Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-05-20T11:06:25Z No. of bitstreams: 2 Dissertação - Rogerio Coutinho das Neves - 2015.pdf: 6048850 bytes, checksum: 1bac6d1d4aa28a3cf0ebbb228b593986 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-05-20T11:12:51Z (GMT) No. of bitstreams: 2 Dissertação - Rogerio Coutinho das Neves - 2015.pdf: 6048850 bytes, checksum: 1bac6d1d4aa28a3cf0ebbb228b593986 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-05-20T11:12:51Z (GMT). No. of bitstreams: 2 Dissertação - Rogerio Coutinho das Neves - 2015.pdf: 6048850 bytes, checksum: 1bac6d1d4aa28a3cf0ebbb228b593986 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Outbreaks caused by atypical mycobacteria are considered as an emerging problem in Brazil and Goias, being frequently associated with invasive procedures, such as laparoscopy, arthroscopy, and aesthetic surgeries among others. Treatment of mycobacteriosis requires highly toxic drugs with doubtful sterilizing effects. Consequently, several studies aim to search for new drugs or molecules with anti-mycobacterial biological activities. The objective of this study was to prospect biomolecules from the venom of the social wasp Polybia paulista (Vespoidea, Vespidae, Polistinae) and from the Tityus sp scorpion with antimycobacteria activity against Mycobacterium abscessus subsp. bolletii (GO06). Peptides from the wasp and scorpion venoms were evaluated in broth microdilution assay and in macrophage infected cultures to determine minimum inhibitory concentration. Peptides with antimycobacterial activity were further evaluated by scanning electron microscopy of M. abscessus subsp. bolletii treated cultures. The peptide toxicities were evaluated on macrophage cultures. Peptides Agelaia-MP, Polybia-MPII, and AVA, derived from wasp and peptides AMP 1, 2, 4, 6, and 8 derived from scorpion presented antimycobacterial activities. AVA and AMP 4 peptides were not toxic for macrophages. In this work three peptides derived from the wasp venom and five peptides derived from the scorpion venom were identified as having antimycobacterial activity. / Surtos causados por micobactérias atípicas são considerados emergentes no Brasil e em Goiás e têm tem sido associados a procedimentos invasivos, como a laparoscopia, artroscopia e cirurgia estética dentre outros. O tratamento de micobacterioses envolve drogas altamente tóxicas que apresentam efeito esterilizante relativo. Portanto, vários estudos têm como alvo a busca de novas drogas ou moléculas biológicas com atividade micobactericida. O objetivo deste estudo foi prospectar biomoléculas do veneno de vespa social Polybia paulista (Vespoidea, Vespidae, Polistinae) e de escorpiões do gênero Tityus com atividade micobactericida in vitro contra Mycobacterium abscessus subsp. bolletii (GO06). Peptídeos derivados do veneno da vespa e do escorpião foram avaliados por microdiluição em caldo, e em culturas de macrófagos infectados determinando a concentração inibitória mínima. Para os peptídeos com atividade micobactericida, o potencial dano causado à parede celular da micobactéria foi analisado por microscopia eletrônica de varredura. A toxicidade de dois peptídeos com potencial uso farmacológico foram avaliados em culturas de macrófagos peritoneais de camundongos BALB/c. Os peptídeos Agelaia-MP, Polybia-MPII e AVA derivados do veneno da vespa e os peptídeos AMP 1, 2, 4, 6 e 8 derivados do veneno de escorpião apresentaram atividade micobactericida. Os peptídeos micobactericidas agem desestruturando os envoltório das bacilos de M. abscessos subsp. bolletti. Os peptídeos AVA e o AMP 4 não apresentaram toxicidade para culturas de macrófagos, e reduziram a carga bacilar de culturas de macrófagos infectadas com M. abscessus subsp. bolletii. Neste trabalho identificou-se três peptídeos derivados da peçonha de vespa e cinco peptídeos derivados de veneno de escorpião com atividade micobactericida.

