Role of zinc in growth, stress response and virulence gene expression of pathogenic Mucorale Rhizopus arrhizus

During COVID-19 pandemic, the cases of mucormycosis increased substantially, with rhino-orbito-cerebral form linked to uncontrolled diabetes being the predominant manifestation. Some clinical and epidemiological studies associated the usage of zinc supplements with the occurrence of COVID-19 associated mucormycosis, but experimental evidence remains limited. This study aimed to elucidate the impact of zinc enrichment on Rhizopus arrhizus , the predominant causative agent of mucormycosis. The effect of zinc supplementation (5 to 150 µM) on fungal growth, metabolic activity, antifungal susceptibility and biofilm formation, along with cell-wall (Congo red), oxidative (hydrogen peroxide) and osmotic (sodium chloride) stress was evaluated in RPMI-1640. Morphological changes were visualized by scanning electron microscopy. Gene expression was monitored by RNA sequencing. Zinc supplementation promoted R. arrhizus growth in a concentration-dependent manner, with the effect being particularly pronounced under less-favorable conditions, such as alkaline conditions resembling the nasal pH of diabetics. Exposure to zinc enhanced the metabolic activity, and partly alleviated the cell-wall and oxidative stress. Susceptibility to amphotericin B and posaconazole was unchanged. Zinc-supplemented cultures over-expressed multiple genes, notably those associated with respiratory electron transport chain, ergosterol biosynthesis, chitosan production, oxidative stress response, mucoricin, high-affinity iron permease, iron transport multi-copper oxidase and ferric-reductase-like protein. Zinc also augmented the growth/metabolism of several other Mucorales but not Aspergillus fumigatus and Candida albicans . The findings suggest that zinc supplementation supports mucoralean growth and enhances the expression of key virulence factors, implying that excessive intake of zinc supplements during COVID-19 pandemic likely contributed to the emergence of mucormycosis.