Mutations in TAC1B drive CDR1 and MDR1 expression and azole resistance in C. auris

Mutations in TAC1B drive CDR1 and MDR1 expression and azole resistance in C. auris | 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 Mutations in TAC1B drive CDR1 and MDR1 expression and azole resistance in C. auris Katherine S. Barker , Darian J. Santana , Qing Zhang , Tracy L. Peters , Jeffrey M. Rybak , Joachim Morschhäuser , Christina A. Cuomo , P. David Rogers doi: https://doi.org/10.1101/2025.02.11.637698 Katherine S. Barker a Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Hospital , Memphis, Tennessee, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Darian J. Santana a Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Hospital , Memphis, Tennessee, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qing Zhang a Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Hospital , Memphis, Tennessee, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tracy L. Peters a Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Hospital , Memphis, Tennessee, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jeffrey M. Rybak a Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Hospital , Memphis, Tennessee, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Joachim Morschhäuser b Institute for Molecular Infection Biology, University of Würzburg , Würzburg, GERMANY Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christina A. Cuomo c Department of Molecular Microbiology and Immunology, Brown University , Providence, Rhode Island, USA d Broad Institute , Cambridge, Massachusetts, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site P. David Rogers a Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Hospital , Memphis, Tennessee, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: dave.rogers{at}stjude.org Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Objective Candida auris has emerged as a fungal pathogen of particular concern owing in part to its propensity to exhibit antifungal resistance, especially to the commonly prescribed antifungal fluconazole. In this work we aimed to determine how mutations in the transcription factor gene TAC1B , which are common among resistant isolates and confer fluconazole resistance, exert this effect. Methods Selected TAC1B mutations from clinical isolates were introduced into a susceptible isolate and reverted to the wild-type sequence in select clinical isolates using CRISPR Cas9 gene editing. Disruption mutants were likewise generated for select genes of interest. TAC1B mutants were subjected to transcriptional profiling by RNA-seq, and relative expression of specific genes of interest was determined by qRT-PCR. Antifungal susceptibilities were determined by modified CLSI broth microdilution. Results TAC1B mutations leading to A640V, A657V, and F862_N866del conferred fluconazole resistance, as well as increased resistance to other triazoles, when introduced into a susceptible isolate. RNA-seq revealed that the ATP-Binding Cassette (ABC) transporter gene CDR1 as well as the Major Facilitator Superfamily (MFS) transporter gene MDR1 were both upregulated by these TAC1B mutations. Disruption of CDR1 greatly abrogated resistance in strains with TAC1B mutations whereas disruption of MDR1 had little to no effect. However, disruption of both CDR1 and MDR1 resulted in an additional reduction in resistance as compared to disruption of either gene alone. Conclusion TAC1B mutations leading to A640V, A657V, and F862_N866del all result in increased resistance to fluconazole and other triazole antifungals, and increased expression of both CDR1 and MDR1 in C. auris . CDR1 is the primary driver of resistance conferred by these TAC1B mutations. Introduction Since its initial identification in 2009, Candida auris has emerged as an important healthcare-associated fungal pathogen causing outbreaks worldwide( Lockhart, Chowdhary, & Gold, 2023 ; Lyman et al., 2023 ). Of particular concern is the prevalence of antifungal resistance among C. auris isolates, including resistance to two and