Orientadores: Carlos Francisco Sampaio Bonafé, Marcelo Lancellotti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T08:59:23Z (GMT). No. of bitstreams: 1 Souza_AncelmoRabelo_M.pdf: 2604011 bytes, checksum: 68977693022bd1bef29d71963a2562d5 (MD5) Previous issue date: 2012 / Resumo: Mycobacterium abscessus é um importante patógeno de origem hospitalar que contamina materiais cirúrgicos e biofarmacêuticos. Estes quando mal esterilizados tornam-se agentes infectantes gerando graves patologias nas pessoas que os utilizam. A crescente incidência desse patógeno, as dificuldades de tratamento, a gravidade clínica e, a dificuldade de esterilizar fômites contaminados com este patógeno motivou a investigação de processos alternativos de esterilização. Atualmente, utiliza-se a alta pressão hidrostática como método bastante adequado para reduzir a carga microbiana e esterilizar materiais hospitalares e biofarmacêuticos que são sensíveis à autoclave. Desta forma, investigou-se a inativação de M. abscessus induzida por pressão em diferentes condições de temperatura e de pH visando progredir em direção a um método de esterilização. De acordo com os resultados obtidos, tratamentos a 250 MPa não inativaram significativamente (5 a 8 ordens de magnitude) a bactéria em até 90 min a 20°C. Entretanto em -15°C a inativação foi completa. O tratamento a 250 MPa a 60ºC por 45 min promoveu significativa inativação bacteriana de até 9 unidades logarítmicas, incluindo o teste com PVC. Além disso, extremos de pH (4 ou 9) também diminuíram acentuadamente o número de bactérias tratados por alta pressão (250 MPa), com inativação completa após 45 min. Assim, este trabalho torna-se de suma importância, uma vez que aponta para o melhoramento de protocolos de descontaminação de fômites hospitalares / Abstract: Mycobacterium abscessus is an important hospital-acquired pathogen which induces infections from medical surgical and biopharmaceutical materials. The increasing incidence of this pathogen, the difficulties of treatment and clinical seriousness motivates the investigation of alternatives in sterilization processes. High hydrostatic pressure is a very adequate method for reducing microbial load and for sterilization of hospital materials and biopharmaceutical that are sensitive to autoclaving. We investigated the pressure-induced inactivation of M. abscessus combined with different temperatures and pH conditions aiming improvements toward a sterilization methodology. According to our findings, treatment at 250 MPa did not inactivate the bacteria significantly (5 to 8 orders of magnitude) up to 90 min at 20 oC, nevertheless at -15°C the inactivation was complete. The treatment at 250 MPa and 60ºC or above by 45 min promoted significant bacteria inactivation, at least 9 logarithmic units, including the test PVC. Extremes of pH values (4 or 9) also decrease very much the bacteria number induced by pressure (250 MPa), with complete inactivation at 45 min. The better knowledge of the effect of high hydrostatic pressure in micobacteria may contribute to improvements in the decontamination of medical materials and pharmaceuticals / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Mycobacterium abscessus est une mycobactérie pathogène émergente à croissance rapidecapable d’induire des infections pulmonaires sévères, notamment chez les patients atteints demucoviscidose. Naturellement résistant à la plupart des antibiotiques disponibles, sa présence peut êtreune contre-indication à la greffe pulmonaire chez ces patients. De plus, l’efficacité des traitementsactuellement préconisés est très fluctuante d’un individu à l’autre et s’accompagne souvent d’effetssecondaires indésirables. Il apparaît donc urgent de développer des stratégies thérapeutiques innovanteset/ou d’identifier de nouvelles cibles pharmacologiques dans la lutte contre ce pathogène. Ce projet dethèse, focalisé sur M. abscessus, est centré sur l’étude mécanistique et fonctionnelle des transporteursmembranaires de la famille des MmpL (Mycobacterial Membrane Protein Large) en rapport avec leur rôledans i) l’élaboration de l’enveloppe mycobactérienne, ii) la physiopathologie infectieuse et iii) leurvalidation en tant que cible d’intérêt thérapeutique.Ainsi, mes travaux ont permis de mettre à jour le rôle clé de MmpL4a dans le transport desglycopeptidolipides (GPL) chez Mycobacterium bolletii, une sous-espèce du complexe M. abscessus et dedémontrer sa participation dans la formation de biofilms, des cordes mycobactériennes et dans la virulencede M. bolletii chez l’embryon de zebrafish. De plus, ces résultats mettent en exergue l’importance derésidus critiques impliqués dans l’établissement de la force proto-motrice, conservés chez toutes les MmpL,et nécessaires à l’activité de ces transporteurs. Enfin, ces