An acquired mechanism of antifungal drug resistance simultaneously enables Candida albicans to escape from intrinsic host defenses

The opportunistic fungal pathogen Candida albicans frequently produces genetically altered variants to adapt to environmental changes and new host niches in the course of its life-long association with the human host. Gain-of-function mutations in zinc cluster transcription factors, which result in the constitutive upregulation of their target genes, are a common cause of acquired resistance to the widely used antifungal drug fluconazole, especially during long-term therapy of oropharyngeal candidiasis. In this study, we investigated if C. albicans also can develop resistance to the antimicrobial peptide histatin 5, which is secreted in the saliva of humans to protect the oral mucosa from pathogenic microbes. As histatin 5 has been shown to be transported out of C. albicans cells by the Flu1 efflux pump, we screened a library of C. albicans strains that contain artificially activated forms of all zinc cluster transcription factors of this fungus for increased FLU1 expression. We found that a hyperactive Mrr1, which confers fluconazole resistance by upregulating the multidrug efflux pump MDR1 and other genes, also causes FLU1 overexpression. Similarly to the artificially activated Mrr1, naturally occurring gain-of-function mutations in this transcription factor also caused FLU1 upregulation and increased histatin 5 resistance. Surprisingly, however, Mrr1-mediated histatin 5 resistance was mainly caused by the upregulation of MDR1 instead of FLU1, revealing a previously unrecognized function of the Mdr1 efflux pump. Fluconazole-resistant clinical C. albicans isolates with different Mrr1 gain-of-function mutations were less efficiently killed by histatin 5, and this phenotype was reverted when MRR1 was deleted. Therefore, antimycotic therapy can promote the evolution of strains that, as a consequence of drug resistance mutations, simultaneously have acquired increased resistance against an innate host defense mechanism and are thereby better adapted to certain host niches.


Introduction
The yeast Candida albicans is a member of the microbiota of the oral cavity and the gastrointestinal and genitourinary tracts in most healthy persons. When host defenses are compromised, C. albicans can also cause symptomatic infections, which range from superficial skin or mucosal infections to life-threatening, disseminated infections. During both colonization and infection, C. albicans must adapt to environmental changes and stressful conditions encountered in its various host niches. To a large extent this is achieved by reversibly regulating gene expression and biochemical activities according to cellular needs [1,2]. In addition, C. albicans also produces genetically altered variants that are better adapted than the originally colonizing strain to long-lasting changes in its habitat or a new host niche [3][4][5]. The generation of such variants is facilitated by the high genomic plasticity of this diploid fungus, which often leads to the amplification or loss of partial or whole chromosomes, especially in response to stress [6]. The increased or decreased copy number of genes that are located on the affected chromosomes of the resulting aneuploid cells may confer a selective advantage under certain adverse conditions [7,8]. Aneuploidies are unstable, and cells can revert to the normal diploid state by chromosome loss or reduplication in the absence of selection pressure [9]. Genetic variants may also arise by simple point mutations that enable the cells to better tolerate harmful conditions. The acquisition of advantageous mutations is frequently followed by loss of heterozygosity for the mutated allele, which can occur by mitotic recombination or loss of the chromosome containing the wild-type allele [10][11][12][13][14][15][16][17][18]. These events are promoted in a stressful environment and further enhance the effect of the mutations [19,20].
A well-documented example of such microevolution within the host is the development of antifungal drug resistance during therapy [21]. Infections by C. albicans are commonly treated with fluconazole, which inhibits the biosynthesis of ergosterol, the main sterol in fungal cell membranes. Mutations in the drug target enzyme sterol 14α-demethylase, encoded by ERG11, result in amino acid exchanges that reduce the affinity of the enzyme for the drug [22]. Similarly, mutations in FKS1, encoding β-1,3-glucan synthase, the target of echinocandin drugs, also result in reduced drug binding [23]. Other point mutations that cause increased drug resistance affect transcription factors and result in permanently changed gene expression programs. The transcription factor Upc2 regulates the expression of ERG11 and other ergosterol biosynthesis genes [24,25]. Gain-of-function (GOF) mutations in Upc2 that result in hyperactivity of the transcription factor cause constitutive upregulation of its target genes and increased fluconazole resistance [15,[26][27][28]. Similarly, GOF mutations in the transcription factors Mrr1 and Tac1, which regulate the expression of the multidrug efflux pumps MDR1 and CDR1/CDR2, respectively, result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates [11-13, 16, 29-31].
