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Aix-Marseille Université Vivek KESHRI Aix-Marseille Université Faculté de Médecine de Marseille Ecole Doctorale des Sciences de la Vie et de la Santé THÈSE DE DOCTORAT Présentée par Vivek KESHRI Date et lieu de naissance: 20-Octobre-1985, Inde Evolutionary Analysis of the β-lactamase Families (Analyse évolutive des familles de β-lactamase) Soutenance de la thèse le 05-Juillet-2018 En vue de l’obtenir du grade de Docteur de l’Université d’Aix-Marseille Membres du jury de la thèse Pr Didier RAOULT Directeur de Thèse Pr Max MAURIN Rapporteur Dr Patricia RENESTO Rapporteur Pr Pierre-Edouard FOURNIER Examinateur Laboratoire d’accueil IHU - Méditerranée Infection, 19-21 Boulevard Jean Moulin, Marseille, France Contents Abstract .......................................................................................................................................... 1 Résumé ........................................................................................................................................... 3 Chapter-1 ....................................................................................................................................... 7 Introduction ................................................................................................................................. 7 Chapter-2 (A) .............................................................................................................................. 13 Phylogenomic analysis of β-lactamase in archaea and bacteria enables the identification of putative new members ............................................................................................................... 13 Chapter-2 (B) ............................................................................................................................... 32 Metallo-β-lactamase enzymes in humans and archae ............................................................... 32 Chapter-3 ..................................................................................................................................... 60 Functional convergence of antibiotic resistance in β-lactamase enzymes is not conferred by simple convergent amino acid substitution ............................................................................... 60 Chapter-4 ..................................................................................................................................... 92 An integrative database of β-lactamase enzymes: sequences, structures, functions and phylogenetic trees ...................................................................................................................... 92 Conclusions ................................................................................................................................ 109 Future Perspective .................................................................................................................... 110 Acknowledgements ................................................................................................................... 114 Abstract This thesis describes three-phase of research investigations. First, Phylogenomic analysis of β- lactamase in archaea and bacteria enables the identification of putative new members followed by hidden sources of metallo-β-lactamases; second, the functional convergent evolution of β- lactamase and third, development of an integrative database of β-lactamase. The β-lactam antibiotics are one of the oldest and widely used antimicrobial drugs. The bacterial enzyme β-lactamase (falls into four molecular classes A, B, C, and D) hydrolyzes the β- lactam antibiotic by breaking the core structure “β-lactam ring”. Antibiotic resistance genes (ARGs) are widely distributed in the natural environment. To identify the novel β-lactamases a comprehensive investigation was performed in different biological databases such as Human Microbiome Project (reference and metagenomic), environmental protein (env_nr), and NCBI’s non-redundant (nr). For identification of putative new sequences and uncovering the diversity, the ancestral sequence and HMM profile of extant β-lactamases were inferred which enables to identify potential homologous sequences from biological databases. The analysis revealed that putative ancestral sequences and HMM profile searches played a significant role in the identification of remote homologous and uncovered the existing β-lactamase enzyme in the metagenomic database as dark-matter. The comprehensive phylogenetic analyses of extant and newly identified β-lactamase represent the novel clades in the trees. Further, the β-lactam antibiotic hydrolysis activity of newly identified sequences (from archaea and human) was investigated in laboratory, which shows β-lactamase activity. The second phase of the investigation was undertaken to examine the functional evolution of β-lactamases. First, 1155 β-lactamase protein sequences were retrieved from ARG- ANNOT database and Minimal Inhibitory Concentration (MIC) values, the measure of antibiotic function, from the corresponding literature. Molecular phylogenetic framework was applied to examine the evolution of β-lactamase functional activity, in particularly the evolutionary association of molecular and phenotypic characteristics. The results revealed that the functional activity of β-lactamase evolved convergently within the molecular class (intra-class level). The third phase of this thesis presents development of an integrative β-lactamase database. The existing public database of β-lactamase has limited information, therefore, an integrative database was developed. The database provides extensive information about the 1 sequences; primary amino acid sequences, the closest structural information in the external structure database- PDB, the functional profile, and phylogenetic trees in a single database. The functional profiles are accessible in the form of Minimum Inhibitory Concentration (MIC) and kinetic parameters. As of now, 1155 antibiotic resistance gene sequences are available in the current database. The database will facilitate to research community working in the field of β- lactam antibiotic resistance. Key words: β-lactam antibiotics, β-lactamase enzymes, ancestral sequence, HMM profile, convergent evolution, β-lactamase database. 2 Résumé Cette thèse décrit trois phases d'enquêtes de recherche. Premièrement, l'analyse phylogénomique de β-lactamases chez les archées et les bactéries permettant l'identification de nouveaux membres putatifs suivis de sources cachées de métallo-β-lactamases; deuxièmement, l'évolution fonctionnelle convergente de β-lactamases et la troisième, le développement d'une base de données intégrative de β-lactamases. Les antibiotiques β-lactamines sont parmi les médicaments antimicrobiens les plus anciens et les plus utilisés. L'enzyme bactérienne β-lactamase (se divise quatre classes moléculaires A, B, C et D) hydrolyse l'antibiotique β-lactame en cassant la structure de base "anneau β-lactame". Les gènes de résistance aux antibiotiques (GRA) sont largement distribués dans l'environnement naturel. Pour identifier les nouvelles β-lactamases, une étude complète a été réalisée dans diverses bases de données biologiques telles que Human Microbiome Project (référence et métagénomique), protéine environnementale (env_nr) et NCBI's non redondant (nr). Pour l'identification de nouvelles séquences putatives et la découverte de la diversité, la séquence ancestrale et le profil HMM de β-lactamases existantes sont déduits, ce qui permet d’identifier des séquences homologues potentielles à partir de données biologiques. L'analyse a révélé que les séquences ancestrales putatives et les recherches de profil HMM jouaient un rôle important dans l'identification de la base de données homologue et métagénomique à distance dans l'enzyme β-lactamase existante comme matière noire. Les larges analyses phylogénétiques des β-lactamases existantes et nouvellement identifiées représentent les nouveaux clades dans les arbres. En outre, l'activité d'hydrolyse des antibiotiques β-lactamines de séquences nouvellement identifiées (provenant d'archées et d'humains) a été étudiée en laboratoire, ce qui montre l'activité de la β-lactamase. La deuxième phase de l'étude a été entreprise pour examiner l'évolution fonctionnelle des β-lactamases. Premièrement, des séquences de protéines ß-lactamase 1155 ont été extraites de la base de données ARG-ANNOT et des valeurs de concentration minimale inhibitrice (CMI), la mesure de la fonction d’antibiotique, d'après la littérature correspondante. Un cadre phylogénétique moléculaire a été appliqué pour examiner l'évolution de l'activité fonctionnelle de la β-lactamase, en particulier l'association évolutive de l'anthrax et des caractéristiques phénotypiques. Les résultats ont révélé que l'activité fonctionnelle de la β-lactamase évoluait de manière convergente au sein de la classe moléculaire (niveau intra-classe). 3 La troisième phase de cette thèse représente le développement d'une base de données intégrative de β-lactamases. La base de données publique actuelle de β-lactamases a des informations limitées, par conséquence, une base de données intégrative a été développée. La base de données fournit des informations générales sur les séquences; séquences d'acides aminés primaires, les informations structurelles les plus proches dans la base de données de structure externe-PDB, le profil fonctionnel et les arbres phylogénétiques dans une base de données unique. Les profils fonctionnels sont accessibles sous forme de
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