Thomas Ipoutcha

Plasmids are key drivers of bacterial adaptation, yet the mechanisms that generate their diversity remain poorly understood. Here, we show that mobile genetic elements (MGEs) orchestrate a fusion-deletion life cycle that repeatedly remodels plasmids in Staphylococcus aureus . Large-scale genomic analyses reveal that multireplicon plasmids are widespread and strongly enriched in transposases. Using experimental assays, we demonstrate that rare MGE-mediated fusion events, via homologous recombination or transposition, combine distinct plasmids into single multireplicon elements, expanding gene content and transfer potential. Antibiotic pressure selectively enriches these fused plasmids, rescuing bacterial populations under stress, whereas opposing selective forces, including phage predation, favour deletion derivatives that preserve essential functions and phage transmissibility. This cyclical process generates dynamic plasmid repertoires with conserved backbones and diverse accessory modules. We propose that MGE-driven fusion-deletion cycles represent a general principle of plasmid evolution, explaining the rapid emergence and persistence of multidrug-resistant plasmids across bacterial pathogens.

Mycoplasma bovis is a minimal bacterium infecting cattle, which causes a wide variety of symptoms and is impacting dairy and beef producers worldwide. Part of the difficulty in research surrounding M. bovis , and other mycoplasmas, is the lack of efficient genome editing tools. As a proof of concept, we previously presented a transposon-based CRISPR-Base Editor system to introduce targeted mutations in M. bovis . In this work, the existing tool has been greatly improved: multi-loci targeting through addition of a second guide RNA; increased number of targetable loci by using an engineered Cas9 with AT-rich PAM specificity, and elimination of the CRISPR-Base Editor from the generated mutants through either transposon excision or use of a curable plasmid. We also propose a dedicated bioinformatic tool to identify target sequences in genes of a given genome. This software was applied to demonstrate the potential of our improved tools in M. bovis and other mycoplasmas of veterinary and human interest that currently lack genome editing methods.

Les systèmes CRISPR/Cas sont largement représentés chez les bactéries et les archées et sont à la base de mécanismes de défense adaptatifs contre l’ADN et l’ARN exogène. De par son intérêt comme outil génétique, le système CRISPR de type II de Streptococcus pyogenes a été particulièrement étudié. Il est composé principalement d’une endonucléase Cas9 et d’un sgRNA qui a pour rôle de guider Cas9 jusqu’à une séquence cible, la spécificité de reconnaissance dépendant également d’une séquence NGG située en aval appelée PAM. Les mollicutes sont des bactéries minimales, dépourvues de paroi et qui utilisent, pour la plupart, un code génétique alternatif. Elles possèdent de petits génomes riches en AT (~70-75%) allant de 0,58 à 2,2 Mpb. La plupart d’entre elles ont un mode de vie parasitique et sont des pathogènes d’une grande diversité d’hôtes (plantes et animaux y compris humains). Chez la plupart des mollicutes, les possibilités d’ingénierie génomique restent limitées car la recombinaison homologue est peu efficace. Le but de ma thèse a été de (1) caractériser les systèmes CRISPR//Cas9 des mollicutes, (2) développer un outil endogène CRISPR/Cas9 pour l’ingénierie de génome de mollicutes et (3) d’étendre cette boite à outils chez certaines espèces d’intérêt pour lesquelles aucun outil génétique de précision n’était disponible.La première partie de mon travail de thèse était l’étude globale des systèmes CRISPR chez les mollicutes. Une reconstruction phylogénétique a permis de mettre évidence une origine commune pour tous les systèmes CRISPR de mycoplasmes et deux origines pour les systèmes de spiroplasmes. L’étude structurale de la protéine Cas9 des mollicutes montre une forte proximité structurale avec la protéine Cas9 de Staphylococcus aureus à l’exception du domaine PI qui intervient dans l’interaction avec la séquence PAM, ce qui suggère que les séquences PAM des systèmes de mollicutes sont sans doute très différentes. Le système CRISPR/Cas9 de Mycoplasma gallisepticum (Mgal), un pathogène d’oiseau, a ensuite été l’objet d’une étude approfondie. Le système naturel a été démontré comme fonctionnel in vivo et le système minimal composé de la MgalCas9 et d’un sgRNA a permis d’induire un clivage de l’ADN in vitro. Une différence de séquence PAM consensus a été observée entre deux souches de Mgal isolées de poulet et de pinson et a pu être mise en corrélation avec le récent changement d’hôte du pathogène et l’émergence de celui-ci dans les populations de pinsons d’Amérique. Un plasmide « all-in-one » contenant le système a été conçu, et la fonctionnalité du système comme outil précis de clivage a été démontré dans différentes espèces, notamment Mgal, Mycoplasma capricolum (Mcap), Mycoplasma pulmonis et Mycoplasma mycoides. Cet outil de contre sélection a été appliqué à Mcap afin de curer son génome d’un élément mobile intégré de 23 kpb (ICE). Finalement, pour pallier à la faible efficacité de recombinaison des mycoplasmes, deux nouveaux outils d’ingénierie génomique ont été mis au point. Un outil « CRISPR Base Editor », composé d’une protéine Cas9 désactivée, fusionnée à une protéine de type déaminase, qui permet de faire des modifications de base de manière très ciblée. La fonctionnalité de cet outil, très efficace et simple d’utilisation, a été validée dans 3 espèces pathogènes majeures de mycoplasmes. Le second outil est basé sur l’importation d’un système de recombinaison RecET issu de phages. Chez Mgal, cet outil d’ingénierie de génome permet ainsi pour la première fois de réaliser des modifications précises à plus grande échelle (délétions, insertions et remplacements).En conclusion, nos travaux ont concerné l’étude fonctionnelle des systèmes CRISPR des mycoplasmes et le développement d’outils d’ingénierie de génomes chez ces mêmes bactéries. Ce travail ouvre de manière significative les possibilités d’étude et d’utilisation des mycoplasmes pour des applications biotechnologiques.