sometimes all three antifungal classes currently available for treatment of serious Candida infections( Ostrowsky et al., 2020 ; Rybak, Cuomo, & Rogers, 2022 ). Strikingly, over 90% of isolates exhibit resistance to the most widely prescribed antifungal worldwide, the triazole antifungal fluconazole. Fluconazole exerts its antifungal activity by competitively binding to and inhibiting sterol demethylase, a key enzyme of the fungal sterol biosynthesis pathway. In Candida species, this results in both reduced production of the major membrane sterol ergosterol as well as accumulation of a toxic sterol intermediate( Kelly, Lamb, Corran, Baldwin, & Kelly, 1995 ). Fluconazole resistance in Candida species can be the result of mutations in the ERG11 gene which encodes sterol demethylase, resulting in altered drug binding or enhanced preference for the natural substrate, leading to reduced enzyme inhibition( Marichal et al., 1999 ). Resistance can also be due to mutations in the genes encoding the transcriptional regulators Tac1, Mrr1, or Upc2 which result in overexpression of genes encoding the ATP Binding Cassette (ABC) transporter Cdr1, the Major Facilitator Superfamily (MFS) transporter Mdr1, or Erg11, respectively( Coste, Karababa, Ischer, Bille, & Sanglard, 2004 ; Dunkel et al., 2008 ; Morschhäuser et al., 2007 ). Rarely, loss-of-function mutations are found in the sterol desaturase gene ERG3 conferring resistance by abrogating the need for sterol demethylase( Kelly, Lamb, & Kelly, 1997 ). Three predominant ERG11 mutations, leading to amino acid substitutions Y132F, K143R, and VF125AL, have been shown to contribute to fluconazole resistance in C. auris ( Rybak et al., 2021 ). The transcription factor Mrr1 and the MFS transporter Mdr1 have also been implicated in resistance in isolates from Clade III( Li, Coste, Bachmann, Sanglard, & Lamoth, 2022 ). We have previously shown that CDR1 deletion in a resistant isolate that overexpresses this gene leads to a significant reduction in resistance to triazole antifungals( Rybak et al., 2019 ). We subsequently identified mutations in the gene encoding the transcription factor Tac1B in resistant isolates that were evolved in vitro in the presence of fluconazole, observed similar mutations among resistant clinical isolates, and demonstrated that the mutation leading to the A640V substitution in and of itself confers increased fluconazole resistance( Rybak et al., 2020 ). However, the relationship between mutations in TAC1B , CDR1 , or other potential resistance effectors remains unclear as the effect of TAC1B mutations on the expression of CDR1 or other genes has yet to be investigated. In the present study we establish that the TAC1B mutations leading to A640V, A657V, and F862_N866del all result in increased resistance to triazole antifungals, as well as increased expression of both CDR1 and MDR1 in C. auris . We also show that CDR1 is the primary driver of resistance conferred by these TAC1B mutations. Methods Isolates, strains, and growth conditions The clinical isolates and strains described in this study are listed in Supplementary Table S1 . Cells were propagated in YPD (1% yeast extract, 2% peptone, 2% dextrose) at 35°C and stored in 40% glycerol at -80°C. Strain construction. C. auris strain construction was based on the methods described previously( Carolus et al., 2024 ; Lombardi, Oliveira-Pacheco, & Butler, 2019 ), and detailed methods are described in Supplementary Methods . Oligonucleotides used in this study are listed in Supplementary Table S2 . Minimum inhibitory concentration (MIC) determinations by broth microdilution MICs for fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole were measured by modified CLSI broth microdilution assays as described previously( Rybak et al., 2021 ). RNA isolation An aliquot of cells from an overnight culture was used to inoculate 10 ml MOPS-buffered RPMI + 2% glucose (pH 7.0) to a OD 600 =0.08-0.12, followed by incubation at 35°C in a shaking incubator (220 rpm) until mid-log phase (6 hrs). Cell cultures were grown in