Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp

Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp Dhanabala-Subhiksha Rajesh-Khanna , Carolina G. Piña Páez , Elora G. Dolan , Kiran S. Mirpuri , View ORCID Profile Jason E. Staijch , View ORCID Profile Deborah A. Hogan doi: https://doi.org/10.1101/2025.05.04.652153 Dhanabala-Subhiksha Rajesh-Khanna a Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth , Hanover, New Hampshire, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Carolina G. Piña Páez b Department of Microbiology & Plant Pathology and Institute for Integrative Genome Biology, University of California—Riverside , Riverside, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Elora G. Dolan a Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth , Hanover, New Hampshire, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kiran S. Mirpuri a Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth , Hanover, New Hampshire, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jason E. Staijch b Department of Microbiology & Plant Pathology and Institute for Integrative Genome Biology, University of California—Riverside , Riverside, California, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jason E. Staijch Deborah A. Hogan a Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth , Hanover, New Hampshire, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Deborah A. Hogan For correspondence: deborah.a.hogan{at}dartmouth.edu Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Infections caused by the emerging pathogenic yeast Clavispora (Candida) lusitaniae can be difficult to manage due to multi-drug resistance. Resistance to the frontline antifungal fluconazole (FLZ) in Candida spp. is commonly acquired through gain-of-function (GOF) mutations in the gene encoding the transcription factor Mrr1. These activated Mrr1 variants enhance FLZ efflux via upregulation of the multi-drug transporter gene MDR1 . Recently, it was reported that, unlike in the well-studied Candida albicans species, C. lusitaniae and Candida parapsilosis with activated Mrr1 also have high expression of CDR1 , which encodes another multi-drug transporter with overlapping but distinct transported substrate profiles and Cdr1-dependent FLZ resistance. To better understand the mechanisms of Mrr1 regulation of MDR1 and CDR1 , and other co-regulated genes, we performed CUT&RUN analysis of Mrr1 binding sites. Mrr1 bound the promoter regions of MDR1 and CDR1 as well as FLU1 , which encodes another transporter capable of FLZ efflux. Mdr1 and Cdr1 independently contributed to the decreased susceptibility of the MRR1 GOF strains against diverse clinical azoles and other antifungals, including 5-flucytosine. A consensus motif, CGGAGWTAR, enriched in Mrr1-bound C. lusitaniae DNA was also conserved upstream of MDR1 and CDR1 across species including C. albicans . CUT&RUN and RNA-seq data were used to define the Mrr1 regulon which includes genes involved in transport, stress responses, and metabolism. Activated and inducible Mrr1 bound similar regions in the promoters of Mrr1 regulon genes. Our studies provide new evolutionary insights into the coordinated regulation of multi-drug transporters and potential mechanism(s) that aid secondary resistance acquisition in emerging Candida . SIGNIFICANCE Understanding antifungal resistance in emerging Candida pathogens is essential to manage treatment failures and guide the development of new therapeutic strategies. Like other Candida species, the environmental opportunistic fungal pathogen Clavispora ( Candida ) lusitaniae can acquire resistance to the antifungal fluconazole by overexpression of the multi-drug efflux pump Mdr1 through gain-of-function mutations in the gene encoding the transcription factor Mrr1. Here, we show that C. lusitaniae Mrr1 also directly regulates CDR1, another major multi-drug transporter gene, along with MDR1. In strains with activated Mrr1, upregulation of MDR1 and CDR1 protects against diverse antifungals potentially aiding the rise of other resistance mutations. Mrr1 also regulates several stress response and metabolism genes thereby providing new perspectives into the physiology of drug-resistant strains. The identification of an Mrr1 binding motif that is conserved across strains and species will advance future efforts to understand multi-drug resistance across Candida species. INTRODUCTION Invasive or systemic candidiasis affects over 1.5 million people each year with high rates of mortality ( 1 ) and localized Candida infections have high economic and quality of life burdens. While Candida albicans is the major causative agent of Candida infections, other non-albicans Candida such as Clavispora ( Candida ) lusitaniae are garnering attention for increased incidence and drug susceptibility profiles ( 2 ). C. lusitaniae can establish difficult-to-treat infections in immunocompromised individuals ( 3 – 9 ). Unlike Candida spp. that are found largely within the human microbiome, C. lusitaniae appears to have a flexible physiology that allows it to occupy environmental, agricultural and human-associated niches ( 10 ). Much like its phylogenetic neighbor Candidozyma ( Candida ) auris , C. lusitaniae also can exhibit resistance to any of the three major antifungal classes – polyenes, echinocandins, and azoles such as fluconazole (FLZ) – within days of treatment ( 9 , 11 – 15 ). C. lusitaniae, like other Candida spp., gains resistance to FLZ through several mechanisms ( 12 ) including mutation of the FLZ target Erg11 or the acquisition of gain-of-function (GOF) mutations in the gene encoding for the multi-drug resistance regulator Mrr1. Mrr1 GOF variants have constitutive activity and upregulate the expression of the multi-drug efflux pump gene MDR1 . The major facilitator superfamily (MFS) transporter Mdr1 is conserved across Candida species and has promiscuity for structurally and functionally distinct substrates including FLZ, bacterial phenazines and salivary antimicrobial peptides like histatins ( 16 – 18 ). Mrr1-dependent transcription of MDR1 and other genes can be induced by xenobiotics such as benomyl ( 19 – 23 ) and the metabolite methylglyoxal ( 24 ). Across Candida species, Mrr1 GOF variants also co-regulate the expression of MDR1 and putative methylglyoxal dehydrogenases ( 23 , 25 – 28 ) and confer fitness advantages outside of FLZ resistance in C. lusitaniae ( 24 ). In Demers et al. ( 21 ), we described the repeated selection for MRR1 GOF mutations in clinical isolates recovered from a chronic lung infection of C. lusitaniae . These mutations evolved in a FLZ-naïve environment suggesting there are unrecognized roles for MRR1 in host adaptation. Genomic analyses of these isolates identified secondary suppressor mutations that either attenuated constitutive activity or restored the inducible Mrr1 phenotype. The regain of inducibility underscores that there are benefits associated with an inducible Mrr1 phenotype as well. The physiology of strains with an inducible and activated Mrr1 seems to be vastly different as over 90 targets including those reported in other species such as MDR1 and methylglyoxal dehydrogenase-encoding MGD1 and MGD2 as well as novel unanticipated targets like CDR1 , encoding for a multi-drug transporter, were differentially expressed in a transcriptomic analysis of the different MRR1 alleles ( 18 , 21 ). The ATP-binding cassette (ABC) superfamily transporter Cdr1 is a well-studied Candida multi-drug efflux pump that is conserved across species and exports a wide range of substrates including Mdr1 targets like FLZ as well as distinct ones like rhodamine-6-G ( 29 – 31 ). In C. albicans , CDR1 expression is regulated by the zinc-cluster transcription factor Tac1 and GOF mutations in the TAC1 gene is another mechanism of FLZ resistance ( 27 , 32 , 33 ). However, recent studies in emerging Candida spp. Including C. lusitaniae and C. parapsilosis have shown