Mrr1, Tac1, and Upc2 belong to the zinc cluster transcription factor family, which is unique to the fungal kingdom and characterized by a well-conserved DNA-binding motif containing six cysteine residues that coordinate two zinc atoms [32]. C. albicans possesses 82 predicted zinc cluster transcription factors, which are involved in the regulation of diverse cellular processes, although the functions of many of them have not yet been studied in detail [33,34]. It is conceivable that GOF mutations like those found in Mrr1, Tac1, and Upc2 may also occur in other members of the family and confer new phenotypes that are advantageous under adverse conditions encountered in some host niches. As many transcription factors are activated in response to specific signals and are often not active under standard growth conditions, deletion of the corresponding genes does not necessarily result in an obvious phenotype when the conditions in which they are required are not known. This was the case for Mrr1 and Tac1, whose ability to confer drug resistance was only uncovered by the identification of GOF mutations in fluconazole-resistant clinical isolates [16,30]. The availability of hyperactive alleles of zinc cluster transcription factors may therefore reveal their biological function and also predict the potential of C. albicans to generate variants with novel phenotypes by acquiring GOF mutations in these transcriptional regulators. Since it cannot be generally predicted which mutations would render a wild-type transcription factor hyperactive, we recently established a method for the artificial activation of zinc cluster proteins by C-terminal fusion with the heterologous Gal4 activation domain and generated a library of C. albicans strains expressing all zinc cluster transcription factors of this fungus in a potentially hyperactive form [34]. Screening of this library showed that one of the artificially activated transcription factors, which was thereafter termed Mrr2, conferred fluconazole resistance by upregulation of the major C. albicans multidrug efflux pump CDR1 [34]. Based on these findings, other investigators searched for naturally occurring gain-of-function mutations in the MRR2 gene in a collection of fluconazole-resistant C. albicans isolates. Indeed, three epidemiologically related isolates with elevated CDR1 expression levels contained a mutated MRR2 allele that caused CDR1 overexpression and increased fluconazole resistance when introduced into a drug-susceptible strain, demonstrating the clinical relevance of the predicted resistance mechanism [35].
In addition to antifungal drugs, which are introduced by medical treatment, C. albicans encounters many other harmful molecules within its host, which may be taken up with the diet, generated by other members of the microbiota, or produced by the host as a defense mechanism against invading pathogens. Humans secrete saliva containing different antimicrobial peptides, including histatins, in order to protect the oral mucosa from pathogenic microbes. Histatins have strong antifungal activity, with histatin 5 (Hst 5) exhibiting the most potent fungicidal activity against C. albicans and other Candida species [36]. Hst 5 is an unusual antimicrobial peptide in that it is not membrane-lytic but rather acts intracellularly to cause cell death [36]. C. albicans possesses several mechanisms to evade killing by Hst 5 and thereby can tolerate the presence of low levels of this antimicrobial peptide. The extracellular glycodomain of the plasma membrane protein Msb2 is shed into the environment and binds Hst 5 as well as other antimicrobial peptides, thereby protecting C. albicans from their action, and extracellular Hst 5 is also proteolytically degraded by secreted aspartic proteases of the fungus [37][38][39]. Furthermore, C. albicans can recover from stresses generated by intracellular Hst 5 by mechanisms that are mediated by MAP kinases [40]. As Hst 5 acts within the cells, preventing its intracellular accumulation is another potential resistance mechanism. It was recently shown that the Flu1 efflux pump transports Hst 5 out of the cells and that mutants lacking FLU1 are hypersusceptible to killing by Hst 5 [41]. We reasoned that FLU1 expression, like that of the drug efflux pumps MDR1, CDR1, and CDR2, might also be regulated by a zinc cluster protein and that C. albicans might acquire Hst 5 resistance by GOF mutations in this transcription factor. We, therefore, set out to identify transcription factors that regulate FLU1 expression and investigate whether C. albicans can develop increased Hst 5 resistance by this mechanism.