triplicate. Cells were collected by centrifugation, supernatants removed, and cell pellets stored at -80°C. RNA was extracted using methods described previously with some modification( Santana & O’Meara, 2021 ). Cell pellets were resuspended in 100 µl FE Buffer (98% formamide, 0.01 M EDTA) at room temperature. Fifty microliters of 1 mm RNase-free glass beads were added to the cells and were subjected to vortex disruption for 5 min at room temperature followed by snap-cooling on ice. The cell lysate was clarified by centrifugation, and the supernatant was DNase-treated for 30 min followed by isopropanol precipitation. RNA integrity was confirmed by agarose gel electrophoresis, and concentrations were approximated by Nanodrop. cDNA synthesis and qRT-PCR cDNA was synthesized from 500 ng RNA using the RevertAid First Strand cDNA Synthesis Kit (Invitrogen/Thermo Fisher) with the provided random primer mix according to the manufacturer’s instructions. qRT-PCR was performed from three biological replicates, each with three technical replicates, using SYBR Green PCR master mix (Bio-Rad). Fold changes were calculated using the ΔΔ CT method with target gene CT values normalized to ACT1 CT values (generating dCT values) and the median 1c dCT value (from the three biological replicates) for each target gene used as the comparator for fold change calculation. RNA sequencing and analysis RNA sequencing was performed using Illumina NextSeq for stranded mRNA. Libraries were prepared with paired-end adapters using Illumina chemistries per manufacturer’s instructions, with read lengths of approximately 150 bp with at least 50 million raw reads per sample. Data were analyzed using the Galaxy web platform public server at usegalaxy.org ( Afgan et al., 2018 ) . Read quality was assessed and reads were quality-trimmed with a PHRED cutoff of 20 using FastP( Chen, Zhou, Chen, & Gu, 2018 ). Reads were then mapped to the C. auris B8441 reference assembly (NCBI GCA_002759435.2) using RNA Star with default parameters ( Dobin et al., 2013 ) followed by quantification using featureCounts ( Liao, Smyth, & Shi, 2014 ) and assessment of differential expression using DESeq2( Love, Huber, & Anders, 2014 ). Expression fold-change and significance cutoffs of >2-fold up- or down-regulated and an adjusted p-value less than 0.05 were established to categorize dysregulated genes. Gene Ontology annotations were retrieved from the Candida Genome Database ( Skrzypek et al., 2017 ) to identify significantly dysregulated GO terms using GoSeq( Young, Wakefield, Smyth, & Oshlack, 2010 ), normalizing for feature lengths extracted from the featureCounts output. Transcriptional overlap between strains was assessed using either UpsetR R Package ( Conway, Lex, & Gehlenborg, 2017 ) or DiVenn 2.0( Sun et al., 2019 ). The GEO accession number was assigned as GSE288372. Results TAC1B mutations observed in clinical isolates confer fluconazole resistance and reduced susceptibility to other triazole antifungals Previously we showed that introduction of the TAC1B mutation leading to the A640V substitution into susceptible clinical isolate AR0387 results in an increase in fluconazole MIC from 1 µg/mL to 8 µg/mL, and correction of this mutation to the wild-type sequence in resistant clinical isolate AR0390 reduces fluconazole MIC from 256 µg/mL to 16 µg/mL( Rybak et al., 2020 ). In order to further investigate the contribution of TAC1B mutations to triazole antifungal resistance, we introduced A640V, A657V, and F862_N866del into C. auris strain 1c. Strain 1c is derived from highly fluconazole-resistant Clade Ic clinical isolate Kw2999 (fluconazole MIC = 256 µg/mL) that harbors a ERG11 mutation leading to the K143R substitution and the TAC1B mutation leading to the A640V substitution( Ahmad, Khan, Al-Sweih, Alfouzan, & Joseph, 2020 ). In strain 1c, both mutations have been corrected to their wild-type sequences resulting in fluconazole MIC of 2 µg/mL. As previously demonstrated in isolate AR0387, reintroduction of the A640V substitution in 1c (strain 1cA640V) resulted in an increase