Dynamique de l'adaptation de Cryptococcus neoformans à l'hôte

Cryptococcosis is an opportunistic infection due to the ubiquitous yeast Cryptococcus neoformans. This pathogen is a facultative intracellular organism. Interaction with immune cells including monocytes/macrophages lineage and dendritic cells are of major importance in the natural history of the infection. In humans, the pathophysiology of the infection evolves in three steps (i) primoinfection in childhood, (ii) dormancy, demonstrated from epidemiological and genotypic data in the lab few years ago (Garcia-Hermoso et al. 1999), (iii) reactivation upon immunosuppression. In terms of disease, clinical presentation and outcome of cryptococcosis are known to be diverse among individuals even those sharing the same underlying diseases. To address the question of the impact of fungal diversity on the natural course of the infection at the macrophage-level (standardized model of yeasts/ j774 macrophages in vitro interaction), murine model-level (murine model of cryptococcosis in OF1 outbred mice) and human-level (CryptoA/D database), we studied (i) the diversity of C. neoformans/macrophages interactions using well characterized clinical isolates (ii) the correlation between in vitro phenotype of the isolate and clinical outcome in humans (iii) the diversity of adaptation to the host. We developed new assays and new tools using flow cytometry I (quantitative flow cytometry, multispectral imaging flow cytometry, sorting), microscopy (dynamic imaging), gene expression analysis (single-cell quantitative real time PCR) to overcome technical issues. We found high variation in phagocytic, 2 hours-, 48hours-intracellular proliferation indexes among the 54 ClinCn compared to H99. No correlation with the genotype was observed. The lack of sterilization at week 2 despite active antifungal therapy was significantly associated with a lower phagocytic index, whereas treatment failure at month 3 and death from cryptococcosis were significantly related to a higher 2 hours-intracellular proliferation. Among 9 selected clinical isolates compared to H99, (i) the virulence in mice was significantly different, intracellular expression of some virulence factors correlated with (ii) intracellular proliferation and (iii) phagocytic indexes. With a focus on multiplication and stress response and considering the H99 reference strain, we observed the appearance of various populations of yeasts during mice and macrophage infections. After sorting yeasts populations, we observed that a specific one was less prone and dependent of serum to grow compared to the other p'opulations. Gene expression analysis revealed that this population had specific metabolic characteristics that could reflect dormancy. We found a high diversity of C. neoformans upon interaction with macrophages considering 54 clinical isolates in correlation with clinical outcome in humans, but also a considerable adaptation to host in our two models considering the reference strains H99. We observed also even more diversity of fungal adaptation to host when clinical isolates were considered. Ail together, these data suggest that cryptococcosis and fungal disease in general could be more complex diseases since diversity, plasticity and adaptation of the fungal organism to hosts is high and heterogeneous.La cryptococcose est une infection opportuniste causée par la levure ubiquitaire Cryptococcus neoformans. L'interaction avec les macrophages est importante pour le développement de la maladie chez l'homme. La physiopathologie évolue en trois étapes: (i) primo-infection, (ii) dormance, (iii) réactivation en cas d'immunodépression cellulaire. La présentation clinique et l'évolution de la cryptococcose sont variables en fonction des individus. L'objectif de mon travail était de comprendre l'impact de la diversité de la levure sur l'évolution de l'infection. Nous avons étudiés (i) les souches cliniques et leur diversité d'interaction avec les macrophages; (ii) la corrélation entre le phénotype in vitro et l'évolution clinique de l'infection chez l'homme; (iii) la diversité d'adaptation à l'hôte. Ainsi, nous avons développés de nouveaux outils (cytométrie de flux quantitative, microscopie en temps réel, single-cell quantitative PCR) pour permettre l'analyse de populations de levures rares. Nous avons trouvés une grande variabilité d'interaction de 54 isolats cliniques avec le macrophage. L'échec mycologique et la mortalité chez l'homme était associés à des phénotypes d'interaction particuliers. A partir de l'analyse de 9 isolats cliniques, nous avons pu mettre en évidence que la virulence des isolats et l'expression de certains gènes de virulence connus étaient variables également. En nous focalisant sur la réponse au stress et la prolifération, nous avons observé l'apparition de différentes populations au cours de l'infection. Après tri des différentes populations, nous avons identifié une population ayant une faible réponse au stress et montré qu'elle était potentiellement dormante. Ces données suggèrent que la cryptococcose et les infections fongiques en générale sont des infections complexes résultants d'une grande diversité, plasticité et adaptation des champignons à l'hôte