Mrr1-dependent changes in CDR1 expression ( 34 , 35 ). Here, we address this altered regulation of CDR1 and its effect on strains with constitutively active Mrr1 in C. lusitaniae . In this study, we report that C. lusitaniae Mrr1 directly regulates both MDR1 and CDR1 and that this coordinate regulation of Mdr1 and Cdr1 contributes to decreased sensitivity to multiple clinical and environmental antifungals. Further, analysis of Mrr1-DNA interactions found that Mrr1 directly regulates genes involved in cellular processes beyond drug transport by binding to a consensus Mrr1 motif that is conserved in different species. We also demonstrate that Mrr1 activation state does not alter its DNA localization at these targets. While this model for MDR1 and CDR1 regulation differs from that which has been described in C. albicans , the findings in C. lusitaniae are consistent with recent reports in diverse Candida species including C. auris . Our findings suggest that the rise of drug resistant lineages may be aided by the coordinated regulation of two drug resistance factors under Mrr1 and that plasticity in drug resistance regulation could be instrumental in the development of multi-drug resistant species. RESULTS C. lusitaniae Mrr1 effects on expression of multiple transporters involved in drug resistance We previously characterized the C. lusitaniae clinical isolate strain U04 and its mrr1 Δ derivative complemented with either MRR1 ancestral , which confers Mrr1 activity typical of most C. lusitaniae isolates, or MRR1 Y813C , which confers constitutive Mrr1 activity that renders cells resistant to FLZ ( 21 ). Published transcriptomic comparisons of these strains revealed significantly higher levels of MDR1 ( CLUG_01938_39 ( 18 )), CDR1 ( CLUG_03113 ( 34 )) and CLUG_05825 ( FLU1 ), all of which encode drug efflux proteins, when Mrr1 was constitutively active ( 21 ) ( Fig 1A ). Here on, we refer to CLUG_05825 as FLU1 , as the protein sequence encoded by this gene had the highest similarity by BLAST to the homolog encoded by C7_01520W_A of C. albicans SC5314. Download figure Open in new tab Figure 1: Biochemical and phenotypic analysis of HF-tagged Mrr1 and binding profiles of constitutively active Mrr1. (A) Log counts per million (CPM) values of MDR1 (CLUG_01938_39) , CDR1 (CLUG_03113) and FLU1 (CLUG_05825) from RNA-seq analysis of Demers et al. ( 21 ) comparing U04 clinical isolate (native allele MRR1 Y813C ) and U04 mrr1 Δ complemented with either MRR1 Y813C or MRR1 ancestral . Ordinary one-way ANOVA and Dunnett’s multiple comparisons testing with a single pooled variance were used to evaluate the statistical significance for each gene. ***, p<0.001; ****, p<0.0001. (B) Western blot of whole cell protein lysates of U04 strains expressing N-terminal 6xHis-3xFLAG-tagged Mrr1 (HF-Mrr1) variants. HF-Mrr1 was probed using an α-FLAG antibody. Mean ± SD of HF-Mrr1 band intensities normalized to total protein (n= 4 biological replicates). (C) FLZ MIC of U04 clinical isolate and U04 mrr1 Δ complemented with untagged or HF-MRR1 was determined by broth microdilution assays. The data represent the mean ± SD from three independent experiments. There were no significant differences observed between data from strains with untagged Mrr1 variants and data from strains with their respective HF-tagged counterparts. (A – C) Strains with constitutive Mrr1 activity are in bold. (D-F) HF-Mrr1 Y813C CUT&RUN read coverage plots normalized per 20 bp bin size. Chromosomal positions of regions containing MDR1, CDR1 and FLU1 and adjacent genes are represented to scale with boxes and arrows. Peaks from HF-Mrr1 Y813C -bound DNA recovered by an α-FLAG antibody and for the non-specific binding control recovered via IgG are shown. Signal indicates the average read density in α-FLAG relative to IgG within the peak region. Two independent experiments were performed and both are shown. Construction and activity of epitope-tagged Mrr1 variants To investigate if C. lusitaniae Mrr1 regulation of these transporters was direct, we analyzed Mrr1-DNA interactions. We first generated N-terminal 6xHis-3xFLAG (HF)-tagged versions of different Mrr1 variants. HF-Mrr1-encoding alleles were expressed from the native MRR1 promoter after introduction into the U04 mrr1 Δ mutant background. We found that HF-Mrr1 ancestral and HF-Mrr1 Y813C were stably produced, and both were detected at a slightly higher molecular weight (150 kDa) than the predicted ∼140 kDa. This band was absent in the western blot of samples from the U04 mrr1 Δ strain ( Fig. 1B ). We did not observe any significant differences in Mrr1 levels between strains expressing HF-Mrr1 ancestral and HF-Mrr1 Y813C ( Fig. 1B ). We compared the activities of the HF-Mrr1 variants to their untagged counterparts by evaluating the minimum inhibitory concentration (MIC) of FLZ in strains with either HF-tagged or untagged Mrr1 variants ( Fig. 1C ). The U04 mrr1 Δ strain with untagged MRR1 Y813C had a MIC that was 64-fold higher than the strain with untagged MRR1 ancestral (FLZ MIC of 32 µg/ml vs 0.5 µg/ml) ( 21 ). The U04 mrr1 Δ strain complemented with HF-MRR1 Y813C had a similarly high MIC relative to the strain with HF-MRR1 ancestral ( Fig. 1C ). Thus, the N-terminal HF-tag did not affect Mrr1 function. Analysis of Mrr1 Y813C -DNA localization in C. lusitaniae We evaluated genome-wide binding of HF-Mrr1 Y813C using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) in two independent experiments ( 36 ). An α-FLAG antibody (Ab) was used for the enrichment of HF-Mrr1 bound DNA, and an IgG Ab was used to assess non-specific binding. The recovered DNA was sequenced and aligned to the genome of C. lusitaniae strain L17 (NCBI accession: ASM367555v2). Both U04 and L17 were isolated from the same clinical sample and differ by only ∼108 Single Nucleotide Polymorphisms and ∼130 Insertions/Deletions ( 18 ). The genome of L17 was utilized as it is a highly accurate genome produced by sequencing and assembly of reads obtained using Oxford Nanopore long-read and Illumina technologies. Genomic regions that showed significant fold enrichment in DNA recovered from the α-FLAG when compared to the IgG control of each sample are represented as peaks and indicate HF-Mrr1 Y813C interaction sites ( Fig. 1D ). The average enrichment of reads in α-FLAG relative to the IgG background within an identified peak region was quantified as peak signal ( 37 ). Peaks were filtered using a peak signal cutoff of 2, a false discovery rate (FDR) of <0.05 and a genomic position within 1 kb of an open reading frame (ORF). Approximately 329 CUT&RUN peaks were identified (File S1). The upstream regions of MDR1 , CDR1 and FLU1 all showed strong evidence for Mrr1 Y813C binding. The upstream region of the MDR1 ORF containing its promoter had a significant HF-Mrr1 Y813C peak with an average signal of 10 ( Fig. 1D ). The HF-Mrr1 Y813C peak associated with the MDR1 ORF spanned ∼1.5 kb and extended into the neighboring coding regions of MDR1 ( Fig. 1D ). Thus, as in C. albicans ( 20 , 38 ), C. lusitaniae Mrr1 bound directly upstream of MDR1 . The regions upstream of CDR1 also had a significantly enriched CUT&RUN peak with an average signal of 6.7 and a peak width of ∼1.2 kb ( Fig. 1E ). An Mrr1 binding peak was similarly found upstream of the gene encoding Flu1 ( Fig. 1F ). The peaks associated with the FLU1 ORF had an average signal of 4.8 and covered a length of ∼0.8 kb ( Fig. 1F ). The signal of the HF-Mrr1 Y813C peak upstream of the MDR1 ORF was 1.5-and 2.1-fold higher than upstream of the CDR1 and FLU1 ORFs. Together, these data are consistent with previous reports of Mrr1 regulation of MDR1 and provide evidence for direct regulation of CDR1 and FLU1 by Mrr1 in C . lusitaniae . Constitutive expression of MDR1 reduces susceptibility to short chain azoles, while CDR1 reduces susceptibility to long chain azoles To investigate the phenotypic consequences of Mrr1 regulation of MDR1 , CDR1 , and FLU1 , we determined the concentrations of various azoles required to inhibit 90% (MIC 90 ) of the growth of strain U04 with Mrr1 Y813C and its mdr1 Δ, cdr1 Δ and flu1 Δ derivatives. As the C. albicans homologs of Mdr1, Cdr1 and Flu1 were all capable of fluconazole (FLZ) efflux ( 29 , 30 , 39 ), we first evaluated the FLZ MIC 90 . The U04 strain expressing Mrr1 Y813C had a 32-fold higher FLZ MIC 90 than the isogenic strain with Mrr1 ancestral ( Fig. 2A & Table 1 ). The Mrr1 Y813C mdr1 Δ mutant exhibited an 8-fold lower FLZ MIC 90 than its parent mrr1 Δ+ MRR1 Y813C strain (4 µg/ml vs 32 µg/ml; Fig. 2A & Table 1 ). Download figure Open in new tab Figure 2: Effects of Mrr1 activity and MDR1, CDR1 and FLU1 on susceptibility to clinically relevant azoles. Structures and MICs (µg/ml) of Fluconazole (FLZ) (A), Voriconazole (VOR) (B), Ketoconazole (KTZ) (C), Itraconazole (ITZ) (D) and Isavuconazole (ISA) (E) are shown. MICs were determined using broth microdilution assays for strain U04 (native allele MRR1 Y813C ) and U04 mutants: mrr1 Δ+ MRR1 ancestral , mrr1 Δ+ MRR1 Y813C and mdr1 , cdr1 and flu1 deletion mutants in the mrr1 Δ+ MRR1 Y813C background. Heatmaps represent the optical density (600 nm) of the azole-treated wells normalized to the respective untreated strain controls (column on right). Drug concentrations in µg/ml are shown on the x-axis. The average from three independent experiments performed on different days is shown. Strains with constitutive Mrr1 activity are in bold. View this table: View inline View popup Download powerpoint Table 1: MIC 90 of clinical azoles. MIC 90 values were calculated from the broth microdilution assays in Figure 2 . MIC 90 was defined as the concentration at which 90% growth was inhibited. Fold differences in MIC 90 relative to the azole-sensitive U04 mrr1 Δ + MRR1 ancestral are presented within parentheses (). FLZ – Fluconazole, VOR – Voriconazole, KTZ – Ketoconazole, ITZ – Itraconazole and ISA-Isavuconazole. Although, the FLZ MIC 90 values were unchanged in a cdr1 Δ and flu1 Δ mutant, the flu1 Δ mutant grew slightly less well than the parent strain across concentrations ( Fig. 2A & Table 1 ). Similar Mdr1-dependent resistance was observed for the other short-tailed azole voriconazole (VOR) in strains with constitutively active Mrr1; the mrr1 Δ+ MRR1 Y813C strain had a 32-fold higher VOR MIC 90 than the mrr1 Δ+ MRR1 ancestral strain (0.5 µg/ml vs 0.0156 µg/ml; Fig. 2B & Table 1 ). While the mdr1 Δ mutation resulted in a 4-fold lower VOR MIC 90 than the mrr1 Δ+ MRR1 Y813C and the U04 WT ( MRR1 Y813C ) strains, no difference in MIC 90 was observed for the cdr1 Δ and flu1 Δ mutants ( Fig. 2B & Table 1 ). Overall, strains expressing constitutively active Mrr1 exhibited similar Mdr1-mediated resistance to the triazoles FLZ and VOR. Interestingly, the MDR1 deletion alone was not sufficient to abrogate resistance as the mdr1 Δ mutant still had a 4-fold higher FLZ (4 µg/ml vs 1 µg/ml) and 8-fold higher VOR (0.125 µg/ml vs 0.0156 µg/ml) MIC 90 values than the mrr1 Δ+ MRR1 ancestral strain ( Fig. 2A, B & Table 1 ) implying that there are redundant activities across other Mrr1-regulated azole resistance factors. Thus, our results suggest that Mdr1-mediates resistance to short-tailed azoles in strains with constitutive Mrr1 activity. We evaluated the susceptibility of the different strains to the long-tailed azoles: ketoconazole (KTZ), itraconazole (ITR) and isavuconazole (ISA). The mrr1 Δ+ MRR1 Y813C strain had a 32-, >8-and >16-fold higher MIC 90 values for KTZ, ITR and ISA respectively, than the mrr1 Δ+ MRR1 ancestral strain ( Fig. 2C-E & Table 1 ). Consistent with prior reports of Cdr1-mediated resistance to long-tailed azoles ( 34 , 40 ), the cdr1Δ strain had a >8-fold reduction in MIC 90 values for KTZ, ITR and ISA than the mrr1 Δ+ MRR1 Y813C parental strain ( Fig. 2C-E & Table 1 ). The mdr1 Δ and flu1 Δ strains were not more susceptible to the tested long-tailed azoles than the parent strain ( Fig. 2C-E & Table 1 ). These data indicate that constitutively active Mrr1 confers resistance to long-tailed azoles via Cdr1. Mrr1-regulated Mdr1 and Cdr1 decrease susceptibility to drugs from diverse classes Transporter-mediated efflux of other antifungal compounds of agricultural and clinical relevance has been demonstrated ( 41 ) and strains with constitutive Mrr1 activity exhibited broad-spectrum resistance against multiple toxic substrates in a Biolog Phenotype Microarray screen ( 21 ). Thus, we evaluated the MICs of 5-flucytosine (5-FC), cycloheximide, myclobutanil, terbinafine and fluphenazine for mrr1 Δ+ MRR1 ancestral and mrr1 Δ+ MRR1 Y813C strains. Here on, MIC was defined as the concentration at which no visible growth was observed. The mrr1 Δ+ MRR1 Y813C strain had a 2-to 32-fold increase in the MIC values of the different tested antifungals compared to the mrr1 Δ+ MRR1 ancestral strain ( Fig. 3A ). Further, in the mrr1 Δ+ MRR1 Y813C strain background, the mdr1 Δ derivative resulted in increased susceptibility to 5-FC, cycloheximide and myclobutanil ( Fig. 3B ). The MIC values of cycloheximide and 5-FC decreased by 4-fold in the mdr1 Δ mutant ( Fig. 3B ); support for Mdr1-mediated resistance against the pyrimidine analog 5-FC has been previously shown in C. lusitaniae ( 12 , 25 , 34 ). While the protein synthesis inhibitor cycloheximide was a substrate of both Mdr1 and Cdr1 ( 29 ) in C. albicans , the cdr1Δ mutation did not alter the cycloheximide resistance of the mrr1 Δ+ MRR1 Y813C strain. For the agricultural triazole myclobutanil, the mdr1 Δ and cdr1 Δ mutants had 2-to 4-fold lower MIC values than the mrr1 Δ+ MRR1 Y813C parental strain (2-4 µg/ml vs 8 µg/ml) ( Fig. 3B ). However, both still had 8-fold higher MIC values than the mrr1 Δ+ MRR1 ancestral strain (2-4 µg/ml vs 0.25 µg/ml) suggesting that other Mrr1 targets contributed to myclobutanil resistance. Download figure Open in new tab Figure 3: Effects of Mrr1 activity and MDR1, CDR1 and FLU1 on susceptibility to broad-spectrum antifungals. (A) Log 2 transformed fold difference of MIC for diverse antifungals for strains U04 (native allele MRR1 Y813C ) and its mrr1 Δ+ MRR1 ancestral and mrr1 Δ+ MRR1 Y813C derivatives determined using broth microdilution assays. Data were normalized to that for mrr1 Δ+ MRR1 ancestral strain. (B) Log 2 transformed fold difference in MIC values of mdr1Δ , cdr1Δ and flu1Δ mutants normalized to their parent U04 mrr1 Δ+ MRR1 Y813C . The data represent the mean ± SD from at least three independent experiments performed on different days. Strains with constitutive Mrr1 activity are in bold. Ordinary one-way ANOVA and Dunnett’s multiple comparisons testing with a single pooled variance were used to evaluate the statistical significance between strains for each antifungal. All significant comparisons are shown; *, p<0.05, **, p<0.01, ***, p<0.001 and ****, p<0.0001. Susceptibility of other antifungals was dependent on Cdr1. The MICs for the allylamine antifungal terbinafine and the antipsychotic fluphenazine were lower in the cdr1 Δ mutant. Since FLU1 deletion made drug-sensitive C. albicans hypersusceptible to the metabolic inhibitor mycophenolic acid (MPA) ( 39 ), we also investigated the MPA susceptibility of our strains. Despite the mrr1 Δ+ MRR1 Y813C having a 2-to 4-fold increase in MPA MIC relative to the mrr1 Δ+ MRR1 ancestral strain, its MIC was not impacted by deletion of FLU1 . The mdr1 Δ and cdr1 Δ mutants were also not more susceptible to MPA ( Fig. 3B ). Taken together, our results show that constitutive Mrr1 activity conferred resistance to a broad spectrum of antifungals, largely through its control of Mdr1-and Cdr1 with evidence for redundancy in Mrr1-regulated antifungal resistance mechanisms. Mrr1 directly regulates genes involved in diverse biological processes To examine other genes that were co-regulated with MDR1 , CDR1 and FLU1 , we identified additional genes that were differentially expressed