Identification of zinc cluster transcription factors that regulate FLU1 expression
If FLU1, like other known C. albicans efflux pumps, is regulated by a zinc cluster protein, a hyperactive form of this transcription factor should cause constitutive FLU1 overexpression and, consequently, increased Hst 5 resistance. flu1Δ mutants not only exhibit increased susceptibility to Hst 5 but are also hypersensitive to mycophenolic acid (MPA), a phenotype that is easily recognizable on agar plates containing a suitable concentration of the drug [42]. We therefore screened our library of C. albicans strains expressing artificially activated forms of all zinc cluster transcription factors for increased MPA resistance. In a preliminary test, the strains were directly transferred from our stock collection in microtiter plates onto agar plates with and without MPA. Candidate strains that grew better than the wild-type control in the presence of the inhibitor were then retested in a more sensitive dilution spot assay. This resulted in the identification of four hyperactive transcription factors (Mrr1, Mrr2, War1, and Zcf35) that caused clearly increased MPA resistance (Fig 1A).
MPA resistance might be brought about by different mechanisms, for example by upregulation of the IMH3 gene, which encodes inosine monophosphate dehydrogenase, the target enzyme of MPA [43]. To investigate if the increased MPA resistance of our strains was caused by FLU1 overexpression, we introduced the artificially activated transcription factors into reporter strains containing GFP under the control of the FLU1 promoter. As can be seen in Fig  1B, basal FLU1 expression levels were detectable with GFP as a reporter gene, because the fluorescence of the wild-type reporter strains was well above background fluorescence. Intriguingly, FLU1 promoter activity was increased by all four hyperactive transcription factors (3-to 6-fold), indicating that Mrr1, Mrr2, War1, and Zcf35 regulate FLU1 expression and that their activation results in overexpression of this efflux pump.
We next tested if the increased MPA resistance conferred by hyperactive forms of Mrr1, Mrr2, War1, and Zcf35 depended on Flu1. For this purpose, we generated two independent series of flu1Δ mutants and complemented strains of the wild-type strain SC5314. In accord with a previous report [42], homozygous flu1Δ mutants were hypersusceptible to MPA, and reintroduction of a functional FLU1 copy into these mutants increased their MPA resistance to the level observed for heterozygous mutants (S1 Fig). The genes encoding the hyperactive transcription factors were then introduced into the homozygous flu1Δ mutants and the MPA susceptibilities of the resulting strains compared with those of the corresponding wild-type strains. Fig 2 shows that none of the hyperactive transcription factors caused a noticeable increase in MPA resistance in the absence of Flu1, indicating that this effect on the phenotype of the cells was entirely Flu1-dependent.
Although FLU1 overexpression in the heterologous host Saccharomyces cerevisiae had previously been found to result in elevated fluconazole resistance (hence the name given to the gene), deletion of FLU1 in a C. albicans laboratory strain had little effect on fluconazole susceptibility [42]. In line with these findings, we did not observe a detectable increase in fluconazole susceptibility of the flu1Δ mutants derived from the wild-type strain SC5314, neither in dilution spot assays on agar plates nor when we determined the minimal inhibitory concentration of fluconazole in a broth microdilution assay (Fig 2). Hyperactive forms of Mrr1, Mrr2, and Zcf35 cause increased fluconazole resistance [34]. For Mrr1 and Mrr2, this was also evident in the dilution spot assays on agar plates containing a defined concentration of fluconazole, whereas the strains with the hyperactive Zcf35 showed even slightly reduced growth on the fluconazole plates, despite the elevated MIC for these strains (Fig 2). The strains containing the hyperactive War1 also showed slightly reduced growth on the fluconazole agar plates, but exhibited increased fluconazole resistance when tested in the MIC assays, a phenotype that was not observed for these strains in our previous study, presumably because a different assay medium was used. The increased fluconazole resistance conferred by the four hyperactive transcription factors was also observed when they were expressed in flu1Δ mutants (Fig 2), demonstrating that FLU1 overexpression did not contribute to the fluconazole-resistant phenotype.
