Echinocandin tolerance and persistence in vitro are regulated by calcineurin signaling in Candida glabrata

Echinocandin tolerance and persistence in vitro are regulated by calcineurin signaling in Candida glabrata | 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 Echinocandin tolerance and persistence in vitro are regulated by calcineurin signaling in Candida glabrata Abigail A. Harrington , Timothy J. Nickels , View ORCID Profile Kyle W. Cunningham doi: https://doi.org/10.1101/2025.08.19.671059 Abigail A. Harrington 1 Department of Biology, Johns Hopkins University , Baltimore, Maryland, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Timothy J. Nickels 1 Department of Biology, Johns Hopkins University , Baltimore, Maryland, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kyle W. Cunningham 1 Department of Biology, Johns Hopkins University , Baltimore, Maryland, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kyle W. Cunningham For correspondence: kwc{at}jhu.edu Abstract Full Text Info/History Metrics Preview PDF ABSTRACT Upon exposure to echinocandins, growing yeast cells begin to accumulate cell wall damage and eventually die, resulting in therapeutic effects. While resistance to echinocandins is well studied, tolerance and persistence mechanisms that may also contribute to clinical failures and relapses remain understudied. In time-kill assays with micafungin in vitro , the opportunistic pathogen Candida glabrata exhibited biphasic kinetics of cell death. Modeling with exponential decay equations distinguished a fast-dying major population from a slow-dying minor population, indicative of persistence. A genome-wide forward-genetic screen revealed dozens of genes that appeared to regulate persistence and/or tolerance, but not resistance. Several of those genes encoded calcineurin and its upstream regulators. Using individual gene knockout mutants and FK506, we show that calcineurin signaling increases the lifespans of most C. glabrata cells through a process that is largely independent of Crz1, one of its downstream effectors. The formation of long-lived persister-like cells (i.e. persistence) was strongly dependent on calcineurin signaling, independent of Crz1. Pre-activation of calcineurin using genetic or chemical stressors, such as manogepix, strongly increased tolerance and persistence in C. glabrata , suggesting antagonism of echinocandin efficacy by this new antifungal. Calcineurin signaling was also necessary for induction of tolerance and persistence in Candida albicans . The findings suggest that short-term administration of FK506 during the earliest stages of echinocandin treatment may improve clinical outcomes while possibly avoiding long-term immunosuppression. IMPORTANCE Treatment of fungal infections is often unsuccessful. Potential causes of antifungal failure include tolerance and persistence, which are poorly understood processes used by fungal pathogens to survive our assaults. This study utilizes detailed experimental protocols and genome-wide screens to discover how Candida glabrata induces tolerance and persistence to a major class of antifungals. The findings suggest that a clinical immunosuppressant may be repurposed to combat tolerance and persistence in this pathogenic yeast as well as Candida albicans and perhaps others. INTRODUCTION At least four distinct processes contribute to antibiotic treatment failure: resistance, heteroresistance, tolerance, and persistence ( 1 – 3 ). Resistance occurs when microbes acquire mutations that alter the expression or function of the drug target, the influx, efflux, sequestration or metabolism of the compound, or otherwise increases the effective dosage of the antimicrobial drug. Common measures of antibiotic resistance include the minimal inhibitory concentration (MIC) or the concentration causing a 50% inhibition of maximal growth (IC50). Resistance mutations are stably inherited by daughter cells and cause infections that require higher doses of the antibiotic or alternative antibiotics to remedy. Heteroresistance occurs when a small subpopulation of cells within a clonal population acquires phenotypic resistance without any stable mutations. Heteroresistant subpopulations have a greatly increased MIC relative to the majority of cells in the population and have been shown to contribute to treatment failure in murine models of infection. Tolerance occurs when the clonal population has acquired mutations that increase the lifespan of the cells, even when exposed to excess antibiotics, without altering the MIC or IC50. To overcome tolerance, the duration of antibiotic treatment must be increased, not the dose. Tolerance is usually assayed using time-kill experiments that measure the number of “viable” cells in the population (colony forming units, or CFU) after transient exposure to supra-MIC doses of antibiotics and estimating the half-life of the population. Persistence refers to an epigenetic regulatory process that produces a small number of highly tolerant “persister cells” amongst the large population of fast-dying susceptible cells. Persister cells are typically quiescent, at least transiently, and are therefore not killable by multiple classes of antibiotics, which can increase the likelihood of relapse. All these processes have been studied thoroughly in many infectious species of bacteria. Antifungal resistance is well-studied in some pathogenic fungi ( 4 , 5 ), but heteroresistance, tolerance, and persistence are just beginning to be distinguished and individually unraveled at the molecular level ( 6 ). Tolerance and heteroresistance to azoles, fungistats targeting ergosterol biosynthesis in the ER, is now being investigated in several pathogenic species of yeasts ( 7 – 11 ) as there is growing evidence of clinical relevance of these processes ( 12 ). Heteroresistance to echinocandins, fungicides targeting the cell wall, was recently associated with breakthrough infections by Candida parapsilosis ( 13 ). A retrospective study found that rare echinocandin-tolerant strains of Candida tropicalis were much more lethal than non-tolerant strains in patients treated for candidemia ( 14 ). Though the molecular mechanisms of echinocandin heteroresistance and tolerance have not been elucidated, an inhibitor of the Ca 2+ /calmodulin-dependent protein phosphatase calcineurin (FK506) abolished the tolerance of C. tropicalis and substantially increased the lifespan of infected animals undergoing echinocandin treatment ( 14 ). A better understanding of the regulatory mechanisms responsible for heteroresistance, tolerance, and persistence could potentially lead to development of antifungal therapies that lack undesirable side-effects on the patient (such as immunosuppression caused by FK506). Candida glabrata (also known as Nakaseomyces glabratus ) is the second most common cause of life-threatening candidemia and candidiasis next to C. albicans ( 15 – 17 ), though it belongs to a genus that is more closely related to Saccharomyces than true Candida ( 18 ). Due to its innate and easily acquired resistance to antifungals and its rising incidence, the World Health Organization has classified C. glabrata as a High Priority Threat ( 19 ). In C. glabrata , azole resistance mutations arise primarily in the target ( ERG11 ) and in a transcription factor gene ( PDR1 ) that regulates expression of transporters that efflux the drug ( 20 ). PDR1 also confers mild resistance to most echinocandins through expression of lipid flippases ( 21 , 22 ). However, strong resistance to echinocandins arises primarily through mutations in the two targets, encoded by FKS1 and FKS2 ( 23 , 24 ), the latter of which depends on calcineurin and the Crz1 transcription factor for maximal expression ( 25 ). When a resistance mutation arises in FKS2 , its impact in vitro can be blocked by calcineurin inhibitors ( 25 , 26 ). Even in the absence of antifungals, calcineurin also promotes virulence of C. glabrata in mouse models of invasive candidiasis through a mechanism that is potentially independent of Crz1 ( 27 , 28 ). Time-kill experiments in vitro have shown most C. glabrata cells die quickly when exposed to high doses of echinocandins while a small and variable sub-population dies more slowly ( 29 , 30 ), suggesting the possible development of long-lived