Nouvelles stratégies d'isolement et de caractérisation des microorganismes intracellulaires associés aux amibes

New strategies for the isolation and characterization of amoeba associated microorganisms

• Auteur / Autrice : Yaacoub Jacques Bou Khalil
• Direction : Bernard La Scola
• Type : Thèse de doctorat
• Discipline(s) : Pathologie humaine. Maladies infectieuses
• Date : Soutenance le 16/06/2016
• Etablissement(s) : Aix-Marseille
• Ecole(s) doctorale(s) : École Doctorale Sciences de la Vie et de la Santé (Marseille)
• Partenaire(s) de recherche : Laboratoire : Unité de recherche sur les maladies infectieuses et tropicales émergentes (Marseille)
• Jury : Président / Présidente : Philippe Colson
• Jury : Examinateurs / Examinatrices : Philippe Colson, Florent Sebbane, Max Maurin
• Jury : Rapporteur / Rapporteuse : Florent Sebbane, Max Maurin

Mots clés

Mots clés contrôlés

Virus

Mimiviridae

Amoeba

Cytométrie en flux

Techniques de coculture

Spécificité d'hôte

Lobosea

Génome bactérien

classification

Chlamydiaceae

Mots clés libres

Nouvelles stratégies

Co-Culture

Microorganismes associés aux amibes

Isolement

Virus géant

Chlamydiaceae

Cytométrie en flux

Haut débit

Tri

Caractérisation

Microscopie électronique

New strategies

Co-Culture

Amoebae-Associated microorganisms

Isolation

Giant viruses

Chlamydiaceae

Flow cytometry

High throughput

Sorting

Characterization

Transmission electron microscopy

Résumé

La co-culture d’amibes est utilisée afin d’isoler des microorganismes. Ainsi le premier virus géant,fut découvert. Cependant, les méthodes de culture sur protozoaires sont délicates et fastidieuses. De ce fait, le développement de ces cultures représente une difficulté pour les microbiologistes, limitant ainsi l’analyse d’un nombre important d’échantillons et la caractérisation de nouveaux virus. De nouvelles stratégies et des améliorations des modèles actuels sont donc nécessaires. Notre travail a été de développer de nouvelles stratégies permettant l’isolement de microorganismes associés aux amibes. Dans la 1ere partie nos travaux ont permis une amélioration des cultures avec le développement de nouveaux milieux de culture et l’utilisation ciblée d’antimicrobiens.La clé de ces stratégies est l’association des techniques rapides aux étapes améliorées de culture et leur application à un large panel de protozoaires pouvant abriter des microorganismes. Les résultats ont permis le développement d’un système d’isolement à haut débit très efficace. Nous avons notamment mis au point des techniques de tri de virus géants par cytométrie.Dans la seconde partie, nos travaux ont porté sur la description et la caractérisation des nouveaux isolats.Les résultats obtenus démontrent l’importance de poursuivre l’isolement et la caractérisation de ces microorganismes afin de mieux appréhender l’évolution de ces microorganismes, leur biotope et leur pathogénicité.De nouveaux outils sont nécessaires,notre manque d’imagination et l’absence de systèmes automatisés seront les limites aux nouvelles stratégies dans le monde de la microbiologie.

Amoebae are predators without distinction and they can also act as hosts to several different microorganisms that may coexist simultaneously. Some protozoa are sources of human pathogens where they act as reservoir of any human pathogens like Legionellae, Chlamydiaceae and others. In addition, the first giant virus, Acanthamoeba Polyphaga imivirus, was discovered using Amoeba as cell host. Since then, many other giant viruses have been isolated. For decades, amoebae were used as cell hosts in the culture- based process to isolate microorganisms, and allowed to recover new giant viruses and bacterial species from human and environmental samples. In contrast the co-culture system with protozoa is tedious and fastidious. Microbiologists are limited to routine culture methods, limiting by this the speed of screening potential samples and the efficiency of yielding new isolates. Much effort and improvement were needed. Our work consisted in the development of new strategies and techniques for the isolation of new microorganisms associated to protozoa. In the first part of this work, we described, all the improvements we brought to the protists culture system for the isolation of intracellular microorganisms especially giant viruses and Chlamydiaceae. Major improvements were based on modified culture enrichment steps, adapted culture media, and targeted use of specific drugs. The key of this new strategy was the implementation of high-throughput technologies to the ameliorated culture based systems, and the application of this later to a wide panel of protozoa used as potential host cells. These presented advances and strategies demonstrated significant time saving, and higher sensitivity than older techniques, they considerably increased the potential of collecting new environmental or clinical isolates and also new undiscovered microorganisms especially new giant virus familiesand particular Chlamydiaceae associated to amoebae. We continued to ameliorate the efficiency of the flow cytometric technology by reviewing its contributions to the virology field, then by applying it to the isolation system by sorting the new isolates as a new strategy for better genomic and proteomic analysis. In the second part of this work, we focused on the characterization of new isolates at the level of developmental cycle and genomic description. We used electron microscopy, and genome sequencing as main tools to describe our newly isolated giant viruses but also report new species of Chlamydiaceae and managed to decipher Chlamydiaceae species with a host dependent replication cycle, an issue that has not, thus far, been observed in protozoa-associated Chlamydiaceae. The strategies and results described herein show the importance of pursuing the isolation of new associatedamoebal microorganisms in order to give rise to new insights into the evolution of these microorganisms, their respective biotopes, and their potential or hidden pathogenicity. The more we need to search the more tools are needed, only our lack of imagination and appropriate automated systems will put limits on any needed strategy in the field of microbiology.