Although FLU1 mediates Hst 5 resistance, its expression is not induced by Hst 5 [41]. Therefore, we investigated if basal FLU1 expression levels depend on any of the identified transcription factors. For this purpose, the P FLU1 -GFP reporter fusion was introduced into mrr1Δ, mrr2Δ, war1Δ, and zcf35Δ mutants. S2 Fig shows that all mutants displayed wild-type FLU1 promoter activity, indicating that none of these transcription factors is required for basal FLU1 expression.

A hyperactive Mrr1 confers increased Hst 5 resistance
As hyperactive forms of Mrr1, Mrr2, War1, and Zcf35 cause FLU1 upregulation, we reasoned that they should also mediate increased resistance to Hst 5. Therefore, we compared the percent killing of the wild-type parental strain SC5314 and derivatives containing the artificially activated transcription factors after 60 min of incubation in the presence of various Hst 5 concentrations (Fig 3). The hyperactive Mrr1 indeed conferred increased resistance to the antimicrobial peptide; in the presence of 30 μM Hst 5, killing was reduced from 80% for the wild type to ca. 54% for strains containing the artificially activated Mrr1. In contrast, strains with the hyperactive Mrr2 and War1 were as efficiently killed by Hst 5 as the wild type, and the strains with the hyperactive Zcf35 showed even enhanced sensitivity. Therefore, despite the increased FLU1 expression levels, artificially activated Mrr2, War1, and Zcf35 were unable to confer Hst 5 resistance. We speculated that these hyperactive transcription factors have additional effects on the cells that increase their susceptibility to Hst 5 and thereby abrogate any advantage conferred by FLU1 overexpression. However, subsequent experiments provided a different explanation for this result (see below).

Naturally occurring GOF mutations in Mrr1 cause FLU1 overexpression and increased Hst 5 resistance
Our finding that an artificially activated form of Mrr1 causes increased Hst 5 resistance was particularly intriguing, since many fluconazole-resistant clinical C. albicans isolates contain GOF mutations in Mrr1 [13,16,44]. Such mutations are selected under antifungal therapy, because the hyperactive transcription factor mediates overexpression of the multidrug efflux pump MDR1 and other genes, and thereby confers fluconazole resistance. We investigated if (ZCF35-GAD). Control strains without GFP: SC5314 (Wild type), SCMRR1GAD1A and -B (MRR1-GAD), SCZCF34GAD1A and -B (MRR2-GAD), SCWAR1GAD1A and -B (WAR1-GAD), SCZCF35GAD1A and -B (ZCF35-GAD). Significant differences from the wild-type control (background-subtracted values) are marked with asterisks (*** P < 0.001; ANOVA). https://doi.org/10.1371/journal.ppat.1006655.g001 Simultaneous acquisition of drug and antifungal peptide resistance by C. albicans such naturally occurring Mrr1 GOF mutations also cause FLU1 overexpression and enhanced Hst 5 resistance. Mrr1 GOF mutations have a much stronger effect on MDR1 expression and fluconazole resistance after loss of heterozygosity for the mutated MRR1 allele [16,45], and we reasoned that the same would be true for any effect on FLU1 expression and Hst 5 resistance.

Fig 3. A hyperactive Mrr1 causes increased Hst 5 resistance.
The wild-type strain SC5314 (WT, black bars) and derivatives (gray bars) containing artificially activated MRR1 (SCMRR1GAD1A and -B), MRR2 (SCZCF34GAD1A and -B), WAR1 (SCWAR1GAD1A and -B), or ZCF35 (SCZCF35GAD1A and -B) were exposed to Hst 5 (3.75 μM to 30 μM) using microdilution plate assays, and the percent killing was calculated as described in Materials and Methods. The four transcription factors were tested on separate occasions, and a different batch of Hst 5 was used for MRR1 and ZCF35 vs. MRR2 and WAR1. Significant differences from the wild-type control (t-test) are marked with asterisks (* P < 0.05, ** P < 0.01, *** P < 0.001); n.s., not significant (P > 0.05). https://doi.org/10.1371/journal.ppat.1006655.g003 Simultaneous acquisition of drug and antifungal peptide resistance by C. albicans scenario, we also constructed strains containing a combination of these hyperactive alleles. In addition, strains in which the endogenous MRR1 alleles were replaced in the same fashion by an unmutated wild-type allele were included in the experiments to make sure that the sequential allele replacement strategy alone did not have a phenotypic effect.