persister cells. Such persister cells serve as a reservoir for acquisition of resistance mutations ( 30 ). After engulfment into phagosomes by macrophages, C. glabrata cells typically remain viable and survive longer when exposed to echinocandins, with an increased number of long-lived cells ( 31 ). Therefore, tolerance and persistence in this species may be clinically important and regulated by unknown mechanisms. This study quantifies tolerance and persistence in C. glabrata exposed to echinocandins in vitro by fitting high resolution time-kill data to exponential decay equations developed previously for antibiotic research ( 2 ). It also implements a genome-wide genetic screen using Tn-seq to identify specific regulators of tolerance and persistence, as well as individual gene knockout experiments. The findings suggest tolerance and persistence are governed by processes distinct from resistance and heteroresistance. Remarkably, C. glabrata , and C. albicans , appeared to induce tolerance and persistence ‘on demand’ through the activation of a calcineurin. Therefore, FK506 and other drugs that block calcineurin signaling in yeasts and other fungi may improve clinical outcomes by lowering tolerance and persister cell development in addition to lowering resistance (expression of echinocandin targets). Conversely, the findings suggest that drugs, mutations, and host conditions that stress fungal cells and pre-activate calcineurin may promote tolerance and persistence, thereby worsening clinical outcomes. RESULTS Quantitation of echinocandin tolerance and persistence in C. glabrata Time-kill assays have been used routinely to discriminate, quantify, and characterize sub-populations of cells that exhibit distinct rates of killing by antibiotics ( 2 ). These assays involve treatment of clonal populations with high doses of cidal drugs for varying lengths of time, plating the treated cells onto drug-free agar media at appropriate dilutions, and then counting the number of colonies that appear after additional incubation. The colony forming units (CFU) per mL of starter culture is then charted over time and, if biphasic, analyzed by fitting to the sum of two exponential decay equations ( 2 ). When 8 independent stationary-phase cultures of wild-type C. glabrata (strain BG14) were diluted into fresh medium containing 125 ng/mL micafungin, biphasic survival kinetics were observed after a brief lag ( Fig. 1 ). When charted individually, the eight cultures all behaved similarly ( Fig. S1 ). The lag disappeared when log-phase cultures were tested, while the biphasic kinetics remained ( Fig S2 ). After excluding the lag and fitting the averaged CFUs to exponential equations ( Fig. 1 , smooth curve), a minor population (9%) of long-lived cells (t ½ = 3.75 hr) could be distinguished from the major population of fast-dying cells (t ½ = 0.75 hr). To test whether stable mutants contributed to the slow-dying sub-populations, eight colonies that arose after 24 hr treatments with micafungin were picked, regrown in fresh medium, and retested individually. All eight cultures again produced similar numbers of long-lived cells ( Fig. 1 , open symbols and dashed curve). These findings show that clonal cultures of C. glabrata consistently and transiently produce two phenotypically distinct populations of cells, the minor one with nearly 5-fold increased half-life in micafungin. Download figure Open in new tab Fig S1. Biological replicates experience similar responses to micafungin and FK506 treatment. Eight single colonies of wild-type BG14 cells were grown to saturation for 72 hours, then diluted 50-fold into media containing micafungin (0.125 µg/mL) medium containing or lacking FK506 (1 µg/mL) and sampled as described in Fig. 1 . Here, individual replicate cultures were counted and plotted (symbols) were plotted with curve fits imported from Fig 1 . Download figure Open in new tab Fig S2. Log-phase populations exhibit no lag phase in time-kill experiments. Four single colonies were grown in fresh SCD medium for 24 hours until log phase phase was achieved. Time-kill assays with micafungin were performed as described in Fig 1 . Download figure Open in new tab Figure 1. Quantifying tolerance and persistence in C. glabrata in response to micafungin treatment. Eight single colonies of wild-type strain BG14 were grown to saturation for 72 hours, then diluted 50-fold into fresh SCD medium containing 125 ng/mL micafungin alone (black) or micafungin plus 1 µg/mL FK506 (red). Cultures were shaken at 30°C, sampled at various timepoints, serially diluted, and plated on drug-free YPD medium. Colony forming units (CFU) were determined and the eight replicates averaged and charted (filled symbols). The best fit of the data to exponential decay equations are shown (solid smooth curves). Eight single colonies that appeared after 24 hours of treatment were picked and retested in the same conditions (Round 2; open symbols and dashed curves). When micafungin was substituted with other glucan synthase inhibitors (e.g. caspofungin, ibrexafungerp), similar numbers of persister cells with similar lifespans were detected, though slight variation was evident ( Fig. S3 ). The half-lives and the population sizes did not change even when the doses of these antifungals were greatly increased ( Fig. S3 ). Additionally, the survival kinetics of several mutants that decreased ( fks2Δ , pdr1Δ ) or increased ( mrp20Δ ) resistance to micafungin ( 21 ) were indistinguishable from those of the BG14 parent strain ( Fig. S4 ). Altogether, these results suggest C. glabrata can produce substantial numbers of long-lived persister-like cells through transient regulatory processes that are distinct from stable mechanisms that control resistance. Further, this mechanism also occurs independent of heteroresistance, as it was not detectable in wild-type C. glabrata strains ( 7 ). In the remainder of this study, the process of producing and maintaining such persister-like cells will be termed ‘persistence’ while the processes governing lifespan of the remaining susceptible cells will be referred to as ‘tolerance’, in accordance with earlier conventions ( 2 ). Download figure Open in new tab Figure S3. Tolerance and persistence to β-1,3-glucan synthase inhibitors is dose- and drug-independent. Wild-type cells were grown and treated with varying concentrations of micafungin (MF), caspofungin (CF), or ibrexafungerp (IBX) in as described Fig 1 . Data points are the averages of four biological replicates and were used to generate the curve fits. Download figure Open in new tab Figure S4. Strains associated with increased and decreased micafungin resistance do not alter tolerance or persistence. (Top Row) Strains were grown and treated with micafungin as described Fig 1 . (Bottom) mrp20Δ mutant was treated with an 8X dose (1 µg/mL) of micafungin to compensate for resistance. Cultures were sampled and CFUs were calculated as described in Fig. 1 . Data points are the averages of four biological replicates and were used to generate the curve fits. A forward genetic screen identifies regulators of tolerance and persistence To identify genes that specifically regulate tolerance and/or persistence in C. glabrata , a genome-wide Tn-seq screen was implemented using a complex pool of transposon insertion mutants in strain BG14 that had been previously analyzed for resistance to micafungin and other antifungals ( 21 , 32 ). Resistance screens employed very low doses of antifungals and long exposure times (48 hr), which would not have uncovered the genes that regulate tolerance and persistence. To elucidate such genes, the transposon pool was exposed to a high, lethal dose of micafungin for a short duration (6 hr) and then the cultures were washed and regrown in drug-free fresh medium before Tn-seq analysis. Mutants with elevated tolerance or persistence would be enriched in this modified protocol (i.e. positive Z-scores) while mutants with diminished tolerance or persistence would be depleted from the general pool (i.e. negative Z-scores). Tolerance/persistence Z-scores were calculated for 5275 annotated genes by comparing the 6 hr and 0 hr exposures to 64 ng/mL micafungin (Table S1). When these Z-scores were charted against resistance Z-scores obtained previously with 8 ng/mL micafungin ( 21 ), the correlation was poor ( Fig. 2 ). Hundreds of mitochondrial genes ( Fig. 2 , yellow symbols) and other genes that significantly increased resistance to micafungin had little impact on tolerance/persistence, as expected from the time-kill analysis of mitochondrial mrp20Δ mutants ( Fig. S4 ). Conversely, dozens of genes with significantly high or low tolerance/persistence Z-scores had resistance Z-scores close to zero ( Fig. 2 ). These findings further suggest that micafungin tolerance and persistence may be controlled by a relatively small number of genes that are largely distinct from those that regulate micafungin resistance. Download figure Open in new tab Figure 2. Genetic regulation of tolerance/persistence differs from that of resistance. A pool of Hermes transposon insertion mutants in the BG14 strain that was previously analyzed for resistance to low micafungin (Resistance Z-score) was reanalyzed in high micafungin for tolerance and persistence to high micafungin (Persistence/Tolerance Z-scores) and charted. A total of 36 genes exhibited diminished tolerance/persistence (Z −3). GO term analysis ( 33 ) indicated significant enrichment (p-value = 5.2E-5; false discovery rate = 6.3E-2) of two small genes ( CNB1, RCN1 ) that encode regulators of calcineurin, a well-studied Ca 2+ /calmodulin-dependent protein phosphatase. An upstream activator of calcineurin in S. cerevisiae and C. albicans ( KCH1 ) ( 34 , 35 ) also was present on this list. However, the CNA1 gene, encoding the catalytic subunit of calcineurin, was not significant (Z = 0.26) possibly due to the small effective size of this gene and the presence of an autoinhibitory domain at the C-terminus. To explore the possible involvement of calcineurin in the regulation of tolerance and persistence, micafungin time-kill experiments were performed on wild-type C. glabrata in the presence of FK506, a specific inhibitor of calcineurin. Strikingly, FK506 caused a 2.5-fold decrease in the half-life of the susceptible cells (i.e. tolerance) and a 270-fold decrease in the number of long-lived persister-like cells in micafungin ( Fig. 1 , red symbols and curves). Of eight cells that survived 24 hr exposure to micafungin plus FK506 and produced colonies in drug-free media, all produced wild-type patterns in time-kill experiments upon retesting in the same conditions ( Fig. 1 dashed curve, Fig. S1 ). These findings using FK506 confirm the Tn-seq results, suggest that calcineurin signaling drives both tolerance and persistence, and demonstrate those behaviors are readily reversible in C. glabrata . As calcineurin signaling becomes activated in response to micafungin and other stressors of the cell wall ( 36 ), tolerance and persistence may be protective behaviors in C. glabrata that are induced in response to stresses. Calcineurin drives tolerance and persistence in C. glabrata independent of Crz1 and Rcn2 The CNA1 and CNB1 genes were each knocked out in the BG14 parent strain and analyzed by time-kill experiments and mathematical modeling. Both the cna1Δ and cnb1Δ mutants exhibited strongly diminished tolerance and persistence relative to the wild-type control ( Fig. 3 , left panel). Though the number of persister-like cells decreased by more than 100-fold in both mutants relative to wild-type, the half-lives of the persister cells remained constant in all strains at approximately 3.2 hr. As expected, the presence of FK506 in the time-kill experiments did not impact the behavior of cna1Δ and cnb1Δ mutants and forced the wild-type parent strain to behave like the calcineurin-deficient mutants ( Fig. 3 , right). These findings further suggest that calcineurin signaling drives both tolerance and persistence upon exposure to micafungin. Download figure Open in new tab Figure 3. Calcineurin promotes tolerance and persistence. Time-kill assays of cna1Δ (blue), cnb1Δ (red), crz1Δ (green), and wild-type parent strain (black) were performed as described Fig 1 . The averages of four biological replicates (symbols) and were fit to exponential decay equations (smooth curves). Experiments containing FK506 (1 µg/mL) were performed in parallel (right panel). An important effector of calcineurin signaling is the transcription factor Crz1 ( 27 ). Though the CRZ1 gene was not significant the Tn-seq screen, a crz1Δ mutant exhibited levels of tolerance and persistence that were intermediate between wild-type and the cna1Δ and cnb1Δ mutants ( Fig. 3 , left). In the presence of FK506, the crz1Δ mutant resembled the calcineurin-deficient strains ( Fig. 3 , right). Thus, Crz1 seemed to be required for a portion of the effects of calcineurin. Targets of Crz1 include FKS2 and RCN2 ( 25 , 28 , 37 ) neither of which were significant in the Tn-seq screen. An fks2Δ mutant was indistinguishable from wild-type in time-kill experiments ( Fig. S4 ), suggesting it is not required for calcineurin-induced tolerance and persistence. An rcn2Δ mutant exhibited wild-type levels of tolerance and persister-like cells, but interestingly the half-life of the persister-like cells increased by ∼1.5-fold ( Fig. S5 ). FK506 decreased the tolerance and persistence of rcn2Δ mutants to the same levels as the calcineurin-deficient mutants ( Fig. S5 ). A plasmid that overexpresses RCN2 from a strong constitutive PDC1 promoter did not alter micafungin tolerance or persistence relative to a control plasmid when introduced into cna1Δ mutants or wild-type cells ( Fig. S5 ). These findings suggest that Rcn2 functions in its canonical role as a feedback inhibitor of calcineurin signaling ( 28 , 37 ) instead of an effector of calcineurin signaling in the regulation of tolerance and persistence. Download figure Open in new tab Figure S5. RCN2 negatively regulates calcineurin in time-kill experiments. (Left) Wild-type and rcn2Δ strains were grown and treated with micafungin containing or lacking FK506 and time-kill experiments were performed as described Fig 1 . Data points are the averages of four biological replicates and were used to generate the curve fits. (Right) Wild-type and cna1Δ strains were transformed with either empty plasmid (+ vector) or pCN-PDC1-RCN2 (+ RCN2 ). Single colonies were grown to saturation in SCD+NAT media for 72 hours to select for plasmid, then washed and diluted into fresh medium containing micafungin and and analyzed as described in Fig. 1 . Data points are the averages of four biological replicates and were used to generate the curve fits. To further explore the role of calcineurin in regulating tolerance and persistence, the effects of manogepix were studied. Manogepix is a preclinical antifungal that blocks GPI anchor biosynthesis in the ER ( 38 ) and produces cellular stresses that very strongly activates calcineurin signaling in C. glabrata ( 36 ). A 2-hour pre-treatment of wild-type cells with manogepix resulted in moderately increased tolerance and persistence to micafungin ( Fig. 4 ). Though no such increases were observed in cnb1Δ mutants, the crz1Δ mutant exhibited a dramatic increase in micafungin tolerance when pre-exposed to manogepix ( Fig. 4 ). Checkerboard assays were utilized to determine whether manogepix increases resistance to micafungin in crz1Δ mutants. Manogepix did not antagonize micafungin in crz1Δ mutants ( Fig. 5 ). However, manogepix did antagonize micafungin in wild-type parent strain, possibly due to calcineurin and Crz1-dependent expression of target genes such as FKS2 . These findings suggest that calcineurin signaling is a key driver of tolerance and persistence in C. glabrata , with a portion of its effects mediated by Crz1 and another portion mediated by unknown effectors. Download figure Open in new tab Figure 4. Manogepix activation of calcineurin induces micafungin tolerance and persistence independent of Crz1. Wild-type (black), cnb1Δ (red), and crz1Δ (purple) strains were grown to saturation and diluted 25-fold into fresh medium containing (dashed lines) or lacking (solid lines) manogepix (0.6 µg/mL). After shaking at 30°C for 2 hours, cultures were diluted 2-fold in fresh medium containing micafungin (125 ng/mL) and analyzed in time-kill experiments as described in Fig. 1 . The averages of four biological replicates (symbols) were used for curve fitting (smooth curves). Download figure Open in new tab Figure 5. Mannogepix activation