Shewanella spp. and transformants.

	Paper VIII. A greater than expected variability among oxa-48-like
	carbapenemases.

Table 1 .

 1 Sequence alignment for the OXA-48-like subfamily of class D β-lactamases.

C.

Analysis of the dimerization of OXA-48.Table 1 .

 1 Kinetic parameters of OXA-48 and OXA-48 with mutations.

Paper IX. SME-4 producing Serratia marcescens from Argentina and comparison with genomes of other SME-producers.

Table 1 .

 1 MIC values of β-lactams for S. marcescens 163, E. coli TOP10 pTOPO-SME-4 and E. coli TOP10.

Table 2 .

 2 Acquired resistance genes of Sm163, SmCH4, SmMex-1, SmS6, SmS8, SmAW, SmUSA-1 and isolates.

Paper X. LMB-1, a novel B3 metalo-β-lactamase from Citrobacter freundii from Argentina. Table 1. Table 1 MIC values of β-lactams for C. freundii 164, E. coli TOP10 pTOPO-CMY-150, E. coli TOP10 pTOPO-CMY-2 and E. coli TOP10. Table 2 Resistance genes as revealed by Resfinder for C. freundii 164 isolate. Table 3. Kinetic parameters of CMY-150 and CMY-2. Table 4 MIC values of β-lactams for C. freundii 164, E. coli TOP10 (pN-LMB-1), E. coli TOP10 (pTOPO-LMB-1) and E. coli TOP10. Table 5. Specific activities for β-lactamases NDM-1 and LMB-1 Paper XI. Escherichia coli clinical isolates harbouring mcr-1 and mcr-1.5 genes recovered from a University Hospital in Buenos Aires, Argentina. Table 1. Characteristics of colistin-resistant E. coli clinical isolates. Table 2. Characteristics of colistin resistant E. coli clinical isolates and their acquired resistance genes. Paper XII. Rapid detection and discrimination of chromosome-and MCRplasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test.

Table 1 .

 1 Detection and characterization of polymyxin resistance using conventional techniques (MICs and PCR) and the MALDIxin test on 78 carbapenemase-producing E. coli strains received by the French NRC during October and November 2016.

Scientific Objectives ................................................................................... 55 Results .......................................................................................................... 57 A. Analysis of the β5-β6 loop of OXA-48 through mutants produced in the laboratory .................................................................................. 58 Paper I. Role of the loop β5-β6 in the substrate specificity of OXA-48 ....... 60 Paper II. Substrate specificity of OXA-48 modified by β5-β6 loop replacement .................................................................................................. 86 B. Analysis of the substrate specificity of natural OXA-48 variants recovered from clinical isolates ........................................................ 107 Paper III. Genetic, biochemical and structural characterization of OXA-517, an OXA-48-like extended-spectrum cephalosporins and carbapenemshydrolyzing β-lactamase ............................................................................ 109 Paper IV. Genetic and biochemical characterization of OXA-519, a novel OXA-48-Like β-lactamase ........................................................................... 131 Paper V. Genetic and Biochemical Characterization of OXA-535, a distantlyrelated OXA-48-like β-lactamase ............................................................... 137

Acknowledgements

There are many who, one way or the other, have helped me to get to this point and I would very much like to thank them like they deserve: I would like to acknowledge Dr. Thierry Naas, first of all for having given me the enormous opportunity to be part of his laboratory. Secondly for entrusting me with this fantastic project with the most amazingly fun enzyme, and last but not least, for guiding me all these years in the scientific field, and also for always having the time to hear me and give me advise. I would also like to acknowledge Dr. Bogdan Iorga for teaching me about the fantastic world of structural biology and having the patience to explain to me and correct me.

I would also like to acknowledge Dr. Neil Woodford and Dr. Jean-Denis Docquier for the honor of having them as reporters of my thesis.

At the same time, I would like to express my sincerest thanks to Dr. Claire Janoir, Dr. Pierre Bogaerts and Dr. Wladimir Sougakoff, for having accepted to be part of the jury.

My particular thanks to Dr. Gabriel Gutkind for being part of the jury and especially for always believing in me and encouraging me to go for more. I'm very grateful towards my collaborators, especially, everyone at Pasteur institute and ICSN, and Dr. Rodriguez, Dra. Nastro and all the Argentinian lab members for giving me strength and for all the advice. I would also like to thank to Agustin Zavala for his UNCONDITIONAL support and for having accepted to embark on this adventure with me. I want also to thank the Zavala family for their support.

My particular thanks to Julie Takissian for always being there to help me, talk and make every day nicer.

A special acknowledgement to Saoussen Oueslati, Drs Bonnin, Dortet, Girlich, and Jousset, for their help, advice and encouraging words.

I also thank all the members of the research unit who have at one time or another helped me, and who have made working in the lab a great experience: Thibaud, Garance, Elodie, Sandrine and Florence. I would also like to acknowledge my friends, the ones here and the ones on the other side of the ocean for giving me the strength and laughs to cope with the stress.

Finally, yet importantly, my family, for teaching me that it is important to grow professionally but most important is growing as a person. Thank you for giving me roots and wings.

Finalmente pero no menos importante, quiero agradecerle a mi familia por haberme enseñado que es importante crecer en lo profesional, pero más importante es crecer como persona. Gracias por darme raíces y alas. Para Papa, Mama, Fran y Lili