études ont abouti à l’identification d’une nouvelleentité chimique très active contre M. abscessus à la fois in vitro et in vivo. Ce composé phare est capable decibler le MmpL3, conduisant à une inhibition du transport des acides mycoliques, connus comme étant descomposants essentiels chez les mycobactéries. Ces travaux représentent le premier exemple d’un inhibiteurciblant spécifiquement le transport d’acides mycoliques chez M. abscessus, ouvrant la voie vers desapplications thérapeutiques prometteuses. / Mycobacterium abscessus is a rapid- growing mycobacterial species and an emerging pathogen,responsible for severe lung infections, particularly in cystic fibrosis patients. Naturally resistant to mostcurrently available antibiotics, its presence can represent a contraindication to subsequent lungtransplantation in these patients. That the currently recommended treatments present fluctuating resultsamong patients and are often associated with important side effects, imposes to develop innovativetherapeutic strategies and/or to identify new pharmacologically relevant targets in the fight against thispathogen. This project, focusing on M. abscessus, is intended to explore mechanistic and functionalaspects of members of the MmpL (Mycobacterial Membrane Protein Large) family of membrane proteinswith respect to i) their role in elaboration of the mycobacterial envelope, ii) their involvement in thephysiopathology of the infection and iii) their validation as potentially attractive therapeutic drug targets.In this context, my studies unraveled the key role of MmpL4a in the transport of glycopeptidolipids (GPL)in Mycobacterium bolletii, a sub-species of the M. abscessus complex and demonstrated its implication inthe formation of biofilms, cording and virulence of M. bolletii in infected zebrafish. Moreover, theseresults put forward the importance of critical residues, conserved in all MmpL members, involved in theestablishment of the proton-motive force required for the activity of these transporters. Finally, thesestudies succeeded in the identification of a new chemical entity exhibiting a potent activity against M.abscessus both in vitro and in vivo. This hit compounds targets MmpL3, leading to the inhibition of thetransport of mycolic acids, known to represent essential components of the mycobacterial cell wall. Theseresults emphasize the mode of action of the first specific inhibitor of mycolic acid transport in M.abscessus, paving the way to promising therapeutic applications.

Mycobacterium abscessus est une mycobactérie à croissance rapide, et un pathogène opportuniste responsable d’infections pulmonaires notamment chez les patients atteints de la mucoviscidose, et d’infections cutanéomuqueuses. La source de contamination pourrait être environnementale mais des contaminations interhumaines ne sont pas exclues. Les amibes environnementales pourraient jouer un rôle de réservoir. M. abscessus est capable de résister aux mécanismes de défense bactéricides des phagocytes environnementaux et humains. Le génome complet de M. abscessus a été séquencé mettant en évidence de nombreux facteurs de virulence non mycobactériens. Certains sont des facteurs de virulence connus dans le monde bactérien, comme la phospholipase C ou le facteur de captation du magnésium MgtC. Ces facteurs ont été montré induits en présence d’amibes, mais ne peuvent à eux seuls expliquer la survie intracellulaire et la virulence de M. abscessus. Nous avons donc, au cours de ce projet, criblé une banque de mutants générée par transposition chez M. abscessus, à la recherche de mutants dénués de croissance intracellulaire en amibes et macrophages. Cette approche a permis d’identifier, de façon majeure, 5 gènes du locus ESX-4 de M. abscessus codant un système de sécrétion de type VII avec tous ces composants cœur conservés. Pour mieux comprendre la contribution d’ESX-4 dans la survie intracellulaire de M. abscessus, un mutant obtenu par double recombinaison au sein du gène eccB4 dans la souche type de M. abscessus (ΔeccB4) a été construit. EccB4 est un élément structurel central du système de sécrétion codé par ESX-4. ΔeccB4 présente un défaut de survie au sein des cellules, lié à déficit de blocage de l’acidification phagosomale ainsi qu’un défaut de dégradation de la membrane phagosomale, empêchant un contact phagosome-cytosol. Ce travail a permis de révéler pour la première fois dans le monde mycobactérien le rôle d’un locus ancestral de sécrétion ESX-4 au sein d’une mycobactérie. L’étude des protéines secrétées par ce locus est actuellement en cours au laboratoire, afin d’envisager des approches thérapeutiques et vaccinales pour contrer cette mycobactérie multirésistante aux antibiotiques. / Mycobacterium abscessus is a fast growing mycobacterium, and an