due to a direct consequence of constitutive Mrr1 activity. There were twenty five genes that were differentially expressed when Mrr1 was constitutively active (Mrr1 Y813C ) compared to mrr1 Δ and low-activity Mrr1 ( 21 ) (FDR < 0.05 and fold change ≥ 1.5) and had a HF-Mrr1 Y813C peak located within 1 kb from their ORF regions including MDR1 , CDR1 , and FLU1 ( Fig. 4A , Table S1A). These 25 genes will be referred to as the C. lusitaniae Mrr1 regulon (Table S1A). Slim Gene Ontology (GO) analysis of the C. albicans homologs of the C. lusitaniae Mrr1 regulon genes found transport, response to chemical, response to stress, and cellular homeostasis as the most enriched biological process terms (Table S1B). The Mrr1 regulon included two putative peptide transporters ( OPT1 and OPT5 ), two extracellular cell wall proteins ( ECM33 and CSA1 ), two involved in metal homeostasis ( CTR2 and CFL4 ), a putative glycerol transporter ( HGT10 / STL1 ), an alternative oxidase ( AOX2 ), and multiple metabolic enzymes or putative oxidoreductases (Table S1A). Of note, the 77 indirect Mrr1 targets ( Fig. 4A ) were further enriched for transport, chemical and stress response processes in a Slim GO analysis of their C. albicans homologs (File S2A-B). Download figure Open in new tab Figure 4: The Mrr1 regulon and the consensus Mrr1-binding DNA motif of C. lusitaniae . (A) Venn diagram shows the overlap between differentially expressed genes from RNA-seq in Demers et al. ( 21 ) and ORFs with HF-Mrr1 Y813C peaks in their intergenic regions from CUT&RUN. The 25 differentially regulated genes that have HF-Mrr1 Y813C peaks in either the 5’ or 3’ regions are listed in Table S1A. (B, C) HF-Mrr1 Y813C CUT&RUN read coverage plots normalized per 20 bp bin size. Chromosomal positions of regions containing MGD1 and MRR1 and adjacent genes are represented to scale with boxes and arrows. Peaks from HF-Mrr1 Y813C -bound DNA recovered by an α-FLAG antibody and for the non-specific binding control recovered via IgG are shown. Signal indicates the average read density in α-FLAG relative to IgG within the peak region. Two independent experiments were performed and both are shown. (D) Sequence logo of the consensus motif detected within 100 bp of CUT&RUN peak summits by STREME. E-value is an estimate of motif significance. The 9-nt c onsensus M rr1- b inding motif (cMBM) is boxed in yellow. (E) cMBM location (blue hatches) in the ∼890 bp upstream intergenic regions of MDR1 in different C. lusitaniae strains. (F) cMBM location (blue hatches) in the 1 kb upstream intergenic regions of the MDR1 homologs of C. parapsilosis CDC317, C. auris B11205 , C. albicans SC5314 and C. lusitaniae ATCC 42720. The C. lusitaniae ATCC 42720 upstream intergenic region is 893 bp. The phylogenetic tree was constructed using the MDR1 nucleotide sequences. We previously showed that the C. lusitaniae Mrr1 induced MGD1 and MGD2 in the presence of exogenous MGO ( 24 ), a toxic 2-oxo-aldehyde released by metabolically dysregulated cells and activated macrophages at sites of infection ( 42 ). Further, upregulation of MGD1 and MGD2 by constitutively active Mrr1 conferred a growth advantage in the presence of MGO ( 24 ). Methylglyoxal dehydrogenases are co-regulated with MDR1 in several other Candida spp. including C. albicans and C. auris ( 18 , 21 , 23 , 25 , 26 , 28 ). Interestingly, despite high expression of both MGD1 and MGD2 transcripts in strains with Mrr1 Y813C ( 21 ), only the promoter regions of MGD1 had a HF-Mrr1 Y813C CUT&RUN peak with an average signal of 6.5 ( Fig. 4B ). A HF-Mrr1 Y813C peak of average signal 2.8 was also present in the promoter regions of MRR1 ( Fig. 4C ) indicating at a mechanism for potential positive self-regulation of MRR1 transcripts which is consistent with previously published RNA-seq data ( 21 ). Three Mrr1 regulon genes ( CLUG_04865, CLUG_01574, and CLUG_04429 ) had no clear homologs in C. albicans , but did have homologs in the more closely related C. auris . Although not differentially expressed in the U04 transcriptome, a putative alcohol dehydrogenase (CLUG_00171 ) and a putative phospholipase C ( CLUG_01152 ) had HF-Mrr1 Y813C peaks in their promoter regions and were upregulated in the clinical C. lusitaniae P3 isolate with a MRR1 V668G GOF allele ( 25 ). Five genes were less abundant in strains with activated Mrr1 (Table S1A); one of these, CLUG _ 01020 ( STL1 ), was the only locus with an HF-Mrr1 Y813C peak in its 1 kb downstream intergenic region with no peak in its upstream region (Table S1A). Definition of an Mrr1-binding DNA motif that is conserved across species To better understand direct Mrr1 regulation of targets, we used the STREME algorithm ( 43 ) to determine if specific motifs were enriched within sequences corresponding to 329 HF-Mrr1 Y813C CUT&RUN peaks (File S1). The 100 bp sequences upstream and downstream of peak summits (the most enriched point within an identified peak) were used as input for discriminative de novo motif discovery ( 43 ). A set of sequences chosen at random from C. lusitaniae L17 genome and matched in length and number was used as background to identify enriched motifs in the input set. A 14-nucleotide (nt) consensus sequence RCGGAGWTARSVNN was the topmost motif predicted by STREME ( Fig. 4D ). When this consensus sequence was scanned for in the upstream intergenic regions of MDR1 from C. lusitaniae L17 and ATCC 42720 (ASM383v1), the motif was observed seven times in the C. lusitaniae L17 MDR1 promoter region and six of these were conserved in C. lusitaniae strain ATCC 42720 (Fig. S1A). The 14-nt consensus sequence had substantial nucleotide ambiguity at both ends (positions 1 and 11-14) ( Fig. 4D ). Therefore, for subsequent motif analyses, we focused on the internal 9-nt CGGAGWTAR motif ( Fig. 4D , boxed). The 9-nt CGGAGWTAR motif and the 14-nt RCGGAGWTARSVNN motif were similarly detected in the promoter regions of MDR1 in both strains (Fig. S1A). Henceforth, we refer to the 9-nt CGGAGWTAR motif as the c onsensus M rr1- b inding DNA m otif (cMBM) ( Fig. 4D ) The analysis of MDR1 promoter sequences from the clinical isolate AR0398 (GCA_032599225.1) and two distantly related environmental isolates 79-1 (GCA_032599145.1) and 76-31 (GCA_032599085.1) found the six cMBM sites detected in strains L17 and ATCC 42720 ( 44 ). Each of the cMBMs were at identical positions and orientations relative to the MDR1 translational start sites across the different strains ( Fig. 4E ). cMBMs were also found upstream of CDR1 and they were again conserved in position in both L17 and ATCC 42720 strains despite differences in the length of the CDR1 adjacent intergenic regions (Fig. S1B). At least one cMBM, and often multiple cMBMs, were found within the peak spanning regions associated with all but two of the genes in the Mrr1-regulon (File S3). We also scanned for the cMBM in the promoter sequences of the MDR1 and CDR1 homologs in Candida spp. At least three copies of cMBM were found in the MDR1 and CDR1 promoter sequences of C. albicans and C. parapsilosis , and one cMBM in C. auris ( Fig. 4F & S1C). In the case of C. albicans , two cMBMs occurred in locations previously annotated to be important for MDR1 transcriptional regulation. These cMBMs were discovered between the-200 to-400 regions which encompassed the benomyl response element (-260 and-296) ( 45 ) and the Mrr1-binding region that contained the C. albicans Mrr1-binding DNA motif DCSGHD (-342 to-492) ( 38 ). In a ChIP-qRT analysis of Mrr1 binding to the C. albicans MDR1 promoter, DNA recovery was highest at these cMBM-containing regions relative to the rest of the MDR1 promoter sequence ( 20 ). Together these data strongly suggest that the consensus Mrr1-binding DNA motif discovered in C. lusitaniae is conserved in other Candida species. Transcription factors of the zinc-cluster family, which includes Mrr1, typically bind to CGG motifs occurring as direct, inverted or everted repeats ( 