Host brain infection induces RNAi epimutation in Mucor circinelloides and enhances blood-brain barrier invasion

Mucorales fungi cause mucormycosis, a rapidly progressive invasive fungal disease with high mortality, particularly in immunocompromised patients and in severe disseminated infections. Central nervous system involvement is among the most devastating manifestations of mucormycosis, yet the mechanisms that enable Mucorales to adapt to the brain environment remain poorly understood. In Mucor species, RNA interference (RNAi) can generate reversible heritable epimutations that silence specific genes without altering DNA sequence, providing a transient and reversible mechanism for antifungal drug resistance. Here, we show that host brain infection induces RNAi-dependent silencing of endogenous fungal loci that promote central nervous system adaptation. Small RNA sequencing of Mucor circinelloides recovered from infected mouse organs identified seven brain epimutation loci, named bepA to bepG, that accumulated small RNAs preferentially after brain infection. Among these, bepA encodes a conserved mitogen-activated protein kinase (MAPK)-like serine/threonine kinase related to cell wall integrity signaling. Deletion of the bepA gene confers resistance to the calcineurin inhibitors FK506 and cyclosporin A, allowing hyphal growth under conditions that impair calcineurin-dependent morphogenesis. BepA modulates FK506-induced MAPK signaling and controls a transcriptional program enriched in cell-wall remodeling genes. To model fungal central nervous system entry by a filamentous fungal pathogen, we established a human blood-brain barrier transwell platform that quantifies Mucor migration across brain endothelial monolayers. This model revealed that bepA mutation enhances fungal migration across brain endothelial monolayers without detectable changes in electrical barrier resistance. These findings identify brain-induced RNAi epimutation as a mechanism of organ-specific fungal adaptation and reveal reversible silencing of a MAPK-like kinase as a route to enhanced central nervous system invasion.