Metformin exerts a dual effect on Mucorales virulence by altering oxidative metabolism through the regulation of rhizoferrin levels.

Mucormycosis, caused by fungi belonging to the order Mucorales, is associated with patients who suffer from uncontrolled diabetes mellitus. Blood glucose management in diabetic patients is performed through multiple strategies, with metformin being a common drug for controlling these levels. In this work, we show that the addition of low or high concentrations of metformin during the culture of Mucor lusitanicus and Rhizopus arrhizus differentially affects the toxicity of their cell-free culture supernatants toward Caenorhabditis elegans. The same effect was observed in infections of Galleria mellonella and mice treated with different metformin concentrations. Virulence induced by low concentrations of metformin correlates with augmented mitochondrial activity, which enhances respiration and increases hydroxyl radical (•OH) levels. Moreover, the SS toxicity towards C. elegans was eliminated by a mitophagy inducer and oxidative metabolism inhibitors, supporting that the effect of low metformin concentration on virulence is mediated by enhancing mitochondrial activity. Meanwhile, high metformin concentrations led to decreased virulence and reduced mitochondrial activity, along with the upregulation of AMP-activated protein kinase (AMPK)-α1-subunit (known also as Snfα-1), a mitophagy marker, catalase, and superoxide dismutase transcripts. Furthermore, deletion of the genes encoding the regulatory subunit of protein kinase A (PkaR1) and rhizoferrin synthetase partially revealed the signaling pathway underlying metformin-induced virulence of M. lusitanicus. In general, we found that metformin concentrations have a dual effect on the virulence of both Mucorales species by regulating oxidative metabolism, which may be relevant to the outcome of mucormycosis in diabetic patients treated with metformin.