Mrr1 regulation of methylglyoxal catabolism and methylglyoxal-induced fluconazole resistance in Candida lusitaniae

In Candida species, the transcription factor Mrr1 regulates azole resistance genes in addition to the expression of a suite of other genes including known and putative methylglyoxal reductases. Methylglyoxal (MG) is a toxic metabolic byproduct that is significantly elevated in certain disease states that frequently accompany candidiasis, including diabetes, kidney failure, sepsis, and inflammation. Through the genetic analysis of Candida lusitaniae (syn. Clavispora lusitaniae) strains with different Mrr1 variants with high and low basal activity, we showed that Mrr1 regulates basal and/or induced expression of two highly similar MG reductases, MGD1 and MGD2, and that both participate in MG detoxification and growth on MG as a sole carbon source. We found that exogenous MG increases Mrr1-dependent expression of MGD1 and MGD2 in C. lusitaniae suggesting that Mrr1 is part of the natural response to MG. MG also induced expression of MDR1, which encodes a major facilitator protein involved in fluconazole resistance, in a partially Mrr1-dependent manner. MG significantly improved growth of C. lusitaniae in the presence of fluconazole and strains with hyperactive Mrr1 variants showed greater increases in growth in the presence of fluconazole by MG. In addition to the effects of exogenous MG, we found knocking out GLO1, which encodes another MG detoxification enzyme, led to increased fluconazole resistance in C. lusitaniae. Analysis of isolates other Candida species found heterogeneity in MG resistance and MG stimulation of growth in the presence of fluconazole. Given the frequent presence of MG in human disease, we propose that induction of MDR1 in response to MG is a novel contributor to in vivo resistance of azole antifungals in multiple Candida species. Author Summary In Candida species, constitutively active variants of the transcription factor Mrr1 confer resistance to fluconazole, a commonly used antifungal agent. However, the natural role of Mrr1 as well as how its activity is modulated in vivo remain poorly understood. Here, we have shown that, in the opportunistic pathogen Candida lusitaniae, Mrr1 regulates expression and induction of two enzymes that detoxify methylglyoxal, a toxic metabolic byproduct. Importantly, serum methylglyoxal is elevated in conditions that are also associated with increased risk of colonization and infection by Candida species, such as diabetes and kidney failure. We discovered that methylglyoxal causes increased expression of these two Mrr1-regulated detoxification enzymes as well as an efflux pump that causes fluconazole resistance. Likewise, methylglyoxal increased the ability of multiple C. lusitaniae strains to grow in the presence of fluconazole. Several other Candida strains that we tested also exhibited stimulation of growth on fluconazole by methylglyoxal. Given the physiological relevance of methylglyoxal in human disease, we posit that the induction of fluconazole resistance in response to methylglyoxal may contribute to treatment failure.