Genomic epidemiology and ecology of cryptococcus neoformans var. grubii in southern Africa

Abstract

Despite the increased availability of antiretroviral therapy, cryptococcal meningitis remains a neglected disease that is responsible for up to 30% of AIDS related deaths. In the light of next generation sequencing technologies, this thesis investigates the molecular epidemiology of Cryptococcus neoformans var. grubii (Cng) in southern Africa. Sampling in the field and laboratory isolation yielded 37 environmental Cng and 38 C. gattii (Cg) from various ecotypes in Zambia and South Africa. In Zambia, the two sister-species were associated with different ecoregions (p <0.001). Cng was predominantly found inhabiting the Zambezi Mopane Wood- lands, a previously described ecological niche for the pathogen, whereas Cg was more frequently isolated from wet Miombo Woodlands. The fungal community structure associated with these two saprophytes was explored using a high-throughput metabarcoding approach. Changes in the microbial assemblages were observed between the different ecoregions (RANOSIM >0.350, p <0.001) and these differences were more pronounced when climatic (temperature and precipi- tation) and spatial factors were considered. This work confirmed the local adaptation of these fungal species to their specific ecoregions, both at the level of the fungal community and their associated environmental variables. The characterisation of Cryptococcus biotic environments will help to understand where the pathogen might emerge and to predict a potential spillover of the pathogen into susceptible host populations as well as uninfected ecoregions. Whole-genome sequencing of 50 environmental Cng genomes revealed the presence of 26 isolates that grouped into lineage VNI, and 24 isolates that grouped into lineage VNB. Genome-scans were performed to uncover genetic ‘outliers’that were putatively under selection within each molecular type. Genomes of environmental VNB isolates were found to group into two statistically-supported clades and 32% of VNB isolates possessed the MAT a mating-type locus. Recombination was shown to occur frequently within the VNB molecular type, whereas VNI isolates displayed a more clonal profile with the MAT a mating-type being much more rare (4%). VNI infections were shown to be more often associated with urban centres while VNB Cng are mainly present across the Colophospermum mopane region, therefore VNB infection most likely occurs in rural settings. Subsequently, a comparison between environmental and clinical VNB isolates was performed to understand whether the genetic makeup of Cryptococcus neoformans isolated in patients is linked to specific environmental genotypes. Genetic variants associated with clinical isolates were uncovered. Nineteen genes with a known-virulence function were found to be un- der selection in the environment and were also found to be also associated with clinical isolates. In vitro phenotypic assays were performed to identify variations between clinical and environ- mental isolates. Clinical isolates were found to be thermotolerant, produced larger capsule and were more easily phagocytosed than environmental isolates. Clinical VNI isolates replicated faster at 37◦ C than clinical VNB isolates (p <0.002). This finding might be a reflection of the evolutionary relationship between the VNI molecular type and its association with domestic and feral pigeons. Finally, the epidemiology of the VNI molecular type with isolates from the African and Asian continents (n=155) was investigated in the light of whole-genome sequence data. Phylogenetic analyses revealed eight genetic clusters. A ten-fold increase in population size was observed in each VNI cluster in the past 200 to 8,000 years, suggesting the role of the domestication of the pigeon in the global spread of the VNI molecular type. These find- ings indicate that each cluster is likely to contribute differently to the neglected epidemic of Cryptococcus neoformans var. grubii worldwide.