We first tested the fluconazole susceptibilities of the strains ( Table 1). As previously reported [20], replacement of the endogenous MRR1 alleles of strain SC5314 by a nonmutated MRR1 copy did not affect drug susceptibility, whereas the P683S mutation caused a 32-fold increase in the MIC of fluconazole (from 0.5 μg/ml to 16 μg/ml). A similar effect was observed for all other GOF mutations, which raised the MIC to 16 or 32 μg/ml. In each case, identical results were obtained for two independently constructed strains. We then introduced the P FLU1 -GFP reporter fusion into the strains with the different MRR1 GOF mutations to determine their effect on FLU1 expression. Fig 4A shows that all hyperactive MRR1 alleles caused elevated FLU1 expression levels, albeit to various degrees. The strongest upregulation (ca. 7-fold) was mediated by the N803D and T360I mutations, and the weakest effect (2-to 3-fold upregulation) was observed for the P683S and T896I mutations. The differences were reproducible, because in all cases the two independently constructed reporter strains exhibited similar fluorescence values. These results demonstrated that the overexpression of FLU1 by a hyperactive Mrr1 is not a peculiar effect of the artificially generated Mrr1-GAD fusion but a general characteristic of naturally occurring activated forms of Mrr1.
We selected six different Mrr1 GOF mutations (G997V, G878E, Q350L, N803D, T360I, T896I) to investigate if they also resulted in increased Hst 5 resistance. As can be seen in Fig  4B, all strains containing hyperactive MRR1 alleles were less efficiently killed by Hst 5 than the parental wild-type strain SC5314 (killing decreased from ca. 90% to ca. 60%), demonstrating that naturally occurring Mrr1 GOF mutations conferred increased Hst 5 resistance. In a further control experiment we confirmed that the strains in which the endogenous MRR1 alleles were replaced by a nonmutated wild-type copy exhibited wild-type Hst 5 susceptibility, as expected (S3 Fig).

Mrr1-mediated Hst 5 resistance does not depend on Flu1
Despite the fact that hyperactive forms of Mrr2, War1, and Zcf35 caused even stronger FLU1 upregulation than the artificially activated Mrr1 (see Fig 1B), only the latter enhanced the resistance of the cells to Hst 5 (Fig 3). We therefore investigated if the increased Hst 5 resistance mediated by a hyperactive Mrr1 was indeed caused by FLU1 overexpression. For this purpose, we compared the percent killing by Hst 5 of wild-type and flu1Δ cells with and without the Simultaneous acquisition of drug and antifungal peptide resistance by C. albicans artificially activated MRR1 allele (Fig 5). Unexpectedly, deletion of FLU1 in the prototrophic wild-type strain SC5314 did not result in hypersensitivity to Hst 5, in contrast to previous observations with the auxotrophic laboratory strain CAF4-2 [41]. Even more surprisingly, the hyperactive Mrr1 reduced the susceptibility of the cells to killing by Hst 5 both in the presence and absence of FLU1 with comparable efficiency (at 30 μM Hst 5, the flu1Δ mutants were even slightly more resistant in this experiment). Therefore, Mrr1-mediated Hst 5 resistance must be caused by other mechanisms, which might also mask any additional contribution of FLU1 overexpression in this strain background.