of calcineurin induces micafungin resistance through Crz1 and Fks2. Growth (green) of wild-type and crz1Δ strains was measured after incubation for 24 hr in medium containing varying concentrations of mannogepix and micafungin. Similar effects were seen in two additional replicates. Chronic Activation of Calcineurin increases tolerance and persistence Some of the genes with positive tolerance/persistence Z-scores in the Tn-seq screen may generate chronic cellular stresses that pre-activate calcineurin when disrupted with transposons. One such gene is FKS1 (Z = 3.38), which encodes the major catalytic subunit of glucan synthase ( 25 , 39 , 40 ). The fks1Δ mutants are FK506-sensitive because they depend on calcineurin signaling, Crz1, and elevated expression of FKS2 for viability ( 25 ). When tested in time-kill assays, fks1Δ mutants exhibit a large increase in tolerance compared to BG14 and the parent strain ( Fig. 6 , Left). Thus, chronic pre-activation of calcineurin through Fks1-deficiency may increase tolerance and persistence much like the acute activator, manogepix. Download figure Open in new tab Figure 6. Genetic activation of calcineurin increases tolerance and persistence. The fks1Δ (green), ipt1Δ (orange), and gtb1Δ (blue) mutants were generated and tested in time-kill experiments as described in Fig. 1 in the absence (smooth curves) and presence (dashed curves) of FK506. Averages of four biological replicates were used to generate the curve fits. Several genes encoding ER proteins (e.g. CNE1 , GTB1 , KEG1 , SKN1 , VMS1 ) exhibited strongly increased tolerance/persistence Z-scores (Table S1, Fig. 2 ). Knockout mutants of GTB1 were generated and tested in time-kill experiments in the presence and absence of FK506. The gtb1Δ mutants exhibited elevated tolerance to micafungin that were strongly blocked by FK506 ( Fig. 6 , Right). The IPT1 gene, which was also highly significant in the Tn-seq screen (Z = 13.0), encodes an enzyme in the Golgi complex that synthesizes the abundant sphingolipid M(IP2)C. In time-kill experiments, an ipt1Δ mutant exhibited elevated tolerance and persistence that was likewise blocked by FK506 ( Fig. 6 , Right). Heteroresistance to micafungin was not increased in the fks1Δ mutant ( Fig. S6 ). These findings suggest that many negative regulators of calcineurin signaling may be among the list of genes with positive Z-scores in micafungin tolerance/persistence screens. However, other gene deficiencies may impact tolerance and persistence through calcineurin-independent effects. Download figure Open in new tab Figure S6. Heteroresistance to micafungin was not observed in C. glabrata strains. Wild-type (BG14) and mutant strains were grown to saturation for 72 hours, diluted into fresh SCD medium, and then plated on YPD agar media containing varying doses of micafungin (0 to 0.015 µg/mL). Plates were incubated for 24 hours at 30°C and colonies were counted using a dissecting microscope. HOG signaling pathway negatively regulates tolerance and persistence independent of calcineurin Calcineurin undoes the effects of serine/threonine protein kinases on shared substrates. Two genes encoding protein kinases ( FPK1 , SSK2 ) exhibited strongly positive Z-scores in the tolerance/persistence screen and therefore could produce phosphoproteins that are directly targeted by calcineurin. However, they were also strongly positive for micafungin resistance ( Fig. 2 ). SSK2 encodes a MAPKKK that phosphorylates and activates Pbs2, a MAPKK, that in turn phosphorylates Hog1, a MAPK, that is well known to respond to high-osmolarity signals ( 41 ). Though PBS2 and HOG1 were near zero in the Tn-seq screens, an upstream regulator of SSK2 ( SSK1 ) also exhibited positive tolerance/persistence and resistance Z-scores. Knockout mutants lacking the SSK2 , PBS2 , and HOG1 genes in the BG14 background were analyzed in time-kill experiments using 8-fold higher doses of micafungin to compensate for their mild resistance to echinocandins. All three mutants exhibited elevated tolerance relative to the wild-type control in the presence of FK506 ( Fig. S7 , Fig. 7 ). These results indicate the HOG signaling pathway normally negatively regulates tolerance and persistence independent of calcineurin. In the absence of FK506, calcineurin signaling strongly increased tolerance and persistence in all three mutants ( Fig. 7 , Fig. S7 ), which indicates that calcineurin promotes tolerance and persistence independent of HOG signaling. The effects of hog1Δ mutations were also observed in a crz1Δ mutant background ( Fig. 7 ). Thus, HOG signaling can negatively regulate tolerance and persistence independent of Crz1 and calcineurin. Download figure Open in new tab Figure S7. CN-dependent tolerance and persistence is conserved in the CBS138 strain of C. glabrata . Strains were grown and treated with micafungin containing or lacking FK506 as described Fig 1 . Data points are the averages of four biological replicates and were used to generate the curve fits. Download figure Open in new tab Figure 7. Hog1 diminishes tolerance and persistence independent of calcineurin and Crz1. The hog1Δ crz1Δ double knockout mutant (blue), the single knockout mutants (green, red), and wild-type control strain (black) were analyzed in time-kill experiments as described in Fig. 1 with the effects of FK506 illustrated separately (right panel). Averages of four biological replicates were used to generate the curve fits. The CBS138 strain of C. glabrata utilizes calcineurin but not HOG signaling to regulate micafungin tolerance and persistence The CBS138 strain of C. glabrata naturally carries a mutation that inactivates the SSK2 component of the HOG signaling pathway ( 41 ). This mutation may be adaptive by providing enhanced tolerance and persistence, resulting in infections that are more difficult to treat. To begin investigating this possibility, time-kill experiments were performed on pbs2Δ , cna1Δ, cnb1Δ, and crz1Δ mutants in the CBS138 strain background. Unlike its effects in the BG14 strain background, the pbs2Δ mutation had no significant impact on tolerance and persistence in the CBS138 strain background, which exhibited somewhat elevated tolerance and persistence even in the presence of FK506 ( Fig. S8 ). The effects of cna1Δ and cnb1Δ , were conserved in CBS138, however the effects of crz1Δ were small relative to BG14 ( Fig. S9 ). The natural deficiency of HOG signaling and other polymorphisms in CBS138 may contribute to its enhanced antifungal tolerance. Interestingly, a recent survey of CBS138 and dozens of additional C. glabrata strains revealed considerable variation in the number of persister-like cells observed after micafungin exposure ( 30 ). Download figure Open in new tab Figure S8. The HOG pathway negatively regulates tolerance and persistence independent of calcineurin. Strains were grown as describe in Fig. 1 , treated with a higher dose of micafungin (1 µg/mL) containing or lacking FK506 (1 µg/mL), and sampled as previously described. Data points are the averages of four biological replicates and were used to generate the curve fits. Download figure Open in new tab Figure S9. CBS138-derived strains naturally lack HOG signaling. Wild-type CBS138-derived with natural ssk2- mutation and a pbs2Δ derivative were grown, treated with a high dose of micafungin (1 µg/mL) containing or lacking FK506 (1 µg/mL), and sampled as described in Fig. 1 . Data points are the averages of four biological replicates and were used to generate the curve fits. Calcineurin signaling promotes tolerance and persistence to micafungin, but not amphotericin B, in C. albicans To test whether calcineurin drives tolerance and persistence in C. albicans , micafungin time-kill experiments were performed on wild-type strain SC5314 in the presence and absence of FK506. Biphasic kinetics of cell death were observed in both scenarios while the loss of calcineurin signaling decreased the half-life of susceptible cells approximately 1.4-fold and decreased the number of persister-like cells approximately 100-fold ( Fig. 8 , black symbols). Similar results were obtained previously using cna1Δ/Δ mutants of C. albicans instead of FK506 ( 42 ). Curiously, the half-life of persister-like cells in C. albicans was much longer than that of C. glabrata , indicating that the