Abstract
BACKGROUND: In humans, Cryptococcus neoformans and Cryptococcus gattii species complexes are the leading cause of fungal meningitis globally. To establish CNS infection, Cryptococcus must breach the BBB, primarily comprised of specialized brain microvascular endothelial cells (BMECs), which is the prerequisite for cryptococci to invade the brain. Despite its clinical impact, the mechanisms underlying host-pathogen interaction at the BBB particularly involving environmental isolates remain under-characterized. OBJECTIVE: This study aimed to investigate the cyto-morphological and transcriptomic responses of HBMECs to infection by clinical and environmental Cryptococcus isolates using a dual approach-ultrastructural electron microscopy and high-throughput dual RNA-Seq. METHODS: HBMECs were infected in vitro with molecularly typed clinical and environmental isolates of C. neoformans and C. gattii at two infection time points (4 hpi and 18 hpi). Transmission electron microscopy was used to visualize host cell ultrastructural alterations, while dual RNA-Seq was performed to assess differential gene expression in both host and pathogen. RESULTS: TEM revealed extensive ultrastructural changes in infected HBMECs, including membrane ruffling, increased microvilli, mitochondrial alterations, ER dilation, Golgi fragmentation, nuclear deformation, and autophagosome formation. Transcriptomic profiling demonstrated functional enrichment of several critical cryptococcal virulence-associated genes linked to immune evasion and stress adaptation including various immune signaling pathways elicited by the HBMECs as a counter measure to the cryptococcal invasion. CONCLUSION: Clinical and environmental Cryptococcus isolates exhibit comparable invasive potential and elicit similar host endothelial responses with consistent effects observed across all isolates and time points. This integrative study combining ultrastructural and transcriptomic analyses highlights conserved host-pathogen interactions at the BBB, identifies potential molecular targets for antifungal therapy and underscores the pathogenic relevance of environmental reservoirs in cryptococcal meningitis. cryptococcal meningitis, blood-brain barrier, invasion, transmission electron microscopy, Dual RNASeq, differential gene expression, host-pathogen interaction, HBMECs, ultrastructural alterations.