opportunistic pathogen responsible for lung infections particularly in patients with cystic fibrosis, and for mucocutaneous infections. The source of contamination could be environmental but human-to-human contaminations are not excluded. Environmental amoeba could play a role as a reservoir. M. abscessus is able to resist to the bactericidal defense mechanisms of environmental and human phagocytes. The complete genome of M. abscessus has been sequenced and presents numerous non-mycobacterial virulence factors. Some are known virulence factors in the bacterial world, such as phospholipase C or the magnesium uptake factor MgtC. These factors have been shown to be induced in the presence of amoeba, but cannot alone explain the intracellular survival and virulence of M. abscessus. We thus, in the course of this project, screened a library of mutants generated by transposition in M. abscessus, in search of mutants lacking intracellular growth in amoeba and macrophages. This approach made it possible to identify, in a major way, 5 genes of the ESX-4 locus of M. abscessus encoding a type VII secretion system with all these conserved core components. In order to better understand the contribution of ESX-4 to the intracellular survival of M. abscessus, a mutant obtained by double recombination within the eccB4 gene in the M. abscessus type strain (ΔeccB4) was constructed. EccB4 is a central structural element of the secretion system encoded by ESX-4. ΔeccB4 has a defect of survival within the cells, linked to deficiency of blockage of the phagosomal acidification as well as a defect of degradation of the phagosomal membrane, preventing phagosome-cytosol contact. This work made it possible to reveal for the first time in the mycobacterial world the role of an ancestral locus ESX-4 secretion within a mycobacterium. The study of the proteins secreted by this locus is currently underway in the laboratory, in order to consider therapeutic and vaccine approaches to counter this multiresistant antibiotic mycobacterium.

Made available in DSpace on 2016-12-23T13:56:10Z (GMT). No. of bitstreams: 1 Dissertacao de Tamea Aparecida Linhares Possa.pdf: 2318772 bytes, checksum: 8fc876ab052a92c8f592b53ce4064216 (MD5) Previous issue date: 2011-08-23 / Mycobacterium abscessus is currently slow-growing mycobacteria considered more pathogenic. Cause infections, especially in patients with cystic fibrosis and has been described with increasing frequency in infections of skin and soft tissues secondary to invasive procedures such as acupuncture, insulin injections, mesotherapy, and laparoscopic and arthroscopic procedures after undergoing cosmetic procedures in plastic surgery like breast implants and implant lipoplasty. Although very lethal, infection has a high morbidity mainly due to the intrinsic resistance to several antimicrobial M. abscessus, considered the toughest mycobacteria. In order to study the clinical, therapeutic and microbiological infections of skin and soft tissue related to surgical procedures for aesthetic M. abscessus, we used a descriptive study with data found in the medical records of 16 patients with infection of skin and soft tissues treated at a university reference state. We analyzed the clinical features of infection during the initial presentation, after removal of the prosthesis implanted in the first query in reference service, during and after the treatment. Given the small sample size was not carried out assessment of statistical significance, but the results show that treatment response is varied, the withdrawal of the implanted prosthesis contributes to initial resolution of symptoms, treatment is prolonged and the recommended treatment schedule is difficult to be performed due to the adverse effects mainly related to amikacin. The clinical evolution does not occur in a linear fashion, even during treatment. There are patients who develop spontaneous resolution of infection after surgical debridement and removal of foreign bodies, patients who respond to treatment patients whose treatment seems not to interfere in the evolution and patients in whom treatment appears to contribute to the intensification of the signs and symptoms . Despite the different types of evolution, all the patients examined were considered cured at the end of the study / O Mycobacterium abscessus é atualmente a micobactéria de crescimento lento considerada mais patogênica. Causa infecções, principalmente em pacientes portadores de fibrose cística e tem sido descrito com frequência cada vez maior em infecções de pele e partes moles secundárias a procedimentos invasivos tais como acupuntura, injeções de insulina, mesoterapia, procedimentos artroscópicos e laparoscópicos e após realização de procedimentos estéticos em cirurgias plásticas como implante de