An evolutionary population genomics approach to determine the genetic basis of virulence in the pathogenic fungus Cryptococcus neoformans

Infectious fungi are causing an increasing burden of disease worldwide. The fungus Cryptococcus neoformans infects individuals with damaged immune systems, such as those suffering from HIV/AIDS, commonly resulting in meningitis. This pathogen has become one of the leading fungal causes of disease and death worldwide, causing a million infections per year and 15-20% of all AIDS-related deaths. Research has shown that the ability to successfully infect, and to cause disease (virulence), largely depends on the DNA sequence (genotype) of the infecting Cryptococcus strain, and that a specific lineage (known as VNI) has undergone an ecological transition from a tree-associated wood-decaying organism to a bird-associated human pathogen. We have dated the emergence of this pandemic lineage out of Africa to colonise the world within the last 5,000 years. There is now an opportunity to determine how this emergence occurred, by sequencing the genomes of many environmental strains of C. neoformans and comparing their genetic diversity to that found causing disease in HIV patients worldwide. Our study will characterise the the genomic diversity of infecting Cryptococcus strains from HIV patients in affected regions of sub-saharan Africa and Southeast Asia. The African patients are being enrolled in a newly-funded MRC trial on the efficacy of antifungal drugs against cryptococcosis. We will collect isolates of C. neoformans from their environmental reservoir, trees, across South East Asia (principally Thailand) and sub-Saharan Africa, and compare their genome sequences to those known to cause disease in human patients. This will allow us to identify the genomic regions that are associated with virulence in humans, and will date major evolutionary events in C. neoformans, including the shuffling of genes through sexual recombination, how many lineages emerged from Africa to colonise the world, and when this occurred. Importantly, we will profile our environmental versus clinical infectious isolates with respect to key virulence determinants including size and shedding of their capsule, their ability to invade and grow within human macrophage cells and their ability to kill the model insect species Galleria mellonella. For isolates that show significant differences in virulence, we will profile patterns of gene expression for isolates cultured under conditions that mimic infection. These results will provide novel insights into diverse virulence determinants, some of which may provide new therapeutic targets against this difficult to treat infection.

We will use our cryopreserved collection of >300 clinical and environmental MLST-profiled Cng isolates from SE Asia and Africa to sequence isolates, including the reference H99, to a depth of ~100x. Reads will be aligned to the Broad Institute Cng VNI H99 reference genome to allow assembly, and base-calling. SNPs that are entirely covered in all isolates with high confidence will be concatenated for final alignment against the H99 chromosomes to produce a super-alignment. Aneuploid contigs will be treated as heterozygotes and detected using a depth-dependent binomial technique. In tandem with bioinformatics development, we will continue to expand our collection of environmental isolates from Southern Africa and SE Asia in collaboration with MRC-funded trials. Isolates will be Illumina-sequenced and sequentially added to the population genomics database. These will then be subjected to a genomics analysis aimed at determining the patterns and processes that have led to contemporary distributions of genetic diversity for global Cng. Associations will be sought between genotype and virulence by using clinical vs environmental isolates and, within clinical isolates, using the detailed clinical outcome data available for the source patients. Functional characterisation of outbreak clinical versus arboreal/environmental isolates will be done using the established Cng virulence factors, and a high-throughput analysis of macrophage parasitism using an intracellular proliferation rate assay for representatives of each Cng genotype. Virulence will also be assessed utilising the waxmoth larvae as a surrogate for murine models. Lineage-specific phenotypes will be analysed and panels of isolates that exhibit virulent versus hypovirulent phenotypes will be subjected to analysis of gene-expression using RNAseq. This will set the stage for downstream QTL-mapping of virulence in this sexually-reproducing fungus and the discovery of novel pathogen effectors.

We are concerned that insufficient development has occurred to appropriately disburse the avalanche of population genomics data that is being released by next-generation technologies, and even major repositories such as the NCBI Short Read Archive are finding it difficult to sustain adequate funding and resources. In order to counter this deficit, and to capitalise on the potential for population genomics datasets to transform evolutionary epidemiology, significant cross-disciplinary work needs to occur between partners for the full impact of next-generation genomics to be manifested. We will capitalise on our extensive expertise in bioinformatics, high-performance computing, statistical genetics, wet-lab and field-epidemiology and clinical trials to provide informatics solutions to allow community-wide access to the datasets and informatics tools that we are developing. As such, the wider epidemiological community stands to benefit from our research as our informatics platforms grow from our current investments.

Government and NGO charities in areas that are struck by the HIV pandemic will make use of our data on the distribution of Cryptococcus pathogenicity. As it is unlikely that people can be protected from aerosol-infection of environmental Cryptocococcus, our work is specifically targeted at identifying the aspects of the cryptococcal genome that are important in virulence, with the hope that we can use this information to develop targeted therapeutics. There are currently only three classes of antifungal agents with activity against Cryptococcus, with only one drug per class in common usage for treatment of cryptococcal meningitis. The newest of these, fluconazole, was licensed in 1993, and is poorly fungicidal and associated with secondary resistance. There is an urgent need for novel antifungal drug targets and adjunctive therapies, as well as for drugs with improved safety and tolerability profiles. Fungal pathogen genetics is currently at a turning point owing to the discovery of previously unsuspected virulence effectors, related to the RxLxE/Q/D motifs recently uncovered in Plasmodium. The potential discovery of similar virulence effectors in Cryptococcus may yield completely novel, untried, antifungal drug targets that could be exploited by industry to add to our scant armamentarium of antifungal drugs.