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Thomas IPOUTCHA

Team Mollicutes

Doctoral specialty: Microbiology - Immunology

at the Haut Carré amphitheater in Talence (33400)

Abstract :

CRISPR/Cas systems are widely represented in bacteria and archaea and provide them an adaptative defense mechanism against exogenous DNA or RNA. Because of its wide use as a genome engineering tool, type II CRISPR system from Streptococcus pyogenes has been extensively studied. The core system includes the endonuclease Cas9 and one sgRNA that drives Cas9 on a target site with a recognition specificity depending on the NGG Protospacer Adjacent Motif (PAM). Mollicutes are minimal bacteria that have no cell wall and most of them use an alternative genetic code. Their genomes are AT rich (~70-75%) with a size ranging from 0.58 to 2.2 Mbp. Most of them have a parasitic lifestyle and are pathogens of a wide host diversity including humans, other animals and plants. For most mollicutes species, genome engineering currently remains challenging mainly because homologous recombination is poorly efficient. The aim of my thesis was to (1) characterize CRISPR/Cas9 systems of mollicutes, (2) develop a genome engineering tool based on endogenous CRISPR/Cas9 system and, (3) extend the toolbox for some mycoplasmas of veterinary interest for which any precise genetics tool was available.
The first part of my thesis work was a global survey of CRISPR systems in mollicutes. Complete or degraded CRISPR systems were found in 21 out of the 52 representatives mollicutes species included in the study. Phylogenetic reconstruction indicated a probable unique origin of mycoplasma CRISPR/Cas9 systems while two different origins were predicted for systems present in spiroplasmas. Comparison of the predicted structures Cas9 from mollicutes and Staphylococcus aureus showed a strong structural proximity except for the PI domain that is involved in the interaction with the PAM sequence. This suggests probable differences of PAM recognition specificity. We next focused on the CRISPR/Cas9 system of the bird pathogen Mycoplasma gallisepticum (Mgal). The functionality of the system was demonstrated in vivo and a minimal system including Cas9 and a sgRNA was designed and evaluated in vitro. An extensive evaluation of the PAM motifs recognized by two MgalCas9 from two distinct strains of Mgal isolated in chicken and house-finch was conducted using an in vitro cleavage assay coupled with a deep sequencing strategy. A difference of PAM consensus was observed between the two strains and was correlated with a recent host jump from chicken to American house-finch. Next, a set of “all in one” plasmids was constructed and MgalCas9 was successfully used as a tool for precise DNA cleavage in different mycoplasma species: Mgal, Mycoplasma capricolum subsp. capricolum (Mcap), Mycoplasma pulmonis et Mycoplasma mycoides subsp. mycoides. Using this counter selection tool, we were able to cure the Mcap genome from a 23 kbp auto-excisable genetic mobile element (ICE). In order to overcome the low efficiency of natural recombination in mycoplasmas that limits genome engineering possibilities, I developed two new generations tools. The first one is a “CRISPR Base Editor” consisting in an inactivated Cas9 protein that is fused to a deaminase protein (pmcDA1). This hybrid enzyme allowed very specific base modification in the mycoplasma genome. This simple tool was demonstrated to be highly efficient in three main pathogenic mycoplasmas, opening new possibilities for their study. The second tool is based on the phage recombination system RecET that was used to drive homologous recombination in mycoplasma. For the very first time in Mgal, this tool allowed us to perform large scale and precise modifications (Insertion, deletion and replacement).

In conclusion, my PhD thesis was dedicated to the studies of CRISPR system of mycoplasma and the development of new precise genome engineering tools for these bacteria. This work opened unprecedent possibilities for the study of pathogenic mycoplasmas and further biotechnological applications.

Jury :

Mme BÉBÉAR, Cécile, PU-PH à l’Université de Bordeaux                                             Présidente
M. BIKARD, David, Chargé de Recherche, Institut Pasteur, Paris                                  Rapporteur
Mme CITTI, Christine, Directeur de Recherche à l’ENVT, Toulouse                               Rapporteure
M. FREMAUX, Christophe, Cadre scientifique à l’IFF, Dangé-Saint-Romain                   Examinateur
Mme LARTIGUE, Carole, Chargée de Recherche, INRAE, Villenave d’Ornon                 Invitée
M. SIRAND-PUGNET, Pascal, Maitre de conférences à l’Université de Bordeaux           Directeur de thèse