próteses mamárias e lipoplastia. Embora de baixa letalidade, a infecção apresenta alta morbidade principalmente devido a resistência intrínseca do M. abscessus a vários antimicrobianos, sendo considerado a micobactéria mais resistente. Com o objetivo de estudar os aspectos clínicos, terapêuticos e microbiológicos das infecções de pele e partes moles relacionados a procedimentos cirúrgicos estéticos por M. abscessus, utilizou-se um estudo descritivo com dados encontrados em prontuário de 16 pacientes com infecção de pele e partes moles tratados no serviço Universitário de Referência Estadual. Foram analisados os aspectos clínicos da infecção durante a apresentação inicial, após retirada das próteses implantadas, na primeira consulta no serviço de referência, durante o tratamento e após o término do tratamento. Devido ao reduzido tamanho da amostra não foi realizado avaliação de significado estatístico, porém os resultados mostram que a resposta ao tratamento é variada, a retirada da prótese implantada contribui para resolução inicial dos sintomas, o tratamento é prolongado e o esquema de tratamento recomendado é difícil de ser realizado devido aos efeitos adversos principalmente relacionados à amicacina. A evolução clínica não ocorre de maneira linear, mesmo na vigência do tratamento. Há paciente que evoluem com resolução espontânea da infecção após debridamento cirúrgico e retirada de corpos estranhos; pacientes que respondem ao tratamento; pacientes em que o tratamento parece não interferir na evolução e pacientes em que o tratamento parece colaborar para intensificação dos sinais e sintomas. Apesar dos diferentes tipos de evolução, todas as pacientes analisadas foram consideradas curadas ao final do estudo

Les phospholipases et en particulier les phospholipases C sont d'importants facteurs de virulence chez de nombreuses bactéries pathogènes (C. perfringens, B. Cereus et P. aeruginosa). Cependant, peu de choses sont connues sur l'implication de ces enzymes dans le processus de virulence des mycobactéries. Bien que l'étude des mutants des phospholipases C de M. tuberculosis dans un modèle d'infection chez la souris ait permis de proposer une implication de ces protéines dans la virulence de ce bacille, leurs propriétés biochimiques, leur mode d'action et leur rôle physiologique exact restent à élucider. Ce manque de données biochimiques sur les phospholipases mycobactériennes peuvent être attribuée à la difficulté à produire et à purifier des quantités importantes de ces enzymes. Dans le but de mieux caractériser le rôle physiologique des phospholipase mycobactériennes, l'objectif de ma thèse a été de mettre au point des conditions d'expression hétérologue permettant la production des phospholipases C mycobactériennes recombinantes (rPLC) dans différents systèmes d'expression (E. coli, Pichia pastoris et baculovirus/cellules d'insectes). Ces systèmes d'expression n'ayant pas donné des résultats satisfaisants, nous avons développé une méthode efficace d'expression de ces protéines en utilisant M. smegmatis.Ce système d'expression nous a permis de produire et de purifier les quate PLC (PLC-A, PLC-B, PLC-C et PLC-D) de M. tuberculosis et la PLC de M. Abscessus sous forme soluble et active. Nous avons pour la première fois montré que ces protéines purifiées avaient un effet cytotoxique sur les macrophages de souris en culture mais ne présentaient aucune activité hémolytique. en utilisant des marquages radioactifs, nous avons confirmé que l'effet cytotoxique observé était lité à l'hydrolyse des phospholipides des membranaires des cellules hôtes. Pour la première fois, nous avons pu confirmer que ces PLC sont directement impliquées dans le processus d'infection et de virulence.Un autre aspect de mon travail de thèse a concerné l'étude de deux autres protéines sécrétées par M. tuberculosis appartenant à la famille des cutinases : la Rv1984c et la Rv3452. Après les avoir produites et purifiées chez E. Coli, nouq avons montré que malgré ces deux protéines présentent 50% d'identité de séquence en acides aminés, elles ont des spécificités de substrat différentes et probablement un rôle physiologique différent. La Rv1984c est une lipase capacle d'hydrolyser des lipides à chaines moyennes, alors que la Rv3452 est une phospholipase de type A2 et est capable d'induire la lyse de macrophage de souris en culture. / Phospholipases, particularly phospholipases C, are important virulence factors in several pathogenic bacteria (C. perfringens, B. cereus, L. monocytogenese and P. aeruginosa). However, little is know on the involvement of thses enzymes in mycobacteria pathogenesis. Although study on M. tuberculosis phospholipases C mutants in a mouse aerosol model of infection gave rise to the contribution of these proteins in virulence process, but