46 ). Constitutively active and low-activity Mrr1 localize to similar genomic regions in C. lusitaniae Previous studies on C. lusitaniae Mrr1 suggested that expression at some loci (e.g. MDR1 and MGD1 ) ( 21 , 24 , 25 ) was repressed by low-activity Mrr1 variants and induced in the presence of benomyl and MGO inducers of Mrr1 or by constitutively activate Mrr1 variants. Thus, we compared DNA localization of the HF-Mrr1 Y813C to the genome-wide binding of low activity HF-Mrr1 ancestral in the absence of Mrr1 inducing stimuli. Using the same parameters as for the analysis of HF-Mrr1 Y813C , we found around 1,276 peaks associated with HF-Mrr1 ancestral –bound DNA (File S4). The MDR1 intergenic region revealed a significant HF-Mrr1 ancestral peak that spanned a region of ∼1.6 kb and had a signal of 15.1 ( Fig. 5A ). HF-Mrr1 ancestral peaks were also found upstream of CDR1 and FLU1 (1.6 and 2.2 peak signal, respectively; Fig. 5B-C ). Comparison of HF-Mrr1 ancestral and HF-Mrr1 Y813C -bound sites upstream of MDR1 , CDR1 and FLU1 exhibited a striking similarity in their peak profiles (Fig. S2A-C). The remarkable overlap of HF-Mrr1 ancestral and HF-Mrr1 Y813C CUT&RUN peaks present in over 930 genomic locations ( Fig. 5D ) suggest that Mrr1-mediated repression and induction are not due to differences in Mrr1 localization to the DNA. Download figure Open in new tab Figure 5: Local and global binding profiles of constitutively active and low-activity Mrr1. (A-C) HF-Mrr1 ancestral CUT&RUN read coverage plots normalized per 20 bp bin size. Chromosomal positions of regions containing MDR1, CDR1 and FLU1 and adjacent genes are represented to scale with boxes and arrows. Peaks from HF-Mrr1 ancestral -bound DNA recovered by an α-FLAG antibody and for the non-specific binding control recovered via IgG are shown. Signal indicates the average read density in α-FLAG relative to IgG within the peak region. (D) Circos plot showing global CUT&RUN-determined Mrr1-binding peaks of HF-Mrr1 Y813C (in blue) and HF-Mrr1 ancestral (in grey) in the C. lusitaniae L17 genome. Mrr1-binding peaks with a signal ≥2-fold compared to their respective IgG backgrounds and up to 1 kb away from the nearest ORF from Experiment 1 were used (see Supplemental Files 1 and 4). The genomic positions of the 25 differentially expressed genes that constitute the Mrr1-regulon are marked with the L17 gene IDs. In Demers et al. ( 21 ), we characterized MRR1 alleles with GOF mutations that resulted in constitutive activity and Mdr1-dependent FLZ-resistance ( Fig. 6A ) as well as alleles with both GOF mutations and secondary suppressor mutations that restored the inducible low activity state such as MRR1 L1191H+Q1197*(L1Q1*) ( Fig. 6A ). The mrr1 Δ+ MRR1 L1Q1* strain had more than a 32-fold lower FLZ MIC value (0.125 µg/ml vs 32 µg/ml) than strains with MRR1 GOF alleles ( MRR1 Y813C and MRR1 L1191H ) ( Fig. 6B ). Since GOF mutations in Mrr1 did not affect DNA localization, we evaluated whether secondary suppressor mutation(s) altered these interactions by performing CUT&RUN on U04 mrr1 Δ strains expressing HF-Mrr1 L1Q1* from its endogenous promoter. Western blot confirmed that the truncated HF-Mrr1 L1Q1* was present at levels similar to that of the full-length HF-Mrr1 ancestral and HF-Mrr1 Y813C (Fig. S3A). The HF-tag did not modify Mrr1 L1Q1* activity as strains expressing tagged Mrr1 L1Q1* exhibited similar 32- to 64-fold lower FLZ MIC as untagged Mrr1 L1Q1* when compared to strains expressing the constitutively active Mrr1 Y813C variant (Fig. S3B). Our CUT&RUN analysis found HF-Mrr1 L1Q1* - bound DNA to be significantly enriched in the upstream intergenic regions of MDR1 , CDR1 and FLU1 ORFs ( Fig. 6C-E ) with a peak profile identical to HF-Mrr1 ancestral and HF-Mrr1 Y813C . The HF-Mrr1 L1Q1* peak recapitulated the 1.5- and 2-fold higher signal upstream of MDR1 relative to CDR1 and FLU1 . Across the entire C. lusitaniae genome, the HF-Mrr1 L1Q1* - bound genomic sites (see File S5 for peaks) were strikingly similar to the HF-Mrr1 ancestral and HF-Mrr1 Y813C -bound sites suggesting that secondary suppressor mutation(s) do not likely impact Mrr1 localization to the DNA (Fig. S4). Hence, our results illustrate that Mrr1 localization at the C. lusitaniae DNA are unaltered by the tested mutations and are independent of Mrr1 activation state. Download figure Open in new tab Figure 6: Evolution of naturally acquired MRR1 mutations and binding profiles of low-activity Mrr1. (A) Schematic of the clinically evolved MRR1 alleles reported in Demers et al. ( 21 ). The asterisk indicates a nonsense mutation. Alleles in blue and orange encode for constitutively active and low-activity Mrr1 variants, respectively. (B) FLZ MIC of U04 clinical isolate (native allele MRR1 Y813C ) and U04 mrr1 Δ complemented with MRR1 L1191H or MRR1 L1Q1* (L1191H + Q1197*) was determined by broth microdilution assays. The data shown represent the mean ± SD from three independent experiments. Strains with constitutive Mrr1 activity are in bold. (C-E) HF-Mrr1 L1Q1* CUT&RUN read coverage plots normalized per 20 bp bin size. Chromosomal positions of regions containing MDR1, CDR1 and FLU1 and adjacent genes are represented to scale with boxes and arrows. Peaks from HF-Mrr1 L1Q1* -bound DNA recovered by an α-FLAG antibody and for the non-specific binding control recovered via IgG are shown. Signal indicates the average read density in α-FLAG relative to IgG within the peak region. DISCUSSION In this study, we demonstrated that constitutively active C. lusitaniae Mrr1 directly upregulates several multi-drug transporter-encoding genes, including MDR1 and CDR1, leading to reduced susceptibility to both short-tailed and long-tailed azoles and other antifungals ( Fig. 1 - 3 ). The coordinated regulation of both MDR1 and CDR1 by Mrr1 in C. luistaniae differs from their regulation in the well-studied species C. albicans wherein Mrr1 is the primary regulator of MDR1 and Tac1 is the main CDR1 transcriptional activator ( 23 , 33 ). We identified a consensus Mrr1 binding motif (cMBM; CGGAGWTAR) that colocalized with Mrr1 CUT&RUN peaks and that was present in multiple positions within the peaks in regions adjacent to C. lusitaniae MDR1 , CDR1 and in almost all other Mrr1-regulated genes ( Fig. 4D & File S2). The cMBM sequences in Mrr1 peak regions were conserved in other C. lusitaniae strains ( Fig. 4E & S1B). Furthermore, the cMBM was also enriched in the regions upstream of MDR1 homologs in C. albicans , C. auris and C. parapsilosis, and in C. albicans , the cMBM was present in regions shown to bind C. albicans Mrr1 ( 20 ). The cMBM was also upstream of C. lusitaniae and C. parapsilosis CDR1 which is consistent with reports that constitutive Mrr1 activity also induces expression of CDR1 in these species. Furthermore, we noted the presence of cMBMs in regions upstream of CDR1 in species that have no reports for Mrr1 regulation of CDR1 including C. albicans and C. auris ( 33 , 47 , 48 ). Consistent with the potential for Mrr1 regulation of CDR1 in C. albicans , a ChIP-ChIP analysis detected Mrr1 in the upstream regions of CDR1 ( 49 ), though CDR1 was not reported as an Mrr1 target because its expression was not increased by constitutively active Mrr1. Studies in C. albicans and recent work in C. auris have shown Tac1 with an activating mutation upregulates CDR1 expression and the C. albicans Tac1 regulates CDR1 by binding a consensus CGGN 4 CGG motif in the promoter region ( 49 ). Though C. lusitaniae has a Tac1 homolog (Clug_02369) ( 34 ) and a CGGN 4 CGG motif at-761 in the CDR1 promoter region (data not shown), strains with low Mrr1 activity and a cdr1 Δ mutant had similar susceptibilities (MIC 12.5 – 25 µg/ml) to the Cdr1 substrate fluphenazine ( Fig. 3 ) ( 22 , 50 ). In fact, while activating mutations in TAC1 have been characterized in FLZ- resistant C. parapsilosis ( 51 ) and C. auris ( 52 ), to our knowledge, there are no reports on