SignificanceDrug-resistant subpopulations of microbes or tumor cells are difficult to detect but can confound disease treatment. In this deep characterization of a chronic fungal infection, we report unprecedented heterogeneity in the drug resistance-related gene MRR1 among Clavispora (Candida) lusitaniae isolates from a single individual. Cells expressing Mrr1 variants that led to drug resistance, by elevated expression of the MDR1-encoded efflux protein, were present at low levels in each sample and thus were undetected in standard assays. We provide evidence that these drug-resistant fungi may arise indirectly in response to other factors present in the infection. Our work suggests that alternative methods may be able to identify drug-resistant subpopulations and thus positively impact patient care.

Transcription factors play key roles in regulating virulence of the human fungal pathogen C. albicans. In addition to regulating the expression of virulence factors, they also control the ability of C. albicans to switch to filamentous hyphal growth, which facilitates biofilm formation on medical devices and invasion into tissues. We therefore used new CRISPR/Cas9 methods to examine the effects of deleting three C. albicans genes (NDT80, REP1, and RON1) that encode transcription factors with similar DNA binding domains. Interestingly, double and triple mutant strains mostly showed the combined properties of the single mutants; there was only very limited evidence of synergistic interactions in regulating morphogenesis, stress resistance, and ability to metabolize different sugars. These results demonstrate that NDT80, REP1, and RON1 have distinct functions in regulating C. albicans virulence functions.

Transcription factor Mrr1, best known for its regulation of Candida azole resistance genes such as MDR1, regulates other genes that are poorly characterized. Among the other Mrr1‐regulated genes are putative methylglyoxal reductases. Methylglyoxal (MG) is a toxic metabolite that is elevated in diabetes, uremia, and sepsis, which are diseases that increase the risk for candidiasis, and MG serves as a regulatory signal in diverse organisms. Our studies in Clavispora lusitaniae, also known as Candida lusitaniae, showed that Mrr1 regulates expression of two paralogous MG reductases, MGD1 and MGD2, and that both participate in MG resistance and MG catabolism. Exogenous MG increased Mrr1‐dependent expression of MGD1 and MGD2 as well as expression of MDR1, which encodes an efflux pump that exports fluconazole. MG improved growth in the presence of fluconazole and this was largely Mrr1‐dependent with contributions from a secondary transcription factor, Cap1. Increased fluconazole resistance was also observed in mutants lacking Glo1, a Mrr1‐independent MG catabolic enzyme. Isolates from other Candida species displayed heterogeneity in MG resistance and MG stimulation of azole resistance. We propose endogenous and host‐derived MG can induce MDR1 and other Mrr1‐regulated genes causing increased drug resistance, which may contribute to some instances of fungal treatment failure.

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...

Candida auris is a fungal pathogen that has spread since its identification in 2009 and is of concern due to its high incidence of resistance against multiple classes of antifungal drugs. In other Candida species, the transcription factor Mrr1 plays a major role in resistance against azole antifungals and other toxins.

Candida auris is an urgent threat to human health due to its rapid spread in healthcare settings and its repeated development of multidrug resistance. Diseases that put individuals at a higher risk for C. auris infection, such as diabetes, kidney failure, or immunocompromising conditions, are associated with elevated levels of methylglyoxal (MG), a reactive dicarbonyl compound derived from several metabolic processes. In other Candida species, expression of MG reductase enzymes that catabolize and detoxify MG are controlled by Mrr1, a multidrug resistance-associated transcription factor, and MG induces Mrr1 activity. Here, we used transcriptomics and genetic assays to determine that C. auris MRR1a contributes to MG resistance, and that the main Mrr1a targets are an MG reductase and MDR1, which encodes an drug efflux protein. The C. auris Mrr1a regulon is smaller than Mrr1 regulons described in other species. In addition to MG, benomyl (BEN), a known Mrr1 stimulus, induces C. auris Mrr1 activity, and characterization of the MRR1a-dependent and independent transcriptional responses revealed substantial overlap in genes that were differentially expressed in response to each compound. Additionally, we found that an MRR1 allele specific to one C. auris phylogenetic clade, clade III, encodes a hyperactive Mrr1 variant, and this activity correlated with higher MG resistance. C. auris MRR1a alleles were functional in Candida lusitaniae and were inducible by BEN, but not by MG, suggesting that the two Mrr1 inducers act via different mechanisms. Together, the data presented in this work contribute to the understanding Mrr1 activity and MG resistance in C. auris.ImportanceCandida auris is a fungal pathogen that has spread since its identification in 2009 and is of concern due to its high incidence of resistance against multiple classes of antifungal drugs. In other Candida species, the transcription factor Mrr1 plays a major role in resistance against azole antifungals and other toxins. More recently, Mrr1 has been recognized to contribute to resistance to methylglyoxal (MG), a toxic metabolic byproduct. Here, we show that C. auris MRR1a, the closest ortholog to MRR1 in other species, contributes to resistance to MG, and that Mrr1a strongly co-regulates expression of MGD1, encoding a methylglyoxal reductase enzyme and MDR1, encoding an efflux protein involved in resistance to azole drugs, antimicrobial peptides and bacterial products. We found that one major clade of C. auris has a constitutively active Mrr1 despite high azole resistance due to other mutations, and that this high Mrr1a activity correlates with higher MG resistance. Finally, we gain insights into the activities of MG and another Mrr1 inducer, benomyl, to better understand C. auris regulation of phenotypes relevant in vivo.