two species may regulate the process in different ways. Download figure Open in new tab Figure 8. Calcineurin induces micafungin tolerance and persistence in C. albicans but has no impact on amphotericin B. Wild-type strains of C. albicans (left) and C. glabrata (right) were analyzed by time-kill experiments using 125 ng/mL micafungin (black lines) or 10 µg/mL amphotericin B (red lines) in the absence or presence of FK506 (dashed lines) as described in Fig. 1 . Averages of four biological replicates were used to generate the curve fits. Amphotericin B, which kills fungal cells by depleting ergosterol and creating pores in the plasma membrane ( 43 ), was also found to kill C. albicans and C. glabrata with biphasic kinetics after a brief lag ( Fig. 8 , red symbols). The half-life of the susceptible population was 2 to 2.5-fold shorter than observed with micafungin and, in both species, the killing kinetics were not altered by FK506 (dashed lines). These findings suggest that calcineurin specifically drives tolerance to echinocandins, but not amphotericin B in these species and conditions. DISCUSSION Though there is concern that tolerance and persistence will contribute to therapeutic failure, relapse, and perhaps even the acquisition of resistance ( 30 , 44 ), the regulatory mechanisms behind these phenomena have been understudied in fungal pathogens of humans. This study shows that calcineurin signaling is a major driver of tolerance and persistence to echinocandins in C. glabrata and C. albicans , in addition to driving resistance. Calcineurin and Crz1 produce mild resistance to echinocandins in C. glabrata by increasing expression of the drug target Fks2 ( 25 ). When strong resistance mutations in FKS2 arise in patients, calcineurin inhibitors greatly diminish their impact in vitro ( 25 , 39 ). Here we show that calcineurin signaling also increases tolerance and persistence largely independent of CRZ1, FKS2 , and RCN2 . Though the target of calcineurin that promotes tolerance and persistence has not yet been identified, the HOG signaling pathway was not required. FK506 still lowered tolerance and persistence in ssk2Δ , pbs2Δ , hog1Δ , and crz1Δ hog1Δ mutants. However, all these mutants still exhibited elevated tolerance and persistence in the absence of calcineurin signaling, again through unknown effectors ( Fig. 7 , Fig. S7 ). Interestingly, the HOG signaling pathway is naturally polymorphic in different strains of C. glabrata , contributing to variation in the resistance to micafungin ( 21 ) and other stressors ( 41 ), as well as variation in tolerance and persistence within the species. Multiple strains of C. glabrata may be needed to obtain a complete picture of resistance, tolerance, and persistence mechanisms in this diverse species. As calcineurin also increases echinocandin tolerance in C. albicans and A. fumigatus ( 45 , 46 ), the mechanism may be broadly conserved among diverse fungal pathogens. A recent study concluded that mitochondrial dysfunction regulates echinocandin tolerance in C. glabrata ( 47 ). However, that study did not make use of time-kill experiments and extensively utilized low-resolution dose-kill experiments, where CFU are quantified at a single time point (24 hr) after exposure to varying doses of echinocandins ( 23 ). The study also used “mitochondrial inhibitors” that either have multiple cellular targets (sodium azide) or have no targets at all in C. glabrata (diphenyleneiodonium chloride, rotenone) and did not match several mutants lacking mitochondrial functions. Nevertheless, after 24 hr exposure to high doses of caspofungin, several mitochondrial mutants produced wild-type CFU, consistent with our mitoribosome-deficient mrp20Δ mutant, which exhibited little or no changes in tolerance and persistence ( Fig. S4 ). Additionally, hundreds of mitochondrial gene deficiencies caused by transposon insertions exhibited near-zero tolerance/persistence Z-scores and strongly positive resistance Z-scores ( Fig. 2 ) due, in part, to their activation of Pdr1 signaling and elevated expression of lipid flippases that weakly increase resistance to most echinocandins ( 21 ). This study highlights the benefits of high resolution time-kill experiments are performed at supra-MIC doses and coupled with conventional mathematical modeling. A noteworthy observation of this study is that the estimated number of persister-like cells (i.e. persistence) correlated with the half-life of susceptible cells (i.e. tolerance) in all the mutant strains tested here. By increasing tolerance in the majority of cells in the population, calcineurin signaling may buy time for persister-like cells to form de novo through other regulatory processes. In the absence of calcineurin signaling, tolerance was low and persister-like cells were rare, but still readily detectable. When calcineurin was pre-activated by mutations or inhibitors that target the ER (e.g. manogepix), tolerance and persistence were elevated ( Fig. 4 ). The ability of FK506 to block these effects suggests that ongoing calcineurin signaling is necessary to maintain tolerance during micafungin exposure and enable persistence to arise. In addition to increasing micafungin tolerance and persistence independent of Crz1, manogepix increased micafungin resistance through Crz1-dependent processes ( Fig. 5 ). Manogepix, a fungistat that is currently in phase 2 clinical trials to treat a range of fungal infections ( 38 ), may therefore produce adverse drug-drug interactions with echinocandins. However, when combined with FK506, manogepix becomes fungicidal against C. glabrata and a wide range of fungal pathogens ( 36 , 48 ). The role of calcineurin in manogepix tolerance may overlap with previously studied calcineurin-dependent tolerance to tunicamycin and to azole-class antifungals, which target N-glycosylation and ergosterol biosynthesis in the ER, respectively ( 49 ). Other environmental conditions may effect calcineurin and HOG signaling pathways leading to impacts on echinocandin tolerance and persistence. Engulfment of C. glabrata cells into phagosomes of macrophages produced elevated micafungin tolerance and persistence ( 31 ). Tolerance to amphotericin B was also observed for engulfed C. glabrata cells ( 31 ). It will be interesting to determine whether calcineurin and/or HOG signaling govern these processes, or whether the longer lifespans of engulfed cells arise simply through slower growth rates or perhaps slower drug access to this intracellular compartment. Calcineurin signaling promotes virulence of C. glabrata ( 27 , 28 ) , C. albicans ( 42 , 50 , 51 ), and other fungal pathogens ( 45 , 52 – 54 ) in mouse models of systemic candidiasis. In C. glabrata , the effects of calcineurin were independent of Crz1 ( 27 , 28 ). A new interpretation of those findings is that host environments produce toxins or hostile conditions to the pathogens and generate stresses, which might normally activate calcineurin, induce tolerance and persistence behaviors, and promote fungal cell survival during infection. Manogepix treatment may further activate calcineurin and augment tolerance and persistence mechanisms, while still exerting a fungistatic effect. On the other hand, FK506 treatment may lower the defenses of C. glabrata and enable much more rapid and complete killing by host-derived assaults as well as echinocandins. Non-immunosuppressive analogs of FK506 that specifically target fungal calcineurin could be highly effective antifungals alone or in combination with existing antifungals ( 55 ). Though the immunosuppressive effects of FK506 would preclude long-term use of this compound as an antifungal co-drug, our findings raise the possibility that short-term or bolus co-administration of FK506 during standard echinocandin therapies could improve outcomes without producing sustained immunosuppression. In a mouse model of invasive candidiasis by C. albicans , the efficacy of fluconazole was increased by co-administration of FK506 ( 56 ). Tolerance to fluconazole and other inhibitors of lanosterol desaturase (Erg11) in C. glabrata , C. albicans , and other yeast species has long been known to depend on calcineurin signaling, but not Crz1 ( 27 , 28 , 49 , 52 ). Calcineurin also promoted tolerance to terbinafine in C. albicans ( 57 ), tunicamycin and