their exact biochemical properties, mechanism of action and physiological role remain to be elucidated. This lack of data on mycobacterial phospholipases is mainly due to the difficulty to produce and purify these enzymes in large scale.With the aim to better characterise the physiological role of mycobacterial phospholipases, the main challenge of my thesis was to develop an efficient method for expression and purification of recombinant mycobacterial phospholipases C. Since no satisfactory results have been obtained with standard expression systems (E. coli, Pichia pastoris and baculovirus / insect cells), we develop a robust expression technique for these proteins using M. smegmatis as expression system.This allowed us to produce and purify all four PLC (PLC-A, PLC-B, PLC-C and PLC-D) of M. tuberculosis and the PLC of M. abscessus in soluble and active form. For the first time, we have show, that purified proteins have cytotoxic effect on mouse macrophages but have not haemolytic activity. Using radiolabelled lipids, we have confirmed that this first direct evidence that PLC are involved in infection and virulence processes. Another aspect of my thesis work concerned the study of two other secreted proteins of M. tuberculosis belonging to the cutinase family : the Rv 1984c ant the Rv3452. Recombinant proteins obtains in E. coli were found to have distinct substrate specificities and most likely distict physiological role, despite showing 50% amino acids sequence identity. Rv1984c is a lipase and is able to hydrolyse lipids with medium chains lengthn whereas Rv3452 is type A2, phospholipase and i able to induce macrophage lysis.

L’émergence de formes multirésistantes de tuberculose et la résistance intrinsèque de Mycobacterium abscessus à de nombreux anti-infectieux imposent l’identification de nouveaux antibiotiques et de nouvelles stratégies thérapeutiques. Les mycobactéries sont naturellement peu sensibles aux β-lactamines par production d’une β-lactamase et de cibles atypiques de faible affinité, les L,D-transpeptidases, qui sont efficacement inactivées par une seule classe de β-lactamines, les carbapénèmes. L’objectif de la thèse est d’étudier le mode d’action des β-lactamines afin de proposer des stratégies permettant d’optimiser ces antibiotiques. Pour comprendre la spécificité des L,D-transpeptidases vis-à-vis des carbapénèmes, nous avons étudié la cinétique et le mécanisme de la réaction d’inactivation de ces enzymes par différentes méthodes de spectroscopie en flux arrêté. Nos résultats indiquent que l’efficacité des carbapénèmes est due à leur capacité à former rapidement un intermédiaire tétrahédrique et à la stabilité de l’acylenzyme. La spécificité des L,D-transpeptidases pour les carbapénèmes ne dépend pas de leurs chaînes latérales, qui pourraient être modifiées pour améliorer les propriétés pharmacologiques de ces antibiotiques. Chez M. abscessus, nous avons identifié un inhibiteur de la β-lactamase, l’avibactam, qui augmente l’activité de certaines β-lactamines in vitro, en intracellulaire et dans un modèle d’infection du poisson zèbre. Nos résultats montrent que les β-lactamines peuvent être optimisées pour le traitement des infections dues aux mycobactéries multirésistantes par l’amélioration de l’inactivation des cibles ou l’inhibition des β-lactamases. / The emergence of multidrug-resistant tuberculosis and the intrinsic resistance of Mycobacterium abscessus to most antibiotics require the identification of new drugs and new therapeutic strategies. Mycobacteria are naturally poorly susceptible to β-lactam antibiotics due to production of a β-lactamase and of atypical low-affinity targets, the L,D-transpeptidases, which are effectively inactivated by a single class of β-lactams, the carbapenems. The aim of the thesis is to study the mode of action of β-lactams to propose strategies for the optimization of these antibiotics. To understand the specificity of L,D-transpeptidase for carbapenems, we have studied the kinetics and mechanism of inactivation of these enzymes using various stopped-flow spectroscopic methods. Our results indicate that the efficacy of carbapenems is due to their ability to rapidly form a tetrahedral intermediate and to the stability of the acylenzyme. The specificity of the L,D-transpeptidases for carbapenems does not depend upon the side chains of the drugs, which may be modified to improve their pharmacological properties. In M. abscessus, we have shown that the β-lactamase inhibitor avibactam increases the activity of various β-lactams in vitro, intracellularly, and in zebrafish model. Our results show that β-lactams can be optimized for the treatment of infections due to multidrug-resistant mycobacteria by improving inactivation of the targets and by inhibiting the β-lactamases.

