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Paysage adaptatif des bêta-lactamases TEM-1 et CTX-M-15 / Adaptative landscape of beta-lactamases TEM-1 and CTX-M-15Birgy, André 25 September 2017 (has links)
De part leur importance médicale, leur capacité à évoluer et leur facilité de manipulations, les bêta-lactamases TEM-1 et plus récemment CTX-M-15, se sont imposées comme des modèles de biochimie mais aussi des modèles pour une étude évolutive des protéines. La caractérisation de la distribution de l'effet des mutations dans une protéine permet d'avancer dans la compréhension des mécanismes moléculaires et des contraintes influant sur l'évolution des gènes de résistance aux antibiotiques et de façon plus globale sur les protéines.Par une approche de mutagénèse exhaustive suivie d’une évolution expérimentale sous sélection antibiotique couplée à du séquençage à haut débit, nous avons pu déterminer et décrire la distribution des effets des mutations dans la protéine TEM-1. Trois catégories de mutants ont été identifiées comme ayant des comportements différents en termes de cinétique de mort face à l'antibiotique. Des études phénotypiques ont permis de proposer un scénario impliquant une saturation progressive des principales protéines liant la pénicilline en fonction de l’activité hydrolytique du mutant de bêta-lactamase. Enfin, un modèle biochimique qualitatif compatible avec cette cinétique est proposé. La comparaison des effets des mutations entre TEM-1 et CTX-M-15, deux bêta-lactamases de la famille des sérines protéases de classe A et homologue à 30%, permet d’étudier la notion de contexte-dépendance des mutations. Notamment, à travers le résidu 251 qui est différent entre ces 2 protéines et intolérant aux mutations, nous replaçons cette problématique dans une perspective évolutive globale. Cela nous permet d’étudier les incompatibilités mutationnelles au sein des protéines et les possibilités de compensation des sites destabilisés. Nous observons une compensation à la fois globale, au travers de l’effet sur la stabilité, mais aussi locale avec une épistasie forte entre le résidu 251 et le site compensateur. Enfin, afin d’étudier de façon exhaustive les intéractions épistatiques dans les protéines, nous nous sommes focalisés sur une hélice alpha de la protéine TEM-1. L’étude de près de 73% des 22000 combinaisons mutationnelles possibles a permis de souligner l’importance de la stabilité thermodynamique, celle-ci expliquant une grande part des effets des mutations. Cependant, un contingent d’interactions ne semble pas expliquable par ce modèle ce qui montre l’importance des interactions locales au sein des hélices. Le couplage de ces approches évolutives quantitatives et mécanistiques permettent à la fois d’avancer dans la compréhension des contraintes qui sous-tendent l’évolution des protéines mais aussi de plonger au cœur de la résistance aux antibiotiques et de ses mécanismes moléculaires. / Beta-lactamases TEM-1 and more recently CTX-M-15 are antibiotic resistance enzymes that combine a medical importance, a fast evolution in the wild and are easy amenable to manipulation in the laboratory. As such, they have become models of biochemistry and also models for the study of protein evolution. The characterization of the distribution of mutational effect within a protein shed light on the molecular mechanisms and the constraints influencing the evolution of proteins.Using an exhaustive mutagenesis approach followed by an experimental evolution under antibiotic selection coupled with high-throughput sequencing, we were able to determine and describe the distribution of the effects of mutations in the TEM-1 protein. Three categories of mutants have been identified as having different behaviors in terms of survival kinetics when facing the antibiotic. Phenotypic studies have allowed us to propose a scenario involving a progressive saturation of the main penicillin-binding proteins as a function of the hydrolytic activity of the beta-lactamase mutant. Finally, a qualitative biochemical model compatible with this kinetic is proposed.I then compared the fitness effects of mutations in TEM-1 and CTX-M-15, which are two beta-lactamases of the class A serine protease family with 30% homology. I focused on the context dependency of mutation effects and concentrated my analysis on residue 251, which harbor a different aminoacid in each of the enzyme that is not functional when inserted in the other protein. I further studied how mutations in the rest of the protein could compensate that incompatibility. Compenstation was found to be associated to mutations acting presumably through on overall effect on protein stability, and on some cases to some local interactions with residue 251.Finally, in order to study exhaustively the epistatic interactions in proteins, we focused one alpha helix of TEM-1. The study of nearly 73% of the 22,000 possible mutational combinations made it possible to underline the importance of thermodynamic stability, which explains a large part of the effects of mutations. However, a contingent of interactions does not seem to be explained by this model, which shows the importance of the local interactions within the helices.The coupling of these quantitative and mechanistic evolutive approaches makes it possible both to advance in understanding the constraints underlying the evolution of proteins but also to plunge into the heart of resistance to antibiotics and its molecular mechanisms.