dithiothreitol in S. cerevisiae ( 58 , 59 ), and to SDZ 90-215 in all these species ( 60 ). These compounds all block essential enzymes in the ER or Golgi complex of the fungal cells and are considered fungistatic in wild-type cells and fungicidal in calcineurin-deficient cells. Because the wild-type yeasts do not lose viability in the presence of these compounds, and often continue to replicate slowly for a few doublings, the degree of tolerance conferred by calcineurin signaling is difficult to quantify with the mathematical models employed in this study. The effectors of calcineurin responsible for tolerance to these fungistats remain unknown. Therefore, it is not yet possible to determine whether the same effectors govern tolerance to these fungistats as well as the fungicidal echinocandins. Our Tn-seq screen has produced dozens of candidate genes that could mediate the effects of calcineurin and potentially serve as targets for development of non-immunosuppressive strategies to increase the efficacies of our most important antifungals. Though calcineurin promoted tolerance to echinocandins and azoles, it did not seem to alter the biphasic kinetics of survival during exposure to amphotericin B in C. glabrata or C. albicans or S. cerevisiae ( 61 ). Amphotericin B kills growing and non-growing fungal cells by sponging essential ergosterols from the plasma membrane and generating lethal pores ( 43 ). The procedures outlined in this study could be adapted for studies of amphotericin B tolerance and persistence mechanisms. As new derivatives of amphotericin B with dramatically less animal toxicity are being developed ( 62 ), a more complete understanding of polyene resistance, tolerance, and persistence mechanisms in fungi could further increase their therapeutic index. METHODS Candida strains and plasmids All strains and their sources are listed in Table S2. C. glabrata strains were derived from BG14, a ura3Δ derivative of wild-type strain BG2 ( 63 ). Individual gene knockouts were constructed using the PRODIGE method ( 64 ) where coding sequences were replaced with the coding sequence of URA3 from S. cerevisiae plasmid pRS406 ( 65 ) using oligonucleotides listed in Table S3. Colony PCR was used for screening validation. The RCN2 gene was PCR amplified from genomic DNA of strain BG14 and cloned into the centromeric plasmid pCN-PDC1 ( 66 ) to generate pCN-PDC1-RCN2 and authenticated via Sanger sequencing. Antifungal Drugs Micafungin (Cat. #18009) and Amphoteracin B (Cat. #11636) were obtained from Cayman Chemicals, caspofungin (Cat. #S3073), tactrolimus (FK506) (Cat. #S5003), and mannogepix (E1210) (Cat. #S0491) from SelleckChem, and ibrexafungerp from Scynexis. TN-seq screen for genes that regulate micafungin tolerance and persistence Pool-3 of Hermes-NAT1 insertion mutants in strain BG14 that had been studied previously for resistance to echinocandins ( 21 ) was thawed from storage at −80°C, grown to saturation, then diluted into fresh SCD-0 medium containing 64 µg/mL micafungin. After 0 and 6 hr of shaking at 30°C, the culture was sampled, chilled on ice, washed once with chilled SCD-0 medium, resuspended in SCD-0, and shaken at 30°C for another 48 hr to allow regrowth of surviving cells. The cells were pelleted, washed, and then genomic DNA was extracted from using the Quick-DNA Fungal/Bacterial Miniprep Kit from Zymo Research (Cat. #D6005). gDNA was fragmented by sonication, repaired, ligated to adapters, size-selected, and then insertion sites were PCR amplified and sequenced on an Illumina MiSeq instrument as described previously ( 21 ). FastQ files were demultiplexed using CutAdapt ( 67 ) and aligned to the BG2v1 reference genome ( 68 ) using Bowtie2 ( 69 ). Mapped reads with low quality (Q<20) or mismatches at position 1 were discarded and the remainder were tabulated for each site in the genome. The total number of sequence reads in each annotated gene were tabulated and Z-scores were calculated for both time points with respect to the starting pool and with respect to each other as before ( 21 ). Gene Ontology analysis was performed on subsets of analyzed genes using GOrilla in S. cerevisiae mode ( 33 ). Time-kill assays and analyses At least four single colonies of each C. glabrata strain were picked and grown to saturation for 72 hours in SCD-0 media at 30°C. Microscopic observations indicated that the C. glabrata cells were monodispersed and not detectably clumped or aggregated. Each replicate culture was diluted 1:50 into fresh media containing antifungals, and shaken at 30°C. Aliquots were periodically removed, serially diluted in SCD-0 medium, and immediately plated onto SCD-0 agar medium using a sterile pinning device or by pipetting. Plates were incubated at 30°C until individual colonies were visible under a dissecting microscope at 10x magnification. Visible colonies were counted manually at one dilution for each sample and CFU per mL of undiluted culture was calculated. Replicates were averaged arithmetically and charted. For curve-fitting, the CFU were log transformed and fit to the equation y = log(m1*exp(-m2*x) + m3*exp(-m4*x)) by non-linear regression in Kaleidagraph (v5.04, Synergy) after exclusion of data points within the lag phase. Parameters m1 and m3 represent sizes of the non-persister and persister cell populations, respectively. Parameters m2 and m4 represent kill constants that were converted to half-lives (= ln2/m2, ln2/m4) of the two populations. Smooth curves were generated using these parameters and overlayed onto the CFU data points. The same procedure was applied for analysis of C. albicans except that YPD medium was used. Population Analysis Profiling (PAP) Assay Single colonies were grown to saturation for 72 hours in SCD-0 media at 30°C. The saturated cultures were diluted 1:50 into fresh SCD-0 media and then serially diluted 1:5. 2 µL of each dilution was spotted onto agar YPD media containing varying doses of micafungin (15 ng/mL to 0.234 ng/mL). Alternatively, when viability was very low, 200 µL of undiluted cultures were plated. Plates were incubated for 24 hours at 30°C and single colonies were counted manually. CFU per mL original culture was then calculated and replicates were averaged. Checkerboard Assay Varying concentrations of micafungin and mannogepix were prepared in SCD-0 and aliquoted to their respective columns and rows in a 96-well dish. Three biological replicates of BG14 and crz1Δ strain were growth to saturation and diluted 3:2000 in fresh SCD-0 media before being aliquoted into 96-well plates one-third of the final well-volume for 1:2000 cell dilution. The 96-well plates were incubated for 24 hours at 30°C, mixed, and optical density at 600 nm was measured (Accuris SmartReader 96T). Data Availability The authors affirm that all data necessary for confirming the conclusions of the article are present within the article, figures, tables, and repository. Raw sequencing reads used in this study were deposited at the NCBI Sequence Read Archive (SRA) with the BioProject ID PRJNA1304976. Micafungin resistance data ( 21 ) were obtained from SRA BioProject ID PRJNA1247003. A tabulation of the chromosomal coordinates and frequency of each mapped transposon insertion site (site count files) and a tabulation of the number of mapped transposon sites that fall within annotated gene boundaries (gene count files) are available upon request. ACKNOWLEDGMENTS The authors thank Dr. Winston Timp for providing access to DNA sequencing instruments and Drs. Joseph Heitman, Karl Kuchler, and Alejandro de las Peñas for providing C. glabrata and C. albicans strains, and Dr. Winston Timp for providing access to critical instruments. This research was supported by grants from the National Institutes of Health (T32-GM007231 to the Cell, Molecular, Developmental Biology, and Biophysics doctoral, training program; R01-AI153414 to KWC). REFERENCES 1. ↵ Kester JC , Fortune SM . 2014 . Persisters and beyond: mechanisms of phenotypic drug resistance and drug tolerance in bacteria . Crit Rev Biochem Mol Biol 49 : 91 – 101 . OpenUrl CrossRef PubMed 2. ↵ Brauner A , Fridman O , Gefen O , Balaban NQ . 2016 . Distinguishing between resistance, tolerance and persistence to antibiotic treatment . Nat Rev Microbiol 14 : 320 – 30 . OpenUrl CrossRef PubMed 3. ↵ Dewachter L , Fauvart M , Michiels J . 