Deciphering the global genomic landscape of C. neoformans: population dynamics, molecular epidemiology and genomic signatures of pathogenicity

Background Cryptococcus neoformans is an opportunistic fungal pathogen that exhibits extensive ecological resilience and is a leading cause of fungal meningitis. Despite its clinical significance, the global population structure, evolutionary dynamics, virulence-associated genome architecture, and antifungal resistance landscape remain poorly understood. This study aimed to investigate the global genomic diversity, molecular epidemiology, phylogenetic relationships, virulome, and resistome of C. neoformans using an integrated comparative genomic approach. Methods In total, we analyzed 139 C. neoformans genomes, comprising 138 publicly available genomes and one laboratory isolate from India. The molecular epidemiological characteristics were analyzed by serotype, molecular type, sequence type and mating type. We performed comparative genomic analyzes to investigate genome-wide similarity, phylogenetic relations and pangenome architecture. The virulome and resistome were characterized by systematic screening of virulence-associated genes and antifungal resistance-associated genes. Representative strains, H99 and Cn, were used to provide further preliminary phenotypic insights through in vitro antifungal susceptibility testing. Result The dataset comprised 126 clinical and 13 environmental isolates. Molecular epidemiological profiling showed a high prevalence of serotype A, molecular type VNI (88%), sequence type ST5 (63%) and mating type MATα (approximately 92%) indicating a highly conserved and largely clonal population structure. Southern Africa was identified as a key region of genomic diversity, with phylogenomic analysis revealing distinct evolutionary lineages, with VNB as the predominant variant among Southern African isolates. High average nucleotide identity (~99.9%) and a closed pangenome structure (Heap’s law exponent = 0.0005) further suggested extensive genomic conservation. Comparative analysis of 17 major virulence-associated genes showed that 11 were part of the core genome. Resistome profiling identified genetic differences in genes related to antifungal resistance, in particular ERG11 , FUR1 , FCY2 and MSH2 , with ERG11 I99V being the most frequent substitution found (56.8%). Conclusion These findings emphasize the worldwide dominance of a hyperclonal VNI-ST5-MATα lineage, a closed pangenome architecture and the conservation of major virulence and antifungal resistance-associated determinants in C. neoformans . This baseline study underscores the need for integrated clinical-genomic surveillance to track hyperclonal dissemination, conserved virulence, and adaptive resistance in this WHO critical-priority pathogen.