Mycobacterium tuberculosis et Mycobacterium abscessus produisent les β-lactamases BlaC et BlaMab qui contribuent à la résistance intrinsèque de ces bactéries aux β-lactamines. Notre objectif est de caractériser l’inhibition de ces β-lactamases par l’avibactam et le clavulanate pour contribuer au développement de nouveaux traitements. Nous avons déterminé le profil de substrat et d’inhibition de BlaMab ainsi que sa structure cristalline, révélant trois différences majeures avec BlaC. BlaMab a une activité supérieure à celle de BlaC pour toutes les β-lactamines sauf la céfoxitine qui est utilisée pour les infections dues à M. abscessus. BlaC est inhibée irréversiblement par le clavulanate et inefficacement par l’avibactam alors que BlaMab présente le comportement inverse impliquant une hydrolyse du clavulanate et une inhibition très rapide par l’avibactam. La structure de BlaMab diffère de celle de BlaC principalement par le remplacement du motif conservé SDN par SDG. L’introduction de SDG dans BlaMab et de SDN dans BlaC a montré que cette différence détermine le profil d’inhibition des β-lactamases. Une seule mutation peut donc entraîner l’émergence d’une résistance aux combinaisons d’une β-lactamine avec le clavulanate ou l’avibactam mais pas avec les deux inhibiteurs. L’avibactam et le clavulanate offrent donc des alternatives thérapeutiques en cas de résistance à l’un des inhibiteurs. Nous nous sommes également intéressés aux β-lactamines partenaires du clavulanate, pour le traitement de la tuberculose et montrer que la structure des carbapénèmes pouvait être optimisée pour améliorer l’inactivation des cibles et diminuer l’hydrolyse par BlaC. / Mycobacterium tuberculosis and Mycobacterium abscessus produce the β-lactamases BlaC and BlaMab that contribute to the intrinsic resistance of those bacteria to β-lactams. Our objective was to characterize the inhibition of these β-lactamases by avibactam and clavulanate in order to contribute to the development of new treatments. We have determined the inhibition and substrate profiles of BlaMab, as well as its crystal structure, revealing three major differences with BlaC. BlaMab is more active than BlaC with respect to hydrolysis of all β-lactams except cefoxitin, which is used for the treatment of infections due to M. abscessus. BlaC is inhibited irreversibly by clavulanate and inefficiently by avibactam. In contrast, BlaMab shows the opposite behavior involving hydrolysis of clavulanate and a rapid inhibition by avibactam. Structurally BlaC differs from BlaMab mainly by the replacement of the conserved motif SDN by SDG. The introduction of SDG in BlaMab and of SDN in BlaC revealed that this difference determines the inhibition profile of the β-lactamases. A single mutation can therefore lead to the emergence of resistance to the association of β-lactam with clavulanate or avibactam, but not to both associations. Thus, avibactam and clavulanate offer therapeutic alternatives in case of resistance to one of the two inhibitors. We have also investigated the β-lactam partners of clavulanate for the treatment of tuberculosis and showed that the structure of carbapenems could be optimized to enhance the inactivation of the targets and to reduce hydrolysis by BlaC.