in fluconazole MIC to 32 µg/mL. Introduction of the A657V substitution (strain 1cA657V) likewise increased the MIC to 32 µg/mL, whereas F862_N866del (strain 1cADdel) increased the MIC to 64 µg/mL ( Table 1 ) . The susceptibilities to voriconazole, isavuconazole, itraconazole, and posaconazole were also reduced ( Table 1 ) . These results indicate that each of these TAC1B mutations confer increased resistance to the triazole antifungals. View this table: View inline View popup Download powerpoint Table 1. Fluconazole MIC values 1 of Parental Strain 1c and its TAC1B Mutant Derivative Strains Mutations in TAC1B drive overexpression of CDR1 and MDR1 As there were similar shifts in azole resistance for each mutant, we investigated whether there was a common transcriptional response. We performed RNA-seq on strain 1c and clone A from each TAC1B mutant strain and established a cutoff for significant gene dysregulation at 2-fold compared to strain 1c with a DeSeq2 adjusted p-value of <0.05. We observed an overlapping set of dysregulated genes among the TAC1B mutant backgrounds, but each also showed substantial differences in transcriptional response. For instance, the A640V mutation resulted in only 5 dysregulated genes, each of which was also represented by either A657V or F862_N866del (ADdel) ( Figure 1A ) . Of the 40 ADdel mutant dysregulated genes, 72.5% were dysregulated in at least one of the other two strains ( Figure 1A ) . Interestingly, the A657V mutation resulted in a large unique transcriptional response, with 358 genes being dysregulated only in this mutant ( Figure 1A ) . Gene Ontology Enrichment analysis revealed similar patterns of enriched gene sets between strains, with common terms surrounding transmembrane transport and xenobiotic detoxification frequently represented, in line with the role of TAC1 homologs in other species in regulating transporter and efflux activity ( Figure 1B ) . Among these were efflux pumps MDR1 , upregulated in all three mutants, and CDR1 , upregulated in the A640V and ADdel mutants. In the A657V mutant, CDR1 showed an 1.7-fold increase in expression. While this didn’t meet the fold-change cutoff for significance, this increase may still be sufficient to functionally explain the observed increase in triazole MIC ( Figure 1B ) . Dozens of other genes with predicted transporter function were also dysregulated in this strain background, raising the possibility of other influential effectors ( Figure 1C ) . Download figure Open in new tab Figure 1. (A) UpSet plot of transcriptional network overlap, demonstrating the intersections of genes that are dysregulated by >2 fold and adjusted p-value of <0.05 compared to the parental isolate for each strain harboring different TAC1B mutations. Each bar in the upper region signifies the number of dysregulated genes in each intersection while the lower region signifies which strains exhibit dysregulation in those genes. (B) Volcano plots demonstrate magnitude of gene dysregulation for each strain, with significantly dysregulated genes colored in red. The top 5 overrepresented Gene Ontology terms in each dataset are listed. (C) All dysregulated genes that match the GOSlim term for Transporter Activity are represented for each strain. Colors represent the directionality of dysregulation. For all panels, strains are indicated by their harbored TAC1B mutation, while “ADdel” refers to the F862_N866del mutation. We then measured CDR1 and MDR1 expression by qRT-PCR in strains 1cA640V, 1c657V, and 1cADdel compared to strain 1c and observed 2.5-, 1.6-, and 6-fold increase in CDR1 expression ( Figure 2A ) and 2.6-, 11.7-, and 3.5-fold increase in MDR1 expression, respectively ( Figure 2B ). We then measured CDR1 and MDR1 expression by qRT-PCR in susceptible clinical isolates AR0387, its derivative carrying the A640V substitution, resistant clinical isolate AR0390, and its derivative where the A640V mutation has been corrected to the wild-type sequence. CDR1 and MDR1 were upregulated 3.2-fold and 3-fold, respectively, in the presence of the A640V