activating mutations in the TAC1 gene leading to FLZ resistance in C. lusitaniae . While we ( 21 ) and others ( 34 ) have shown that Mrr1 is sufficient to upregulate C. lusitaniae CDR1 , Tac1 may induce CDR1 under conditions not tested in this study. For instance, estradiol is an inducer of Tac1-mediated CDR1 expression in C. albicans ( 53 , 54 ).Together, these data underscore the evolutionary plasticity in transporter regulation in Candida spp. through the adoption of targets from one transcriptional circuit to another ( Fig. 7A ; ( 55 – 57 )). In the case of C. lusitaniae , the coordinated regulation of drug efflux proteins may be a mechanism for cross-resistance to multiple antifungals and may promote the development of other resistance mutations through a reduction in drug susceptibility. Download figure Open in new tab Figure 7: Models for Mrr1 regulation in Candida spp.. (A) Evolution of transporter regulation in Candida spp. Genes having characterized GOF mutations are in blue. The shapes indicate the type of experimental data used to support the model including protein-DNA studies (this study; 20, 38, 49), expression and phenotypic studies ( 18 – 21 , 23 – 28 , 32 – 35 , 40 , 47 , 48 , 50 – 52 ). (B) Possible mechanism(s) for gene induction by Mrr1 based on published studies ( 20 , 38 , 46 , 54 , 58 – 60 ). The mechanisms that impact Mrr1-mediated gene expression may vary between promoters and conditions within a strain, and there may be differences across strains and species. Our data on Mrr1 levels and Mrr1 variants binding to upstream, or in some cases downstream, regions of Mrr1-regulated genes provide insight into Mrr1 regulation. First, we found that Mrr1 variants with differing activities did not have differences in total protein levels (Fig. S3A). Second, activated Mrr1 and inducible but inactive Mrr1 had indistinguishable localization at all cMBMs (Fig. S4) which is consistent with ChIP-qRT analysis of Mrr1 interactions with the MDR1 promoter region in C. albicans ( 20 ). Third, the subset of genes repressed by inducible but inactive Mrr1 had similar Mrr1 localization in their promoter regions as those genes that were not repressed by Mrr1 (Fig. S2A). Thus, Mrr1 is likely regulated through mechanisms such as induced conformational change by ligand, co-factor binding ( 58 ), phosphorylation ( 59 ) or differential activity of co-regulatory proteins. These mechanisms are not mutually exclusive ( 46 ). In C. albicans , changes in activity of coregulatory proteins (Cap1 or Mcm1 ( 38 , 60 )), mediator ( 20 , 54 ) or chromatin remodeling complexes like the Swi/Snf complex influence Mrr1 induction of MDR1 and other genes ( 20 ) ( Fig. 7B ). The >1 kb width of our CUT&RUN peaks is consistent with the presence of multiple cMBMs in regions adjacent to Mrr1-regulated genes and may also reflect the presence of co-regulators or chromatin remodeling complexes that could influence micrococcal nuclease access to DNA. The involvement of multiple regulatory mechanisms allows for the controlled and differential expression of unique gene subsets in different strain backgrounds ( 12 ) in response to environmental cues that may be present in an infection environment (e.g. decreased nutrient availability or metabolites like methylglyoxal or inflammatory molecules) The presence of diverse regulatory mechanisms may promote survival under diverse conditions and may also promote the evolution of novel regulatory circuits across species and even strains ( Fig. 7A ). For many azoles, there was an 8- to 16-fold increase in the MIC values of strains with activated Mrr1 variants compared to strains with low-activity Mrr1 and these differences were dependent on either Mdr1 (FLZ and VOR) or Cdr1 (KTZ, ITR and ISA) ( Fig. 2 & Table 1 ). In the C. lusitaniae clinical isolate P3 which has activated Mrr1, the mdr1 Δ cdr1 Δ double mutant was even more susceptible to azoles (FLZ, VOR, ITR) than their single deletion mutants ( 34 ). Redundancy in transporter efflux was also observed in the case of other broad-spectrum antifungals. In addition to Mdr1 and Cdr1, susceptibility to the tested antifungals could also be mediated by other efflux pumps in the Mrr1 regulon including the MFS family transporter Flu1. While FLU1 is a conserved Mrr1 target in other Candida spp. such as C. albicans ( 17 ) and C. parapsilosis ( 35 ), the promiscuity for substrates between transporters may have concealed any apparent contribution of Flu1 to efflux in a MDR1 / CDR1 overexpression strain ( Fig. 2 , 3 & Table 1 ). Beyond drug efflux, the C. lusitaniae Mrr1 regulon (Table S1A) included genes involved in other transporter activities like oligopeptide transport ( OPT1 ), and chemical and stress response which is consistent with published Mrr1 regulons of C. albicans ( 23 ) and C. parapsilosis ( 28 , 35 ). In C. auris , the OPT1 homolog is upregulated in response to stress such as antifungal exposure ( 61 ) or macrophage phagocytosis ( 62 ). While OPT1 may be involved in nutrient uptake under stress conditions ( 63 ), other metabolic factors including aldehyde and methylglyoxal dehydrogenases and aldo-keto reductases are speculated to protect cells from reactive molecules generated by azole stress ( 29 ). Thus, the Mrr1-regulated metabolic and stress response genes may be important for the persistence of the MRR1 GOF mutants in vivo or could lead to the selection for MRR1 GOF mutants in drugless conditions ( 18 ). Understanding Mrr1 regulation of these additional targets across Candida spp. can provide insights into the mechanisms that change multi-drug transporter regulation in Candida . Materials and Methods Strains and growth conditions Strains used in this study are listed in Table S2. All strains were stored as frozen stocks with 25% glycerol at-80°C and maintained regularly on YPD (1% yeast extract, 2% peptone, 2% glucose, 1.5% agar) plates incubated at 30°C then stored at room temperature. Strains were grown in YPD liquid medium (5 ml) at 30°C on a roller drum for ∼16 h prior to inoculation into specified culture conditions. For drug susceptibility assays, cells were grown in RPMI-1640 (Sigma, containing L-glutamine, 165 mM MOPS, 2% glucose, pH 7) liquid, as noted. Escherichia coli strains were grown in LB with either 150 µg/ml carbenicillin or 15 µg/ml gentamicin as necessary to maintain plasmids. Strain construction Gene replacement constructs for knocking out MRR1 ( CLUG_00542 , as annotated in ( 18 )) and MDR1 ( CLUG_01938/9 ( 18 )) were generated by fusion PCR, as described in Grahl et al. ( 64 ). All primers (IDT) used are listed in Table S3. Briefly, 0.5 to 1.0 kb of the 5’ and 3’ regions flanking the gene was amplified from U04 DNA, isolated using the MasterPure Yeast DNA Purification Kit (epiCentre). The nourseothricin ( NAT1 ) or hygromycin B (HygB) resistance cassette was amplified from plasmids pNAT ( 65 ) and pYM70 ( 66 ) respectively. Nested primers within the amplified flanking regions were used to stitch the flanks and resistance cassette together. Gene replacement constructs for knocking out CDR1 ( CLUG_03113 ) and FLU1 ( CLUG_05825 ) were generated by introducing 30- to 50-bp of the 5’ and 3’ regions flanking the gene of interest into the replacement NAT1 cassette using PCR. PCR products for transformation were purified and concentrated with the Zymo DNA Clean & Concentrator kit (Zymo Research) with a final elution in molecular biology grade water (Corning). Plasmids for complementation of MRR1 Plasmids for complementing untagged MRR1 were created as described in Biermann et al. ( 24 ). Plasmids for complementing N-terminal HF-tagged MRR1 were made as follows. We amplified i) the 6xHis-3xFLAG-tag from an HF-MRR1 tagged C. albicans strain DH2561 using primers ED207 and ED208, ii) the ∼1150 bp upstream region of the MRR1 gene for homology, from the respective MRR1 allele complementation plasmids, using primers ED103 and ED206 and iii) ∼1500 bp of the MRR1 gene