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

Introduction. During the coronavirus disease 2019 (COVID-19) pandemic, the cases of mucormycosis increased substantially, with rhino-orbito-cerebral form linked to uncontrolled diabetes being the predominant manifestation. Several risk factors were implicated in the emergence of COVID-19-associated mucormycosis (CAM), including inappropriate corticosteroid usage and COVID-19-associated glycaemic imbalance. Gap statement. Some clinical and epidemiological studies associated the usage of zinc supplements with the occurrence of CAM, but experimental evidence remains limited. Aim. This study aimed to elucidate the impact of zinc enrichment on Rhizopus arrhizus, the predominant causative agent of mucormycosis. Methodology. 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 Roswell Park Memorial Institute -1640 medium. Morphological changes were visualized by scanning electron microscopy. Gene expression and intracellular zinc levels were determined by RNA sequencing and inductively coupled plasma mass spectrometry, respectively. The cytotoxic potential of R. arrhizus pre-cultured with and without zinc supplementation was assessed in the human embryonic kidney 293 cell line. Results. Zinc supplementation promoted R. arrhizus growth in a concentration-dependent manner, with the effect being particularly pronounced under less-favourable conditions, such as alkaline conditions resembling the nasal pH of diabetics. The results further suggested a zinc-specific effect: supplementation with another trace element (copper) suppressed R. arrhizus growth, and the zinc chelator (1,10-phenanthroline) dose-dependently abrogated the zinc-induced growth enhancement. Exposure to zinc increased the metabolic activity, and partly alleviated 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. R. arrhizus cytotoxicity was also significantly enhanced upon pre-culturing with zinc supplementation. Additionally, zinc augmented the growth/metabolism of several other Mucorales but not Aspergillus fumigatus and Candida albicans . Conclusion. Zinc supplementation promotes mucoralean growth, increases cytotoxic potential and enhances the expression of key virulence factors, including mucoricin and iron transporters. The findings contribute experimental evidence supporting the proposed association of zinc over-availability with the emergence of CAM.