Mrr1 regulation of methylglyoxal catabolism and methylglyoxal-induced fluconazole resistance inCandida lusitaniae

Abstract

22In Candida species, the transcription factor Mrr1 regulates azole resistance genes in addition to the 23 expression of a suite of other genes including known and putative methylglyoxal reductases. 24Methylglyoxal (MG) is a toxic metabolic byproduct that is significantly elevated in certain disease states 25 that frequently accompany candidiasis, including diabetes, kidney failure, sepsis, and inflammation. 26Through the genetic analysis of Candida lusitaniae (syn. Clavispora lusitaniae) strains with different Mrr1 27 variants with high and low basal activity, we showed that Mrr1 regulates basal and/or induced expression 28 of two highly similar MG reductases, MGD1 and MGD2, and that both participate in MG detoxification 29 and growth on MG as a sole carbon source. We found that exogenous MG increases Mrr1-dependent 30 expression of MGD1 and MGD2 in C. lusitaniae suggesting that Mrr1 is part of the natural response to 31 MG. MG also induced expression of MDR1, which encodes a major facilitator protein involved in 32 fluconazole resistance, in a partially Mrr1-dependent manner. MG significantly improved growth of C. 33 lusitaniae in the presence of fluconazole and strains with hyperactive Mrr1 variants showed greater 34 increases in growth in the presence of fluconazole by MG. In addition to the effects of exogenous MG, 35we found knocking out GLO1, which encodes another MG detoxification enzyme, led to increased 36 fluconazole resistance in C. lusitaniae. Analysis of isolates other Candida species found heterogeneity 37 in MG resistance and MG stimulation of growth in the presence of fluconazole. Given the frequent 38 presence of MG in human disease, we propose that induction of MDR1 in response to MG is a novel 39 contributor to in vivo resistance of azole antifungals in multiple Candida species. 41Author Summary 42In Candida species, constitutively active variants of the transcription factor Mrr1 confer resistance to 43 fluconazole, a commonly used antifungal agent. However, the natural role of Mrr1 as well as how its 44 activity is modulated in vivo remain poorly understood. Here, we have shown that, in the opportunistic 45 pathogen Candida lusitaniae, Mrr1 regulates expression and induction of two enzymes that detoxify 46 methylglyoxal, a toxic metabolic byproduct. Importantly, serum methylglyoxal is elevated in conditions 47 that are also associated with increased risk of colonization and infection by Candida species, such as 48 diabetes and kidney failure. We discovered that methylglyoxal causes increased expression of these two 49 Mrr1-regulated detoxification enzymes as well as an efflux pump that causes fluconazole resistance. 50Likewise, methylglyoxal increased the ability of multiple C. lusitaniae strains to grow in the presence of 51 fluconazole. Several other Candida strains that we tested also exhibited stimulation of growth on 52 fluconazole by methylglyoxal. Given the physiological relevance of methylglyoxal in human disease, we 53 posit that the induction of fluconazole resistance in response to...

Balancing positive and negative selection:in vivoevolution ofCandida lusitaniae MRR1

“…Constitutively active Mrr1 also appears to repress expression of 42 genes (fold change ≥ 1.5, FDR<0.05), including multiple iron and/or copper transporters and reductases, and sugar transporters (Table S1). These data combined with Bierman et al, which showed that C. lusitaniae Mrr1 is induced by the spontaneously formed stress signal methylglyoxal (33), imply that Mrr1 may play a larger role in a generalized metabolic or stress response, beyond what has been previously studied in response to FLZ and xenobiotic stressors.…”

“…The expression of MGD1 and MGD2, two C. lusitaniae genes shown to be Mrr1-regulated, correlated with the expression of MDR1 ( Fig. 1D and Table S1) (10,13,33). Gene expression differences between U04 (MRR1 Y813C ), U04 mrr1∆, and U04 mrr1Δ +MRR1 Y813C found that mrr1∆ is fully complemented upon return of MRR1 Y813C to the native locus ( Fig.…”

“…As in C. albicans, C. lusitaniae Mrr1 regulates the expression of the methylglyoxal reductase encoded by MGD1 (CLUG_01281 or GRP2) (10,29,33,38) and the multidrug efflux pump encoded by FLU1 (CLUG_05825) (10,39,40) (Table S1 and Fig. S3A).…”

Fructose Induces Fluconazole Resistance in Candida albicans through Activation of Mdr1 and Cdr1 Transporters

“…However, fructose has been described to also be metabolized to a toxic glycolytic byproduct called methylglyoxal (MG), the elevated levels of which are responsible for hepatotoxicity in diabetic patients [ 40 ]. In Candida lusitaniae , the MG metabolism is believed to be mediated by Mgd1 and Mgd2 reductases, the expression of which is controlled by Mrr1p, which, in turn, is inducible by MG [ 41 ]. The homologous protein in C. albicans ( Ca Mrr1p) is a major transcriptional inductor of MDR1 [ 42 ].…”