Summary: Plasmids are key drivers of bacterial adaptation, yet the mechanisms that generate their diversity remain poorly understood. Here, we show that mobile genetic elements (MGEs) orchestrate a fusion-deletion life cycle that repeatedly remodels plasmids during evolution in Staphylococcus aureus . Large-scale genomic analyses reveal that multireplicon plasmids are widespread and strongly enriched in transposases. Using experimental assays, we demonstrate that rare MGE-mediated fusion events, via homologous recombination or transposition, combine distinct plasmids into single multireplicon elements, expanding gene content and transfer potential. Antibiotic pressure selectively enriches these fused plasmids, rescuing bacterial populations under stress, whereas opposing selective forces, including phage predation, favour deletion derivatives that preserve essential functions and phage transmissibility. This cyclical process generates dynamic plasmid repertoires with conserved backbones and diverse accessory modules. We propose that MGE-driven fusion-deletion cycles represent a general principle of plasmid evolution, explaining the rapid emergence and persistence of multidrug-resistant plasmids across bacterial pathogens.

Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are bacterial defences that target bacteriophages and mobile genetic elements. How these defences evolve in novel host environments remains largely unknown. We studied the evolution of the CRISPR-Cas system in Mycoplasma gallisepticum (also named Mycoplasmoides gallisepticum), a bacterial pathogen of poultry that jumped into a passerine host ~30 years ago. Over the decade following the host shift, all isolates displaying a functional CRISPR-Cas system were found not only to harbour completely new sets of spacers, but the DNA protospacer adjacent motif recognized by the main effector M. gallisepticum Cas9 (MgCas9) was also different. These changes in CRISPR-Cas diversity and specificity are consistent with a change in the community of phages and mobile elements infecting M. gallisepticum as it colonized the novel host. In the years following the host shift, we also detected a gradual rise in isolates displaying non-functional MgCas9. After 12 years, all circulating isolates harboured inactive forms only. This loss of CRISPR-Cas function comes at a time when the passerine host is known to have evolved widespread resistance, which in turn drove the evolution of increasing M. gallisepticum virulence through antagonistic coevolution. Such striking concordance in the rise of inactivated forms of CRISPR-Cas and the evolution of host resistance suggests that the inactivation of the CRISPR-Cas system was necessary for enabling adaptive bacterial responses to host-driven selection. We highlight the need to consider both host and pathogen selection pressures on bacteria for understanding the evolution of CRISPR-Cas systems and the key factors driving the emergence of a pathogenic bacterium in a novel host.