substitution in the AR0387 background ( Figure 2C and 2D ). Likewise, expression of these genes was reduced to wild-type levels when the A640V substitution was corrected to the wild-type sequence ( Figure 2C and 2D ). These data indicate that TAC1B mutations drive CDR1 and MDR1 overexpression in C. auris resistant isolates. Download figure Open in new tab Figure 2. Fold change in CDR1 (A) and MDR1 (B) RNA expression for 1c TAC1B mutant strains and CDR1 (C) and MDR1 (D) transcript abundance for the AR0387 and AR0390 TAC1B allele swap strains was measured by qRT-PCR. In each graph, the median dCT value (calculated from the average ACT1 gene CT value from three technical replicates subtracted from the average target gene CT value from three technical replicates) from three biological replicates of strain 1c or isolate AR0387, for panels A and B and panels C and D, respectively, was the comparator. TAC1B -mediated fluconazole resistance is driven predominantly by CDR1 overexpression with a lesser contribution from MDR1 In order to further examine the contribution of CDR1 and MDR1 to fluconazole resistance due to TAC1B mutations, we disrupted these genes in resistant clinical isolate Kw2999 and strain 1c. CDR1 disruption in Kw2999 resulted in a reduction in fluconazole MIC from 256 µg/mL to 4 µg/mL, and disruption in 1c resulted in a reduction in fluconazole MIC from 2 µg/mL to 0.25 µg/mL. MDR1 disruption in isolate Kw2999 or strain 1c, however, had no effect on fluconazole MIC ( Figure 3 ). Disruption of CDR1 in strain 1cA640V resulted in a decrease in fluconazole MIC from 32 µg/mL to 0.5 µg/mL, disruption in 1c657V reduced the MIC from 32 µg/mL to 1 µg/mL, and disruption in 1cADdel reduced the MIC from 64 µg/mL to 0.5-1 µg/mL. As in Kw2999 and 1c, MDR1 disruption had no effect on fluconazole MIC in any of these three strains ( Figure 3 ). Download figure Open in new tab Figure 3. Fluconazole MIC were measured by broth microdilution in strains in which CDR1 and MDR1 were truncated by premature stop codon individually or in combination in Kw2999, 1c, or derivative backgrounds harboring indicated TAC1B mutations. Two independent clones were generated for each mutant combination. Each point represents a distinct MIC experimental replicate. We also disrupted MDR1 in the CDR1 -disrupted derivatives of 1cA640V, 1cA657V, and 1cADdel and observed further reduction in fluconazole MICs for all strains tested. We observed a reduction from 0.5 µg/mL to 0.125 µg/mL in the 1cA640V background, 1 µg/mL to 0.125 µg/mL in the 1cA657V background, and 0.5-1 µg/mL to 0.0625 µg/mL in the 1cADdel background ( Figure 3 ). In addition, disruption of MDR1 in CDR1 -disrupted mutants of Kw2999 and 1c resulted in a further MIC decrease (from 8 µg/mL to 1 µg/mL and from 0.25 µg/mL to 0.0625 µg/mL, respectively). Similar trends for all strains were observed for itraconazole, posaconazole, voriconazole, and isavuconazole ( Supplementary Figure S1 ). These data indicate that the majority of fluconazole resistance conferred by mutations in TAC1B are driven by overexpression of CDR1 , whereas MDR1 makes a slight contribution to fluconazole resistance in isolates carrying such mutations. Discussion Our findings reveal similarities and differences between genes influenced by TAC1B in C. auris and TAC1 in the somewhat distantly related species C. albicans . In C. albicans Tac1 regulates the genes encoding the ABC transporters Cdr1 and Cdr2, both of which contribute to fluconazole resistance through activating mutations in TAC1 leading to upregulation of these genes( Coste et al., 2004 ). In C. albicans Mrr1 regulates expression of the gene encoding the MFS transporter Mdr1 which also contributes to fluconazole resistance( Morschhäuser et al., 2007 ). In C. auris , TAC1A and TAC1B have been identified as homologs of TAC1 and are situated in tandem with 488 bp between the ORFs on chromosome 5 of the C. auris genome( Mayr, Ramírez-Zavala, Krüger, & Morschhäuser, 2020 ). Only mutations in TAC1B have been implicated in fluconazole resistance, and such mutations