using primers ED209 and ED132. The 6xHis-3xFLAG-tag is placed after the first codon of MRR1 . PCR products were cleaned up using the Zymo DNA Clean & Concentrator kit (Zymo Research). The amplified PCR products were assembled into a pMQ30 vector using the S. cerevisiae recombination technique described in Shanks et al. ( 67 ). Plasmids created in S. cerevisiae were isolated using a yeast plasmid miniprep kit (Zymo Research) and transformed into High-Efficiency NEB®5-alpha competent E. coli (New England BioLabs). E. coli containing pMQ30-derived plasmids were selected for on LB containing 15 µg/ml gentamicin. Plasmids from E. coli were isolated using a Zyppy Plasmid Miniprep kit (Zymo Research) and subsequently verified by Sanger sequencing. MRR1 complementation plasmids were linearized with the NotI-HF restriction enzyme (New England BioLabs), cleaned up using the Zymo DNA Clean & Concentrator kit (Zymo Research) and eluted in molecular biology grade water (Corning) before transformation of 2 µg into C. lusitaniae strain U04 mrr1 Δ as described below. Strain construction Mutants were constructed as previously described in Grahl et al. using an expression-free ribonucleoprotein CRISPR-Cas9 method ( 64 ). One to 2 µg of DNA for gene knockout constructs generated by PCR or 2 µg of digested plasmid, purified and concentrated with a final elution in molecular biology grade water (Corning), was used per transformation. E. coli strains containing the complementation and knockout constructs and crRNAs are listed in Table S2 and Table S3 respectively. Transformants were selected on YPD agar containing 200 µg/ml nourseothricin or 600 µg/ml hygromycin B. Mutants for CDR1 and FLU1 were generated using a microhomology-mediated end-joining (MMEJ) repair method as described in Al Abdallah et al. ( 68 ). One to 2 µg of DNA for gene knockout constructs generated by PCR were used for transformation. crRNAs (IDT) used to target the 5’ and 3’ end of the gene of interest are listed in Table S3. CDR1 and FLU1 knockout transformants were selected on YPD agar containing 200 µg/ml nourseothricin. Protein Isolation Overnight cultures were back diluted into 50ml YPD and grown to exponential phase (∼5 h) at 30°C. Harvested cells were snap-frozen using ethanol and dry ice and stored at-80°C. Thawed cell pellets were resuspended in a homogenization buffer (10 mM Tris-HCl, 150 mM NaCl, and 5 mM EDTA, adjusted to pH 7.4 and 10% sucrose) with protease inhibitor (2x Halt protease, Thermo Scientific) and mixed with an equal volume of 1:1 of 0.5- and 1-mm silica bead mix in a bead beating tube (VWR). Bead beating was carried out for six cycles at a speed of 5.65 for 20 seconds, with a one-minute rest on ice between each cycle. The top liquid phase was collected and centrifuged to remove any cell debris. Supernatants were transferred to new tubes and stored at-80°C. Protein concentrations were determined using the Bradford assay (Quick Start Bio-Rad) with a standard curve generated using serial dilutions of 2 mg/ml bovine serum albumin (BSA). Western Blot for HF-Mrr1 detection Samples were diluted to equal concentrations in sample buffer (3.78% Tris, 5% SDS, 25% sucrose at pH 6.8 and 0.04% bromophenol blue prepared as a 5x stock solution. β-mercaptoethanol (0.05%) was freshly added). Samples were heated for 10 min at 95°C and loaded into 6.5% SDS page gels along with a BioRad All Blue Precision Plus MW marker. The gel was run for ∼40 min at 180 V. The BioRad Turboblot semi-dry transfer system with custom settings (1.3A constant and 25V for 15 min) was used to transfer the protein bands to a LF-PVDF membrane (Immobilon Product IPFL00010). The blots were processed using the standard LICOR protocol for Western blotting, including the optional drying step after transfer and REVERT total protein staining. A milk-based blocking buffer was used instead of the Odyssey blocking buffer. The α-FLAG monoclonal antibody (1mg/mL) (Sigma-Aldrich M2 or ThermoFisher FG4R) was diluted 3000-fold in blocking buffer with 0.1% Tween-20. The goat α-mouse secondary antibody (1 mg/mL) labelled with IRDye 700CW was diluted 15,000-fold in blocking buffer with 0.1% Tween-20. Blots were imaged using an Odyssey CLX scanner (LICOR) and analyzed using the Empiria software (LICOR). CUT&RUN experimental setup and sequencing Overnight cultures were back diluted into 50 ml YPD and grown to exponential phase (∼5 h) at 30°C. Samples were processed using the Epicypher CUT&RUN kit (Epicypher) as per the protocol described in Qasim et al . ( 69 ). Briefly, yeast nuclei were isolated from the thawed cell pellets using Zymolase 100T (Zymoresearch). Digitonin (0.01%) was added to all buffers used hereafter to permeabilize nuclei and prevent bead clumping. The isolated nuclei were bound to activated concanavalin A (ConA)-coated magnetic beads. The nuclei-bound ConA-beads were then split and incubated overnight at 4°C with either 1:100 IgG or α-FLAG primary antibody (Sigma-Aldrich M2 for experiment-1 and ThermoFisher FG4R for experiment-2). After washing to remove unbound primary Ab, pAG-MNase was added to the nuclei and incubated for an hour. Targeted chromatin digestion by pAG-MNase was initiated by adding CaCl 2 and stopped after 30 mins with stop buffer spiked with 50 ng of E. coli DNA. The supernatant with the pAG-MNase digested DNA was then collected and purified using an Epicypher DNA cleanup column. DNA libraries were prepared using the NEB Ultra II protocol kit, with slight modifications as recommended in the Epicypher CUTNRUN kit. Our pilot experiment (Experiment 1) was set up with U04 strains expressing one of the three alleles ( HF-MRR1 ancestral , HF-MRR1 Y813C and HF-MRR1 L1Q1* ) and sequenced using paired-end 150-bp reads on the Illumina Nextseq 2000 platform to achieve a sequencing depth of 10M per sample. Based on the pilot study results, the sequencing depth was adjusted to 5-6M per sample for the subsequent experiment (Experiment 2) including two biological replicates of U04 strain expressing HF-MRR1 Y813C , which were sequenced using paired-end 50 bp reads on the Illumina Nextseq 2000 platform. CUT&RUN data analysis Raw read quality was evaluated using FastQC (v0.12.1) prior to read trimming with Cutadapt (v.4.4) for adapter sequences with additional parameters “--nextseq-trim 20-- max-n 0.8--trim-n-m 1”. Reads were mapped to Clavispora ( Candida ) lusitaniae strain L17 (NCBI accession: ASM367555v2) with Bowtie2 (v2.4.2) using parameters “--local-- no-mixed--no-discordant”. Alignments were sorted coordinate with Samtools (v1.11), filtered for unmapped or multi-mapping reads using sambamba (v0.8.0), and downsampled to 3 million reads per sample to ensure equal sensitivity for peak calling across samples. MarkDuplicates (Picard Tools) was used to identify and remove duplicate reads. Fragment size distributions of individual samples were visualized using deepTools (v3.5.1) command “bamPEFragmentSize”. Peaks were called using the MACS2 (v2.2.7.1) command “callpeak” in narrowpeak mode using IgG IP samples as controls with parameters “-f BAMPE--keep-dup all-g 11999093-q 0.05”. Significant peaks were further filtered to keep only those with 2-fold or greater signal increase relative to control (IgG) samples. The fraction of reads in peaks (FRiP) was calculated for each sample to assess individual quality. The BEDTools (v 2.31.1) command “merge”, with the parameter “-c” for averaging peak signal value, was used to merge peaks with a 2-fold or greater signal from all replicates of experiment-2. BEDTools (v 2.31.1) command “intersect” with the parameter “-a” was used to identify a set of reproducible overlapping peaks between HF-Mrr1 Y813C from Experiment 1 and Experiment 2. Since the nuclei isolation step was not controlled in our CUT&RUN experiments ( 36 , 69 ), it limited our ability to perform differential peak analysis between replicates and across strains expressing different MRR1 alleles. Drug