The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa , a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa . As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa . We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages. IMPORTANCE: Pseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

The rise in frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa , a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically-relevant strains of Pseudomonas aeruginosa . As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination (TAR) cloning and rebooted these two phage genomes in two different strains of P. aeruginosa . We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step towards the construction of clinically-relevant, engineered P. aeruginosa phages. Importance: Pseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically-relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

CRISPR-Cas systems are bacterial defences that target bacteriophages and mobile genetic elements. How these defences evolve in novel host environments remains, however, unknown. We studied the evolution of the CRISPR-Cas system in Mycoplasma gallisepticum , a bacterial pathogen of poultry that jumped into a passerine host ∼30 years ago. Over the decade following the host shift, all isolates displayed a functional CRISPR-Cas system were found not only to harbour completely new sets of spacers, but the DNA protospacer adjacent motif (PAM) recognised by the main effector MgCas9 was also different. These changes in CRISPR-Cas diversity and specificity are consistent with a change in the community of phages and mobile elements infecting M. gallisepticum as it colonised the novel host. In the years following the host shift, we also detected a gradual rise in isolates displaying non-functional MgCas9. After 12 years, all circulating isolates harboured inactive forms only. This loss of CRISPR-Cas function comes at a time when the passerine host is known to have evolved widespread resistance, which in turn drove the evolution of increasing M. gallisepticum virulence through antagonistic coevolution. Such striking concordance in the rise of inactivated forms of CRISPR-Cas and the evolution of host resistance suggests that inactivation of the CRISPR-Cas system was necessary for enabling adaptive bacterial responses to host-driven selection. We highlight the need to consider both host and pathogen selection pressures on bacteria for understanding the evolution of CRISPR-Cas systems and the key factors driving the emergence of a pathogenic bacterium in a novel host. Data summary: The authors confirm all supporting data and protocols have been provided within the article or through supplementary data files available in the online version of this article. GenBank accession numbers of all publicly available M. gallisepticum genomes are listed in Table S3. Sequences of the CRISPR locus of other strains are also provided in Table S3. Impact statement: Mycoplasma are minimal bacteria involved in many diseases affecting humans and a wide diversity of animals. In this paper, we report the evolution of the Type II CRISPR-Cas system of the bird pathogen, Mycoplasma gallisepticum , following an host jump from its original poultry host into its novel house finch host in the early 90’s. Instances in which bacterial pathogens have been documented to jump into and subsequently adapt to a new host are rare, and the well documented case of M. gallisepticum is a unique model to evaluate the effect of any dramatic host environmental change on bacterial CRISPR-Cas defence systems. First, we performed in silico analyses on an extended set of 98 M. gallisepticum genomes to better understand the evolution of the CRISPR-Cas9 system in the novel finch host. We documented several evolutionary events leading to the drastic divergence of spacer sets present in poultry and house finch arrays, as well as the progressive inactivation of the CRISPR-Cas system after 12 years in the novel finch host. Second, using in vitro and in vivo assays, we demonstrated that the evolution of the MgCas9 PI domain, involved in the protospacer adjacent motif (PAM) recognition has led to a major change in the defence system, with a modification of the recognized PAM in the novel host. Such radical change in the CRISPR-Cas defence system of M. gallisepticum may have implications for the its rapid adaptation to its novel host. Together, our results highlight the need to consider not only the host-driven selection pressures a bacterium experiences, but also the complex interplay between phages and defence systems for better understanding the key factors driving the emergence of a pathogenic bacterium in a novel host.

The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of in-yeast cloning strategies, a new approach combining two already described methods, the Fusion cloning and the CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves cloning efficacy.

Mycoplasmas are minimal pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors.