Genome-wide transcriptional profiling identifies HNV1 as a hydroxyurea-responsive negative regulator of virulence in Candida albicans.

Precise DNA replication is essential for maintaining genomic stability, ensuring cellular homeostasis and faithful genetic transmission. Nevertheless, intrinsic and extrinsic factors frequently compromise replication fidelity, leading to replication stress-induced genomic instability, a potential contributor to pathogen virulence. In the fungal pathogen Candida albicans, the global transcriptomic adaptations underpinning the replication stress response and their potential contributions to fungal pathogenicity remain poorly understood. This study employed genome-wide RNA sequencing to map transcriptome dynamics in C. albicans exposed to hydroxyurea (HU), which induces DNA replication stress. Our analysis revealed significant transcriptional reprogramming under HU treatment, identifying 565 differentially expressed genes (DEGs) enriched in DNA replication/repair, cell cycle, oxidoreductase activity, lyase function, and cofactor binding. Notably, HNV1 (Orf19.4872), a previously uncharacterized gene, exhibited robust induction by HU, methyl methanesulfonate (MMS), and H₂O₂. Functional characterization demonstrated that HNV1 deletion conferred enhanced resistance to oxidative stress and hypervirulence in both Galleria mellonella and murine systemic infection models. Mechanistically, Hnv1 functions as a negative regulator of virulence by suppressing extracellular protease secretion through upregulation of SAP4, SAP5 and SAP6, while also modulating antioxidant defense pathways (e.g., CAT1, FDH1, HPD1). CRISPRi-mediated repression of these antioxidant genes reversed the hypervirulent phenotype. Collectively, this work identifies Hnv1 as a critical hub linking DNA replication stress to redox homeostasis and virulence in C. albicans, providing new mechanistic insights into fungal pathogenesis.