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Evolutionary innovations and dynamics in Wagner's model of Genetic Regulatory NetworksWang, Yifei January 2016 (has links)
The gene regulatory network (GRN) controls the expression of genes providing phenotypic traits in living organisms. In particular, transcriptional regulation is essential to life, as it governs all levels of gene products that enable cell survival and numerous cellular functions. However, there is still poor understanding of how shifts in gene regulation alter the underlying evolutionary dynamics and consequently generate evolutionary innovations. By employing Wagner's GRN model, this dissertation investigates how the interplay of simple evolutionary forces (mutation and recombination) with natural selection acting on gene regulatory dynamics can generate major evolutionary innovations. In this dissertation, firstly, I review all currently available research papers using Wagner's GRN model, which is also employed as the computational model used extensively in the remaining chapters. I then describe how Wagner's GRN model and its variants are implemented. Finally, network properties such as stability, robustness and path length in initial populations are investigated. In the first study, I explore the characteristics of compensatory mutation in the context of genetic networks. Specifically, I find that 1) compensatory mutations are relatively insensitive to the size and connectivity of the network, 2) compensatory mutations are more likely to occur in genes at or adjacent to the site of a previous deleterious mutation and 3) compensatory mutations are more likely to be driven by mutations with a relatively large regulatory impact. In the second study, I further investigate the evolutionary consequences of the properties of compensatory mutation discovered previously. Specifically, I find that 1) compensatory mutations can occur regardless of patterns of selection, 2) networks with compensatory mutations exhibit proportionately higher robustness when compensatory mutations interact closely with deleterious mutations or have large effects on gene regulation, and 3) regulatory complexity can arise as a consequence of the propensity for co-localised and large-effect compensatory mutations. In the third study, I provide a mechanistic understanding of how recombination benefits sexual lineages. Specifically, I find that 1) recombination together with selection for developmental stability can drive populations towards the optimum, 2) recombination does not frequently disrupt well-adapted lineages as conventionally expected, and 3) recombination facilitates finding good genetic combinations which are robust to disruption, although it also rapidly purges weaker configurations. In the final study, I show that the selection pressure acting on rewiring gene regulation is critical to increasing benefits for sexual lineages whilst mitigating costs of sex and recombination. Specifically, I find that 1) strong selection strength can greatly benefit low-fitness sexual lineages, especially at the early stage, 2) recombination is initially costly, but it can rapidly evolve to compensate for costs of sex and recombination, and 3) sexual lineages with low levels of sex and recombination can outcompete strictly asexual populations under higher selection pressure and lower mutation rates. The results presented for all of the studies are important for mechanistically understanding evolutionary innovations through altering transcriptional regulatory dynamics. These innovations include 1) facilitating alternative pathway evolution, 2) driving regulatory complexity, 3) benefiting sexual reproduction, and 4) resisting invasion against asexual lineages.
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Mecillinam Resistance in E. coli : fitness, compensation, and resistance in different environmentsEkstrand, Emelie January 2017 (has links)
The global increase of antibiotic resistant bacteria threatens the modern health care and challenges the therapeutic effects of available antibiotics. The b-lactam mecillinam (Mec) is an exception to this due to a stable clinical resistance prevalence resistance of approximately 3%. It is only used to treat uncomplicated urinary tract infections (UTIs), mainly caused by E. coli. Mecillinam resistance (MecR) is easily selected for in laboratory settings and linked to >40 genes, including the mrdA gene encoding the Mec target penicillin-binding protein 2. A majority of the known MecR mutations confer a severe fitness cost. Fitness is important for bacteria to survive in the bladder and clinical isolates have been shown to have high fitness. These isolates contain loss-of-function mutations in the cysB gene, which encode a positive regulator of cysteine biosynthesis. In a previous evolution experiment, fitness cost of cysB and mrdA MecR mutations was compensated and the compensatory mutations were identified. Here the compensatory mutations were reconstructed into wildtype (WT) E. coli strain MG1655, and cysB and mrdA backgrounds to study the impact of the mutations on resistance and fitness, using MIC tests and Bioscreen C assays. Our results show that the mrdA mutants only had partial fitness compensation (significantly lower fitness than WT) for all strains and all strains also increased their MecR. The low fitness is possibly an explanation for the lack of mrdA mutants outside laboratories. Of the clinically relevant cysB mutants the majority lost their resistance when increasing growth rate, some even to levels significantly higher than WT, indicating that DcysB mutations are easier to compensate for. One strain (ydjNmx2) however, had a significantly higher growth rate while remaining clinically MecR.