<p>Mycobacterium abscessus (Mab) is a non-tuberculous mycobacterium that is highly resistant to many antibiotics. Mab causes pulmonary infections in immunocompromised individuals. The presence of efflux pumps to pump out antibiotics and its ability to form biofilms makes Mab a virulent pathogen. Studies have been done on Mab antibiotic tolerance but there are still a lot of gaps in knowledge about the effects of efflux pump inhibitors (EPIs) on antibiotic resistance and lipid biosynthesis in this bacterium during biofilm formation. In this study, we investigated the effects of the EPIs chlorpromazine (CPZ), 1-(1-naphthylmethyl)-piperazine (NMP), thioridazine (TRZ), Phenylalanine-arginine β-naphthylamide (PBN) and plumbagin (PLU) on antibiotic resistance, efflux, biofilm formation and lipid biosynthesis associated with log-phase growth and biofilm formation in Mab. We used the resazurin assay to determine the minimum inhibitory concentration (MIC) of the EPIs. We investigated the effects of the EPIs during biofilm-forming growth conditions on the MICs of antibiotics such as clarithromycin, amikacin, cefoxitin, ciprofloxacin which are the frontline antibiotics used to treat non-tuberculous mycobacterial infections. We also assessed the effects of the EPIs on the accumulation and efflux activities of the Mab cells through ethidium bromide (EtBr) assay. Furthermore, we evaluated the effects of the EPIs at sub-MIC concentrations on Mab biofilm formation under normoxic and hypoxic conditions. We utilized metabolic radiolabeling methods using 14C-palmitic acid and 14C- acetic acid which are precursors of lipid biosynthesis and analyzed lipids by silica-thin layer chromatography and autoradiography. We observed that Mab cells developed higher tolerance to the EPIs in a biofilm-stimulating medium. Furthermore, a decrease in the MICs of antibiotics was observed in the presence of the EPIs. Also, in the presence of the EPIs, there was less efflux activity within the Mab cells. In addition, EPIs inhibited biofilm formation significantly. We also noticed that NMP and PBN inhibited 14C-palmitic acid and 14C- acetic acid incorporation into polar lipids such as glycopeptidolipids, trehalose monomycolate, phosphatidylethanolamine, phosphatidylglycerol/cardiolipin, phosphatidylinositol mannosides at specific tested conditions. Our findings suggest that the EPIs inhibited the activities of the efflux pumps associated with the efflux of the antibiotics and lipid biosynthesis involved in biofilm formation. In conclusion, the results from this study gives insights on possible therapeutic opportunities.</p>

<p dir="ltr">Mycobacterium abscessus (Mab) is a type of mycobacterium that is known for its remarkable resistance to a variety of antibiotics. This pathogen poses a significant risk for individuals with weakened immune systems as it can cause skin and soft tissue infections, pulmonary disease and disseminated infections. Mab's ability to expel antibiotics through efflux pumps and form strong biofilms makes it even more challenging to treat infections. Lipids form a major part of the extracellular matrix of Mab biofilms. Efflux pumps have been shown to export lipids to the cell surface. Despite ongoing research into Mab's antibiotic tolerance, there is still much to learn about the impact of efflux pump inhibitors (EPIs) on antibiotic resistance and lipid biosynthesis during biofilm development in Mab. In this study, we investigated the impact of the EPIs; CCCP (carbonyl cyanide m-chlorophenyl hydrazone), piperine (PIP), reserpine (RES), berberine (BER), and verapamil (VER) on efflux activity, biofilm formation, antibiotic resistance, and lipid biosynthesis in Mab during planktonic and biofilm growth conditions. We found that Mab cells had a higher tolerance to EPIs in biofilm-stimulating medium and that the presence of EPIs led to a decrease in minimum inhibitory concentrations of frontline antibiotics, reduced efflux activity within Mab cells, and significantly inhibited biofilm formation. We examined the effects of EPIs that inhibited biofilm formation on lipid metabolism in Mab using radiolabeling with 14C?palmitic acid and 14C-acetic acid which are precursors of lipid biosynthesis. We observed that the EPI berberine inhibited the incorporation of 14C-palmitic acid into glycopeptidolipids in the surface lipids of planktonic cells and increased cellular glycopeptidolipid (GPL) in biofilm cells. Verapamil-treated cells showed a 55 % increase in cellular trehalose monomycolate (TMM) compared to controls. Piperine-treated cells exhibited a 50 % increase in cardiolipin. The incorporation of 14C-acetate into biofilm cells showed that piperine-treated biofilm cells showed a 146 % increase in surface glycopeptidolipids. Overall, our study enhances our understanding of lipid biosynthesis in Mab, the effects of EPIs on Mab biofilms, efflux mechanisms, and antibiotic resistance and offers insights for combating Mab-related infections.</p>