susceptibility assays Minimum inhibitory concentration (MIC) was determined using a broth microdilution method as previously described ( 70 ). Briefly, 2×10 3 cells were added to a two-fold dilution series of the drug prepared in RPMI-1640 then incubated at 37 °C. The MIC was defined as the minimum drug concentration that abolished visible growth compared to a drug-free control. The MIC 90 was defined as the minimum drug concentration that led to a 90% or greater decrease in growth relative to a drug-free control. No more than a 2-fold difference was observed between MICs recorded at 24 and 48 h; data from the 24 h timepoint was reported unless otherwise noted. The concentration range used for azoles were FLZ: 64 to 0.0625 µg/ml, VOR: 4 to 0.004 µg/ml, KTZ: 1 to 0.004 µg/ml, ITR: 0.4 to 0.003125 µg/ml and ISA: 1 to 0.04 µg/ml. For the broad-spectrum antifungals, the following concentration ranges were used; Myclobutanil: 32 to 0.0625 µg/ml, Terbinafine: 64 to 0.125 µg/ml, Cycloheximide: 32 to 0.0625 µg/ml, 5-FC: 4 to 0.008 µg/ml, Fluphenazine: 200 to 0.39 µg/ml and Mycophenolic acid: 256 to 0.5 µg/ml. MOTIF analysis Sequences spanning ±100 bp around the peak summits identified from CUT&RUN data were extracted from the L17 genome (NCBI accession: ASM367555v2) using BEDTools v2.30.0 ( 71 ). To establish a background control, we used BEDTools random to retrieve randomly selected 200bp sequences from the genome of L17. STREME ( 43 ), part of the MEME Suite (streme--verbosity 1--oc streme_results--dna--totallength 4000000--time 14400--minw 6--maxw 20--thresh 0.05--align center--p around_peaks.fasta --n random_sequences.fasta) was employed for motif discovery and enrichment analysis. Motif scanning across C. lusitaniae strains (L17, AR0398, ATCC 42720, 79-1, and 76-31) and multiple related Candida species ( C. albicans SC5314 (ASM18296v3), C. parapsilosis CDC317 (ASM18276v2), C. auris B11205 (ASM1677213v1) or B8441 (GCA_002759435.3), and C. lusitaniae ATCC 42720) was conducted using FIMO ( 72 ), part of the MEME Suite (fimo --oc fimo_results --verbosity 1 --bgfile --nrdb --thresh 1.0E-3 motif1.meme target_seqs.fasta). MDR1 and CDR1 gene IDs and their translational start site coordinates used for the sequence retrieval of the upstream regions are listed in Table S4. For the phylogenetic gene trees, nucleotide sequences of the respective genes were extracted and aligned using MAFFT v7 ( https://mafft.cbrc.jp/alignment/server/index.html ) with default parameters. A neighbor-joining tree was then constructed based on the aligned DNA sequences, with 1000 bootstrap replicates to assess phylogenetic relationships. The results were visualized using the ggmotif v0.2.0 R package ( 73 ) and FigTree v1.4.4 ( http://tree.bio.ed.ac.uk/software/figtree/ ). Statistical analysis and figure design Ordinary one-way ANOVA and Dunnett’s multiple comparisons testing, with a single pooled variance, were used for statistical evaluation. P values <0.05 were considered significant for all analyses performed and are indicated with asterisks: *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Fig. 6A and 7 were created in BioRender https://BioRender.com/39wn5ht . Data availability The raw sequence reads from CUT&RUN analysis have been deposited into NCBI sequence read archive under Bioproject PRJNA1251050.The data from this study are available within the paper and in the supplemental material, and accessible in OSF through the following link https://osf.io/4zbv8/ and will be made public upon publication. Figure S1: Consensus Mrr1-binding DNA motif in the promoter regions of Mrr1 targets. (A) The positions of the 14-nt (orange hatches) and 9-nt Mrr1-binding motifs ( c onsensus M rr1- b inding m otif or cMBM; blue hatches) in the ∼890 bp upstream intergenic regions of MDR1 in C. lusitaniae L17 and ATCC 42720 strains. (B) cMBM location in the 1 kb upstream intergenic regions of CDR1 from C. lusitaniae L17 and ATCC 42720. (C) cMBM location in the 1 kb upstream intergenic regions of the CDR1 homologs of C. parapsilosis CDC317, C. auris B8441 , C. albicans SC5314 and C. lusitaniae ATCC 42720. The phylogenetic tree was constructed using the CDR1 nucleotide sequences. (B, C) The intergenic region upstream of CDR1 in ATCC 42720 is 326 bp. Grey arrow indicates the adjacent ORF CLUG_03114 . Figure S2: Comparison of binding profiles of constitutively active and low-activity Mrr1. (A-C) Comparison of data from Figures 1D-F and Figures 5A-C to highlight the similarities in peak profiles. HF-Mrr1 Y813C (in blue) and HF-Mrr1 ancestral (in grey) CUT&RUN read coverage plots normalized per 20 bp bin size. Chromosomal positions of regions containing MDR1, CDR1 and FLU1 and adjacent genes are represented to scale with boxes and arrows. Peaks from HF-Mrr1-bound DNA recovered by an α-FLAG antibody and for the non-specific binding control recovered via IgG are shown. Signal indicates the average read density in α-FLAG relative to IgG within the peak region. Figure S3: Biochemical and phenotypic analysis of HF-tagged Mrr1 variants. (A) Western blot of whole cell protein lysates of U04 strains expressing N-terminal 6xHis-3xFLAG-tagged Mrr1 (HF-Mrr1) variants. HF-Mrr1 was probed using an α-FLAG antibody. Mean ± SD of HF-Mrr1 band intensities normalized to total protein (n= 4 biological replicates). (B) FLZ MIC of U04 clinical isolate (native allele MRR1 Y813C ) and U04 mrr1 Δ complemented with untagged or HF-MRR1 was determined by broth microdilution assays. The data shown represent the mean ± SD from three independent experiments. There were no significant differences observed between data from strains with untagged Mrr1 variants and data from strains with their respective HF-tagged counterparts. Strains with constitutive Mrr1 activity are in bold. Figure S4: Global binding profiles of constitutively active and low-activity Mrr1. Circos plot showing global CUT&RUN-determined Mrr1-binding peaks of HF-Mrr1 Y813C (in blue), HF-Mrr1 ancestral (in grey) and HF-Mrr1 L1Q1* (in orange) in the C. lusitaniae L17 genome. Mrr1-binding peaks with a signal ≥2-fold compared to their respective IgG backgrounds and up to 1 kb away from the nearest ORF from Experiment 1 (see Supplemental Files 1, 4 and 5) were used. The genomic positions of the 25 differentially expressed genes that constitute the Mrr1-regulon are marked with the L17 gene IDs. Acknowledgements We thank Owen Wilkins and Noelle Kosarek for CUT&RUN data analysis, Mohammad Qasim, Fred Kolling, Heidi Trask and Jen Spengler for guidance with CUT&RUN pilot experiments and Stacie Stuut for azole structures. Research reported in this publication was supported by National Institutes of Health (NIH) grant R01 AI127548 to D.A.H. This work was also supported by the Cystic Fibrosis Foundation Research Development Program (CFFRDP) STANTO19R0 for the Translational Research Core. Equipment used was supported by the NIH NIGMS grant to Dartmouth BioMT P20-GM113132. J.E.S. and C.G.P.P. were supported by R01 AI130128. J.E.S. is a CIFAR Fellow in the program Fungal Kingdom: Threats and Opportunities. Sequencing was carried out in the Genomics and Molecular Biology Shared Resource (RRID:SCR_021293) at Dartmouth which is supported by NCI Cancer Center Support Grant 5P30CA023108 and NIH S10 (1S10OD030242) awards. 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Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp Dhanabala-Subhiksha Rajesh-Khanna , Carolina G. Piña Páez , Elora G. Dolan , Kiran S. Mirpuri , Jason E. Staijch , Deborah A. Hogan bioRxiv 2025.05.04.652153; doi: https://doi.org/10.1101/2025.05.04.652153 Share This Article: Copy Citation Tools Coordinated regulation of Mdr1- and Cdr1-mediated protection from antifungals by the Mrr1 transcription factor in emerging Candida spp Dhanabala-Subhiksha Rajesh-Khanna , Carolina G. Piña Páez , Elora G. Dolan , Kiran S. Mirpuri , Jason E. Staijch , Deborah A. 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