2019 . Bacterial Heterogeneity and Antibiotic Survival: Understanding and Combatting Persistence and Heteroresistance . Mol Cell 76 : 255 – 267 . OpenUrl CrossRef PubMed 4. ↵ Berman J , Krysan DJ . 2020 . Drug resistance and tolerance in fungi . Nat Rev Microbiol 18 : 319 – 331 . OpenUrl CrossRef PubMed 5. ↵ Lee Y , Robbins N , Cowen LE . 2023 . Molecular mechanisms governing antifungal drug resistance . NPJ Antimicrob Resist 1 : 5 . OpenUrl PubMed 6. ↵ Yang F , Berman J . 2024 . Beyond resistance: antifungal heteroresistance and antifungal tolerance in fungal pathogens . Curr Opin Microbiol 78 : 102439 . OpenUrl CrossRef PubMed 7. ↵ Ben-Ami R , Zimmerman O , Finn T , Amit S , Novikov A , Wertheimer N , Lurie-Weinberger M , Berman J. 2016 . Heteroresistance to Fluconazole Is a Continuously Distributed Phenotype among Candida glabrata Clinical Strains Associated with In Vivo Persistence . mBio 7 . 8. Murphy SE , Bicanic T . 2021 . Drug Resistance and Novel Therapeutic Approaches in Invasive Candidiasis . Front Cell Infect Microbiol 11 : 759408 . OpenUrl PubMed 9. Iyer KR , Revie NM , Fu C , Robbins N , Cowen LE . 2021 . Treatment strategies for cryptococcal infection: challenges, advances and future outlook . Nat Rev Microbiol 19 : 454 – 466 . OpenUrl CrossRef PubMed 10. Gautier C , Maciel EI , Ene IV . 2024 . Approaches for identifying and measuring heteroresistance in azole-susceptible Candida isolates . Microbiol Spectr 12 : e0404123 . OpenUrl 11. ↵ Shor E , Perlin DS , Kontoyiannis DP . 2025 . Tolerance and heteroresistance to echinocandins in Candida auris : conceptual issues, clinical implications, and outstanding questions . Msphere 10 . 12. ↵ Su Y , Li Y , Yi Q , Xu Y , Sun T , Li Y . 2025 . Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance . J Fungi (Basel) 11 . 13. ↵ Zhai B , Liao C , Jaggavarapu S , Tang Y , Rolling T , Ning Y , Sun T , Bergin SA , Gjonbalaj M , Miranda E , Babady NE , Bader O , Taur Y , Butler G , Zhang L , Xavier JB , Weiss DS , Hohl TM . 2024 . Antifungal heteroresistance causes prophylaxis failure and facilitates breakthrough Candida parapsilosis infections . Nat Med 30 : 3163 – 3172 . OpenUrl PubMed 14. ↵ Wu Y , Zou Y , Dai Y , Lu H , Zhang W , Chang W , Wang Y , Nie Z , Wang Y , Jiang X . 2025 . Adaptive morphological changes link to poor clinical outcomes by conferring echinocandin tolerance in Candida tropicalis . PLoS Pathog 21 : e1013220 . OpenUrl PubMed 15. ↵ Katsipoulaki M , Stappers MHT , Malavia-Jones D , Brunke S , Hube B , Gow NAR . 2024 . Candida albicans and Candida glabrata: global priority pathogens . Microbiol Mol Biol Rev 88 : e0002123 . OpenUrl CrossRef PubMed 16. Pfaller MA , Diekema DJ , Turnidge JD , Castanheira M , Jones RN . 2019 . Twenty Years of the SENTRY Antifungal Surveillance Program: Results for Candida Species From 1997-2016 . Open Forum Infect Dis 6 : S79 – S94 . OpenUrl CrossRef PubMed 17. ↵ Bays DJ , Jenkins EN , Lyman M , Chiller T , Strong N , Ostrosky-Zeichner L , Hoenigl M , Pappas PG , Thompson Iii GR . 2024 . Epidemiology of Invasive Candidiasis . Clin Epidemiol 16 : 549 – 566 . OpenUrl 18. ↵ Gabaldon T , Martin T , Marcet-Houben M , Durrens P , Bolotin-Fukuhara M , Lespinet O , Arnaise S , Boisnard S , Aguileta G , Atanasova R , Bouchier C , Couloux A , Creno S , Almeida Cruz J , Devillers H , Enache-Angoulvant A , Guitard J , Jaouen L , Ma L , Marck C , Neuveglise C , Pelletier E , Pinard A , Poulain J , Recoquillay J , Westhof E , Wincker P , Dujon B , Hennequin C , Fairhead C . 2013 . Comparative genomics of emerging pathogens in the Candida glabrata clade . BMC Genomics 14 : 623 . OpenUrl CrossRef PubMed 19. ↵ Fisher MC , Denning DW . 2023 . The WHO fungal priority pathogens list as a game-changer . Nat Rev Microbiol 21 : 211 – 212 . OpenUrl CrossRef PubMed 20. ↵ Whaley SG , Rogers PD . 2016 . Azole Resistance in Candida glabrata . Curr Infect Dis Rep 18 : 41 . OpenUrl CrossRef PubMed 21. ↵ Nickels TJ , Cunningham KW. 2025 . Tn-Seq screens in Candida glabrata treated with echinocandins and ibrexafungerp reveal pathways of antifungal resistance and cross-resistance mSphere submitted . 22. ↵ Nickels TJ , Gale AN , Harrington AA , Timp W , Cunningham KW . 2024 . Transposon-sequencing (Tn-seq) of the Candida glabrata reference strain CBS138 reveals epigenetic plasticity, structural variation, and intrinsic mechanisms of resistance to micafungin . G3 (Bethesda) 14 . 23. ↵ Healey KR , Perlin DS. 2018 . Fungal Resistance to Echinocandins and the MDR Phenomenon in Candida glabrata . J Fungi (Basel) 4 . 24. ↵ Ksiezopolska E , Schikora-Tamarit MA , Beyer R , Nunez-Rodriguez JC , Schuller C , Gabaldon T . 2021 . Narrow mutational signatures drive acquisition of multidrug resistance in the fungal pathogen Candida glabrata . Curr Biol 31 : 5314 – 5326 e10. OpenUrl CrossRef PubMed 25. ↵ Katiyar SK , Alastruey-Izquierdo A , Healey KR , Johnson ME , Perlin DS , Edlind TD . 2012 . Fks1 and Fks2 are functionally redundant but differentially regulated in Candida glabrata: implications for echinocandin resistance . Antimicrob Agents Chemother 56 : 6304 – 9 . OpenUrl Abstract / FREE Full Text 26. ↵ Singh-Babak SD , Babak T , Diezmann S , Hill JA , Xie JL , Chen YL , Poutanen SM , Rennie RP , Heitman J , Cowen LE . 2012 . Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata . PLoS Pathog 8 : e1002718 . OpenUrl CrossRef PubMed 27. ↵ Miyazaki T , Yamauchi S , Inamine T , Nagayoshi Y , Saijo T , Izumikawa K , Seki M , Kakeya H , Yamamoto Y , Yanagihara K , Miyazaki Y , Kohno S . 2010 . Roles of calcineurin and Crz1 in antifungal susceptibility and virulence of Candida glabrata . Antimicrob Agents Chemother 54 : 1639 – 43 . OpenUrl Abstract / FREE Full Text 28. ↵ Chen YL , Konieczka JH , Springer DJ , Bowen SE , Zhang J , Silao FG , Bungay AA , Bigol UG , Nicolas MG , Abraham SN , Thompson DA , Regev A , Heitman J . 2012 . Convergent Evolution of Calcineurin Pathway Roles in Thermotolerance and Virulence in Candida glabrata . G3 (Bethesda) 2 : 675 – 91 . OpenUrl CrossRef 29. ↵ Smith RP , Baltch A , Bopp LH , Ritz WJ , Michelsen PP . 2011 . Post-antifungal effects and time-kill studies of anidulafungin, caspofungin, and micafungin against Candida glabrata and Candida parapsilosis . Diagn Microbiol Infect Dis 71 : 131 – 8 . OpenUrl CrossRef PubMed 30. ↵ Arastehfar A , Daneshnia F , Floyd DJ , Jeffries NE , Salehi M , Perlin DS , Ilkit M , Lass-Floerl C , Mansour MK . 2024 . Echinocandin persistence directly impacts the evolution of resistance and survival of the pathogenic fungus Candida glabrata . mBio 15 : e0007224 . OpenUrl CrossRef PubMed 31. ↵ Arastehfar A , Daneshnia F , Cabrera N , Penalva-Lopez S , Sarathy J , Zimmerman M , Shor E , Perlin DS . 2023 . Macrophage internalization creates a multidrug-tolerant fungal persister reservoir and facilitates the emergence of drug resistance . Nat Commun 14 : 1183 . OpenUrl CrossRef PubMed 32. ↵ Gale AN , Pavesic MW , Nickels TJ , Xu Z , Cormack BP , Cunningham KW . 2023 . Redefining pleiotropic drug resistance in a pathogenic yeast: Pdr1 functions as a sensor of cellular stresses in Candida glabrata . mSphere doi: 10.1128/msphere.00254-23:e0025423 . OpenUrl CrossRef 33. ↵ Eden E , Navon R , Steinfeld I , Lipson D , Yakhini Z . 2009 . GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists . BMC Bioinformatics 10 : 48 . OpenUrl CrossRef PubMed 34. ↵ Stefan CP , Zhang N , Sokabe T , Rivetta A , Slayman CL , Montell C , Cunningham KW . 2013 . Activation of an essential calcium signaling pathway in Saccharomyces cerevisiae by Kch1 and Kch2, putative low-affinity potassium transporters . Eukaryot Cell 12 : 204 – 14 . OpenUrl Abstract / FREE Full Text 35. ↵ Stefan CP , Cunningham KW . 2013 . Kch1 family proteins mediate essential responses to endoplasmic reticulum stresses in the yeasts Saccharomyces cerevisiae and Candida albicans . J Biol Chem 288 : 34861 – 70 . OpenUrl Abstract / FREE Full Text 36. ↵ Pavesic MW , Gale AN , Nickels TJ , Harrington AA , Bussey M , Cunningham KW . 2024 . Calcineurin-dependent contributions to fitness in the opportunistic pathogen Candida glabrata . mSphere 9 : e0055423 . OpenUrl CrossRef PubMed 37. ↵ Miyazaki T , Izumikawa K , Nagayoshi Y , Saijo T , Yamauchi S , Morinaga Y , Seki M , Kakeya H , Yamamoto Y , Yanagihara K , Miyazaki Y , Kohno S . 