have been widely identified across resistant clinical isolates in C. auris Clades I and IV and to a more limited extent in Clades II and III ( Rybak et al., 2020 ). We have previously shown that the A640V substitution in TAC1B leads to an increase in fluconazole MIC when introduced into a susceptible isolate and a reduction in MIC when the sequence is corrected to the wild-type sequence in a resistant isolate carrying this mutation( Rybak et al., 2020 ). In the present study we have confirmed the contribution of the A640V substitution and have shown that A657V and F862_N866del, have similar effects on fluconazole susceptibility. While ABC transporter gene CDR1 overexpression in response to TAC1B mutations has not been previously established, the finding that this gene was upregulated among strains engineered to express these TAC1B mutations was not surprising given the relationship between TAC1 and CDR1 in C. albicans and other Candida species. The overexpression of MDR1 in strains carrying TAC1B mutations was unexpected and is reminiscent of the regulation of both CDR1 and MDR1 by Mrr1 in C. lusitaniae and both CDR1B and MDR1B by Mrr1 in C. parapsilosis ( Demers et al., 2018 ; Doorley et al., 2022 ). Similar to C. albicans , to date, upregulation of MDR1 in C. auris has only been associated with a mutation in MRR1 present in all isolates of Clade III leading to a N647T substitution. Introduction of this mutation into a susceptible Clade IV isolate resulted in a nearly 80-fold increase in MDR1 expression accompanied by an increase in fluconazole MIC from 4 µg/mL to 16 µg/mL and a similar fold increase in voriconazole MIC( Li et al., 2022 ). Deletion of the mutant MRR1 in a clade III isolate resulted in a reduction in MIC from 512 µg/mL to 256 µg/mL and a similar fold reduction in voriconazole MIC. A similar effect was observed upon MRR1 deletion in a different clade III isolate( Mayr et al., 2020 ). We observed at most an 11.7-fold increase in expression of MDR1 in response to mutations in TAC1B . Given that disruption of MDR1 alone in strains carrying TAC1B mutations had little to no effect on fluconazole MIC, and only a modest effect when disrupted in combination with CDR1 , this comparatively modest level of MDR1 overexpression appears not to be sufficient to have a substantive effect on fluconazole resistance. Our work establishes the role of CDR1 regulation through TAC1B mutations in fluconazole resistance in C. auris and clarifies the role of both CDR1 and MDR1 in isolates carrying such mutations. A more complete understanding of the repertoire of TAC1B and other mutations that contribute to fluconazole resistance in C. auris may one day lead to genetic tools to more rapidly and accurately predict patient responses to guide selection of antifungal therapy. Moreover, understanding how these mutations lead to CDR1 upregulation and fluconazole resistance could point to therapeutic strategies for impeding expression of this efflux pump, thereby enhancing the activity and reclaiming the utility of fluconazole against C. auris . Acknowledgements This work was supported by NIH NIAID grant R01 AI169066 awarded to P.D.R. and C.A.C, NIAID grant U19AI110818 to the Broad Institute (C.A.C.), NIH NIAID grant T32 AI106700 (D.J.S.), and in part by the National Cancer Institute of the National Institutes of Health under Award Number P30 CA021765 awarded to the Hartwell Center at St. Jude Children’s Research Hospital. References ↵ Afgan , E. , Baker , D. , Batut , B. , van den Beek , M. , Bouvier , D. , Cech , M. , . . . Blankenberg , D. ( 2018 ). The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update . Nucleic Acids Res ., 46 ( W1 ), W537 – W544 . doi: 10.1093/nar/gky379 OpenUrl CrossRef PubMed ↵ Ahmad , S. , Khan , Z. , Al-Sweih , N. , Alfouzan , W. , & Joseph , L . ( 2020 ). Candida auris in various hospitals across Kuwait and their susceptibility and molecular basis of resistance to antifungal drugs . Mycoses , 63 ( 1 ), 104 – 112 . doi: 10.1111/myc.13022 . 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