Alteration of Fermentative Metabolism Enhances Mucor circinelloides Virulence.

The fungus Mucor circinelloides undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in M. circinelloides virulence. A mutant in the adh1 gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-adh1+) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-adh1+ strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H2O2, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by M. circinelloides contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in M. circinelloides, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.

Alcohol dehydrogenase 1 participates in the Crabtree effect and connects fermentative and oxidative metabolism in the Zygomycete Mucor circinelloides.

Mucor circinelloides is a dimorphic Zygomycete fungus that produces ethanol under aerobic conditions in the presence of glucose, which indicates that it is a Crabtree-positive fungus. To determine the physiological role of the alcohol dehydrogenase (ADH) activity elicited under these conditions, we obtained and characterized an allyl alcohol-resistant mutant that was defective in ADH activity, and examined the effect of adh mutation on physiological parameters related to carbon and energy metabolism. Compared to the Adh+ strain R7B, the ADH-defective (Adh-) strain M5 was unable to grow under anaerobic conditions, exhibited a considerable reduction in ethanol production in aerobic cultures when incubated with glucose, had markedly reduced growth capacity in the presence of oxygen when ethanol was the sole carbon source, and exhibited very low levels of NAD+-dependent alcohol de-hydrogenase activity in the cytosolic fraction. Further characterization of the M5 strain showed that it contains a 10-bp deletion that interrupts the coding region of the adhl gene. Complementation with the wild-type allele adh1+ by transformation of M5 remedied all the defects caused by the adh1 mutation. These findings indicate that in M. circinelloides, the product of the adh1 gene mediates the Crabtree effect, and can act as either a fermentative or an oxidative enzyme, depending on the nutritional conditions, thereby participating in the association between fermentative and oxidative metabolism. It was found that the spores of M. circinelloides possess low mRNA levels of the ethanol assimilation genes (adl2 and acs2), which could explain their inability to grow in the alcohol.