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Mechanisms of Adaptation to Deformylase InhibitorsZorzet, Anna January 2010 (has links)
Antibiotic resistance is a growing problem on a global scale. Increasing numbers of bacteria resistant toward one or multiple antibiotics could return us to the high mortality rates for infectious diseases of the pre-antibiotic era. The need for development of new classes of antibiotics is great as is increased understanding of the mechanisms underlying the development of antibiotic resistance. We have investigated the emergence of resistance to peptide deformylase inhibitors, a new class of antibiotics that target bacterial protein synthesis. The fitness of resistant mutants as well as their propensity to acquire secondary compensatory mutations was assessed in order to gain some insight into the potential clinical risk of resistance development. Most of this work was done in the bacterium Salmonella typhimurium, due to the availability of excellent genetic tools to study these phenomena. In addition, we have studied the bacterium Staphylococcus aureus as peptide deformylase inhibitors have been shown to have the greatest effect on Gram-positive organisms. In the course of this work we also examined the mechanistic aspects of translation initiation. Using a cell-free in vitro translation system we studied the effects of various components on translation initiation. These results have been combined with results obtained from resistant and compensated bacterial strains in vivo to gain new insights into the mechanisms of translation initiation.
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An integrative approach to understanding the fitness cost of rifampicin resistance in Pseudomonas aeruginosaQi, Qin January 2014 (has links)
Antibiotic resistance in bacteria is acquired through spontaneous chromosomal mutations or horizontal gene transfer. In the absence of antibiotics, resistant mutants generally show reduced fitness due to compromised growth rate, competitive ability and virulence compared to their antibiotic-sensitive ancestors. The focus of my research is to dissect the molecular underpinnings of the variations in the fitness cost of chromosomal antibiotic resistance using a systems-level approach. From an evolutionary perspective, my research aims are to understand how the fitness cost influences adaptation in resistant populations in an antibiotic-free environment. Using rifampicin resistance in Pseudomonas aeruginosa as a model, my work shows that most of the variation in the fitness cost of rifampicin resistance can be attributed to the direct effect of rifampicin resistance mutations on transcriptional efficiency. Through RNA-Seq transcriptome profiling, I demonstrate that global changes in gene expression levels associated with resistance mutations are surprisingly subtle, suggesting that the transcriptional regulatory network of P. aeruginosa is robust against compromised transcriptional efficiency. Using experimental evolution and whole-genome sequencing, my work reveals a systematic difference in the genetic basis of adaptation in mutants that were propagated in the absence of antibiotics. During compensatory adaptation, resistant mutants can recover the fitness cost of resistance by fixing second-site mutations that directly offset the deleterious effects of resistance mutations. Amongst resistant mutant populations with low fitness costs, general adaptation limits compensatory adaptation, which is most likely to be due to the rarity of compensatory mutations and clonal interference. Far from being the most ubiquitous mechanism in the evolution of resistance, compensatory adaptation is the exception that is more likely to be observed in resistant mutants with high fitness costs. In addition, I applied key elements of the integrative experimental approach developed in this work to dissect the molecular basis of the fitness cost associated with carriage of the pNUK73 small plasmid in P. aeruginosa, which carries the rep gene encoding a plasmid replication protein. My results confirmed that rep expression generates a significant fitness cost in P. aeruginosa and demonstrate how the molecular origins of the fitness cost of resistance can be dissected in a different biological context.
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The Structural Basis for the Interdependence of Drug Resistance in the HIV-1 ProteaseRagland, Debra A. 13 December 2016 (has links)
The human immunodeficiency virus type 1 (HIV-1) protease (PR) is a critical drug target as it is responsible for virion maturation. Mutations within the active site (1°) of the PR directly interfere with inhibitor binding while mutations distal to the active site (2°) to restore enzymatic fitness. Increasing mutation number is not directly proportional to the severity of resistance, suggesting that resistance is not simply additive but that it is interdependent. The interdependency of both primary and secondary mutations to drive protease inhibitor (PI) resistance is grossly understudied.
To structurally and dynamically characterize the direct role of secondary mutations in drug resistance, I selected a panel of single-site mutant protease crystal structures complexed with the PI darunavir (DRV). From these studies, I developed a network hypothesis that explains how mutations outside the active site are able to perpetuate changes to the active site of the protease to disrupt inhibitor binding.
I then expanded the panel to include highly mutated multi-drug resistant variants. To elucidate the interdependency between primary and secondary mutations I used statistical and machine-learning techniques to determine which specific mutations underlie the perturbations of key inter-molecular interactions. From these studies, I have determined that mutations distal to the active site are able to perturb the global PR hydrogen bonding patterns, while primary and secondary mutations cooperatively perturb hydrophobic contacts between the PR and DRV. Discerning and exploiting the mechanisms that underlie drug resistance in viral targets could proactively ameliorate both current treatment and inhibitor design for HIV-1 targets.
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