2011 . Functional characterization of the regulators of calcineurin in Candida glabrata . FEMS Yeast Res 11 : 621 – 30 . OpenUrl CrossRef PubMed Web of Science 38. ↵ Hodges MR , Ople E , Wedel P , Shaw KJ , Jakate A , Kramer WG , Marle SV , van Hoogdalem EJ , Tawadrous M. 2023 . Safety and Pharmacokinetics of Intravenous and Oral Fosmanogepix, a First-in-Class Antifungal Agent, in Healthy Volunteers . Antimicrob Agents Chemother 67 : e0162322 . OpenUrl PubMed 39. ↵ Healey KR , Paderu P , Hou X , Jimenez Ortigosa C , Bagley N , Patel B , Zhao Y , Perlin DS . 2020 . Differential Regulation of Echinocandin Targets Fks1 and Fks2 in Candida glabrata by the Post-Transcriptional Regulator Ssd1 . J Fungi (Basel) 6 . 40. ↵ Gonzalez-Jimenez I , Keniya MV , Aptekmann AA , Quinteros C , Wilkerson A , Arastehfar A , Daneshnia F , Perlin DS , Shor E . 2025 . Expression of 1,3-beta-glucan synthase subunits in Candida glabrata is regulated by the cell cycle and growth conditions and at both transcriptional and post-transcriptional levels . Antimicrob Agents Chemother 69 : e0050025 . OpenUrl PubMed 41. ↵ Gregori C , Schuller C , Roetzer A , Schwarzmuller T , Ammerer G , Kuchler K . 2007 . The high-osmolarity glycerol response pathway in the human fungal pathogen Candida glabrata strain ATCC 2001 lacks a signaling branch that operates in baker’s yeast . Eukaryot Cell 6 : 1635 – 45 . OpenUrl Abstract / FREE Full Text 42. ↵ Sanglard D , Ischer F , Marchetti O , Entenza J , Bille J . 2003 . Calcineurin A of Candida albicans:involvement in antifungal tolerance, cell morphogenesis and virulence . Mol Microbiol 48 : 959 – 76 . OpenUrl CrossRef PubMed Web of Science 43. ↵ Anderson TM , Clay MC , Cioffi AG , Diaz KA , Hisao GS , Tuttle MD , Nieuwkoop AJ , Comellas G , Maryum N , Wang S , Uno BE , Wildeman EL , Gonen T , Rienstra CM , Burke MD . 2014 . Amphotericin forms an extramembranous and fungicidal sterol sponge . Nat Chem Biol 10 : 400 – 6 . OpenUrl CrossRef PubMed 44. ↵ Wuyts J , Van Dijck P , Holtappels M. 2018 . Fungal persister cells: The basis for recalcitrant infections? PLoS Pathog 14 : e1007301 . OpenUrl CrossRef PubMed 45. ↵ Steinbach WJ , Cramer RA , Jr. , Perfect BZ , Henn C , Nielsen K , Heitman J , Perfect JR . 2007 . Calcineurin inhibition or mutation enhances cell wall inhibitors against Aspergillus fumigatus . Antimicrob Agents Chemother 51 : 2979 – 81 . OpenUrl Abstract / FREE Full Text 46. ↵ Fortwendel JR , Juvvadi PR , Perfect BZ , Rogg LE , Perfect JR , Steinbach WJ . 2010 . Transcriptional regulation of chitin synthases by calcineurin controls paradoxical growth of Aspergillus fumigatus in response to caspofungin . Antimicrob Agents Chemother 54 : 1555 – 63 . OpenUrl Abstract / FREE Full Text 47. ↵ Garcia-Rubio R , Jimenez-Ortigosa C , DeGregorio L , Quinteros C , Shor E , Perlin DS . 2021 . Multifactorial Role of Mitochondria in Echinocandin Tolerance Revealed by Transcriptome Analysis of Drug-Tolerant Cells . mBio 12 : e0195921 . OpenUrl PubMed 48. ↵ Liston SD , Whitesell L , Kapoor M , Shaw KJ , Cowen LE . 2022 . Calcineurin Inhibitors Synergize with Manogepix to Kill Diverse Human Fungal Pathogens . J Fungi (Basel) 8 . 49. ↵ Marchetti O , Moreillon P , Glauser MP , Bille J , Sanglard D . 2000 . Potent synergism of the combination of fluconazole and cyclosporine in Candida albicans . Antimicrob Agents Chemother 44 : 2373 – 81 . OpenUrl Abstract / FREE Full Text 50. ↵ Bader T , Bodendorfer B , Schroppel K , Morschhauser J . 2003 . Calcineurin is essential for virulence in Candida albicans . Infect Immun 71 : 5344 – 54 . OpenUrl Abstract / FREE Full Text 51. ↵ Blankenship JR , Wormley FL , Boyce MK , Schell WA , Filler SG , Perfect JR , Heitman J . 2003 . Calcineurin is essential for Candida albicans survival in serum and virulence . Eukaryot Cell 2 : 422 – 30 . OpenUrl Abstract / FREE Full Text 52. ↵ Chen YL , Brand A , Morrison EL , Silao FG , Bigol UG , Malbas FF , Jr. , Nett JE , Andes DR , Solis NV , Filler SG , Averette A , Heitman J . 2011 . Calcineurin controls drug tolerance, hyphal growth, and virulence in Candida dubliniensis . Eukaryot Cell 10 : 803 – 19 . OpenUrl Abstract / FREE Full Text 53. Chen YL , Yu SJ , Huang HY , Chang YL , Lehman VN , Silao FG , Bigol UG , Bungay AA , Averette A , Heitman J . 2014 . Calcineurin controls hyphal growth, virulence, and drug tolerance of Candida tropicalis . Eukaryot Cell 13 : 844 – 54 . OpenUrl Abstract / FREE Full Text 54. ↵ Odom A , Muir S , Lim E , Toffaletti DL , Perfect J , Heitman J . 1997 . Calcineurin is required for virulence of Cryptococcus neoformans . Embo J 16 : 2576 – 89 . OpenUrl Abstract / FREE Full Text 55. ↵ Yadav V , Heitman J . 2023 . Calcineurin: The Achilles’ heel of fungal pathogens . PLoS Pathog 19 : e1011445 . OpenUrl CrossRef PubMed 56. ↵ Marchetti O , Entenza JM , Sanglard D , Bille J , Glauser MP , Moreillon P . 2000 . Fluconazole plus cyclosporine: a fungicidal combination effective against experimental endocarditis due to Candida albicans . Antimicrob Agents Chemother 44 : 2932 – 8 . OpenUrl Abstract / FREE Full Text 57. ↵ Karababa M , Valentino E , Pardini G , Coste AT , Bille J , Sanglard D . 2006 . CRZ1, a target of the calcineurin pathway in Candida albicans . Mol Microbiol 59 : 1429 – 51 . OpenUrl CrossRef PubMed Web of Science 58. ↵ Bonilla M , Nastase KK , Cunningham KW . 2002 . Essential role of calcineurin in response to endoplasmic reticulum stress . Embo J 21 : 2343 – 2353 . OpenUrl Abstract / FREE Full Text 59. ↵ Kim H , Kim A , Cunningham KW . 2012 . Vacuolar H+-ATPase (V-ATPase) Promotes Vacuolar Membrane Permeabilization and Nonapoptotic Death in Stressed Yeast . J Biol Chem 287 : 19029 – 39 . OpenUrl Abstract / FREE Full Text 60. ↵ Snyder NA , Kim A , Kester L , Gale AN , Studer C , Hoepfner D , Roggo S , Helliwell SB , Cunningham KW . 2019 . Auxin-Inducible Depletion of the Essentialome Suggests Inhibition of TORC1 by Auxins and Inhibition of Vrg4 by SDZ 90-215, a Natural Antifungal Cyclopeptide . G3 (Bethesda) 9 : 829 – 840 . OpenUrl Abstract / FREE Full Text 61. ↵ Bojsen R , Regenberg B , Gresham D , Folkesson A . 2016 . A common mechanism involving the TORC1 pathway can lead to amphotericin B-persistence in biofilm and planktonic Saccharomyces cerevisiae populations . Sci Rep 6 : 21874 . OpenUrl CrossRef PubMed 62. ↵ Maji A , Soutar CP , Zhang J , Lewandowska A , Uno BE , Yan S , Shelke Y , Murhade G , Nimerovsky E , Borcik CG , Arango AS , Lange JD , Marin-Toledo JP , Lyu Y , Bailey KL , Roady PJ , Holler JT , Khandelwal A , SantaMaria AM , Sanchez H , Juvvadi PR , Johns G , Hageman MJ , Krise J , Gebremariam T , Youssef EG , Bartizal K , Marr KA , Steinbach WJ , Ibrahim AS , Patterson TF , Wiederhold NP , Andes DR , Pogorelov TV , Schwieters CD , Fan TM , Rienstra CM , Burke MD . 2023 . Tuning sterol extraction kinetics yields a renal-sparing polyene antifungal . Nature 623 : 1079 – 1085 . OpenUrl CrossRef PubMed 63. ↵ Cormack BP , Falkow S . 1999 . Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata . Genetics 151 : 979 – 87 . OpenUrl Abstract / FREE Full Text 64. ↵ Edlind TD , Henry KW , Vermitsky JP , Edlind MP , Raj S , Katiyar SK . 2005 . Promoter-dependent disruption of genes: simple, rapid, and specific PCR-based method with application to three different yeast . Curr Genet 48 : 117 – 25 . OpenUrl CrossRef PubMed 65. ↵ Sikorski RS , Hieter P . 1989 . A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae . Genetics 122 : 19 – 27 . OpenUrl Abstract / FREE Full Text 66. ↵ Zordan RE , Ren Y , Pan SJ , Rotondo G , De Las Penas A , Iluore J , Cormack BP. 2013 . Expression plasmids for use in Candida glabrata . G3 (Bethesda) 3 : 1675 – 86 . OpenUrl CrossRef 67. ↵ Martin M . 2011 . Cutadapt removes adapter sequences from high-throughput sequencing reads . EMBnetjournal 17 : 3 . OpenUrl 68. ↵ Xu Z , Green B , Benoit N , Sobel JD , Schatz MC , Wheelan S , Cormack BP . 2021 . Cell wall protein variation, break-induced replication, and subtelomere dynamics in Candida glabrata . Mol Microbiol 116 : 260 – 276 . OpenUrl PubMed 69. ↵ Langmead B , Salzberg SL . 2012 . Fast gapped-read alignment with Bowtie 2 . Nat Methods 9 : 357 – 9 . OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted August 19, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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