Comparative Proteomics of Extracellular Vesicles of Two Species of Mucorales Reveals Species-Specific Cargo.

Rhizopus arrhizus is one of the most common etiological agents of mucormycosis worldwide, whereas Mucor irregularis has been reported with a relatively high frequency in China, exhibiting clear regional characteristics. However, the molecular basis underlying their differential host responses remains incompletely understood. Fungal extracellular vesicles (EVs) play diverse biological roles across fungal species and have emerged as important carriers of bioactive cargos that modulate host-pathogen interactions. In this study, we compared features of EVs derived from R. arrhizus and M. irregularis and evaluated their immunomodulatory effects. EVs were isolated from PBS suspensions collected from fungal cultures via differential ultracentrifugation, followed by physical and proteomic characterization. Quantitative proteomics via DIA (data independent acquisition) identified 246 EV-associated proteins showing species-specific detection or quantitative abundance differences between the two species. In the murine footpad infection model, R. arrhizus induced more severe local lesions than M. irregularis, whereas EVs from M. irregularis elicited substantially higher pro-inflammatory cytokine expression in RAW 264.7 macrophages in vitro. Collectively, our results demonstrate marked divergence between R. arrhizus and M. irregularis in EV size, protein composition, and macrophage responses, providing a comparative resource for further investigation of EV-associated factors in Mucorales-host interactions.