Overexpression of the multidrug efflux pump MDR1 contributes to Mrr1-mediated Hst 5 resistance As hyperactive forms of Mrr1 cause overexpression of many genes [16,45], we searched the known Mrr1 target genes for candidates that might promote Hst 5 resistance. TPO2 encodes another putative efflux pump that is closely related to Flu1 [41], and it is bound and upregulated by a hyperactive Mrr1 [45]. Mdr1 also has high similarity to Flu1 [42], and MDR1 is one of the most strongly upregulated genes in strains with Mrr1 GOF mutations [16,45]. Although no role of TPO2 and MDR1 in Hst 5 resistance had been found in a previous study [41], it seemed possible that these Mrr1 target genes could mediate Hst 5 resistance when they are overexpressed. Northern hybridization experiments showed that both MDR1 and TPO2 are expressed only at low levels in the wild-type strain SC5314 (Fig 6, left lanes). Interestingly, only Mrr1, but not the other artificially activated transcription factors, Mrr2, War1, and Zcf35, caused upregulation of MDR1 and, more weakly, TPO2 (Fig 6A), providing a potential explanation why the latter did not cause Hst 5 resistance. Most naturally occurring GOF mutations in Mrr1 had an even stronger effect on MDR1 and TPO2 expression than the artificially activated Mrr1, although expression levels strongly depended on the specific allele ( Fig 6B). As FLU1, MDR1, and TPO2 all are upregulated by a hyperactive Mrr1, it was conceivable that each of these three related transporters contributes to Mrr1-mediated Hst 5 resistance and the importance of a single efflux pump would not be easily detectable. We therefore constructed flu1Δ mdr1Δ tpo2Δ triple mutants and introduced the G997V GOF mutation, which caused an efficient upregulation of all three genes (see Fig 4A and Fig 6B), into both MRR1 alleles of the mutants. The deletion of the transporters in the wild-type parental strain, in which they were not or only weakly expressed, did not result in hypersusceptibility to Hst 5 (S4 Fig). However, the increased Hst 5 resistance conferred by the hyperactive Mrr1 was reduced, albeit not abolished, in the triple mutants, indicating that one or more of the transporters, but also additional Mrr1 target genes, promote Hst 5 resistance (Fig 7A). To assess the relative contribution of each individual transporter, we introduced the G997V mutation also into both MRR1 alleles of flu1Δ, mdr1Δ, and tpo2Δ single mutants. Deletion of FLU1 or TPO2 did not or only slightly reduce the Hst 5 resistance of cells containing the MRR1 G997V mutation (Fig 7B and 7D). In contrast, deletion of MDR1 increased the Hst 5 susceptibility of cells with the hyperactive Mrr1 (killing increased from ca. 60% to ca. 80% in the presence of 30 μM Hst 5), although the cells were still more resistant than the wild type (ca. 90% killing) (Fig 7C). These results demonstrated that overexpression of MDR1 is one mechanism that contributes to the increased Hst 5 resistance of cells with Mrr1 GOF mutations.

Fluconazole-resistant C. albicans isolates with Mrr1 GOF mutations have acquired increased Hst 5 resistance
We next investigated if fluconazole-resistant clinical C. albicans isolates that have acquired MRR1 GOF mutations during antimycotic therapy also exhibit increased Hst 5 resistance. For this purpose, we selected five matched pairs of fluconazole-susceptible and -resistant isolates Simultaneous acquisition of drug and antifungal peptide resistance by C. albicans from AIDS patients with different MRR1 GOF mutations. We had previously generated mrr1Δ mutants from the five resistant isolates [13,16], which allowed us to assess the contribution of Mrr1 to their phenotypes. To compare FLU1 expression levels, we introduced the P FLU1 -GFP reporter fusion into all these strains. Two independent reporter strains were derived from each clinical isolate and one from both independently constructed mrr1Δ mutants of each resistant isolate to ensure the reproducibility of the results (one of the mrr1Δ mutants of isolate 2271 exhibited a high unspecific autofluorescence and could not be used for these experiments). As Simultaneous acquisition of drug and antifungal peptide resistance by C. albicans can be seen in S5 Fig, FLU1 expression was moderately increased in all five resistant isolates compared to the matched susceptible isolates (2-to 5-fold), and the elevated expression levels returned to those observed in the susceptible isolates when MRR1 was deleted. These and previous results [13,16] demonstrate that Mrr1 GOF mutations cause concomitant upregulation of both efflux pumps, MDR1 and FLU1, in fluconazole-resistant clinical C. albicans isolates.
We then assessed if the Mrr1 GOF mutations also resulted in enhanced Hst 5 resistance of the clinical isolates. Fig 8 shows that killing by Hst 5 was reduced in all five fluconazole-resistant isolates with Mrr1 GOF mutations compared to their matched susceptible isolates, but to different degrees. A relatively minor but significant reduction in killing (from 94% to 84%) was observed for isolate B4 compared to isolate B3 (Fig 8B), whereas isolate DSY2286 was highly resistant, as hardly any killing of this isolate was observed at the tested Hst 5 concentration (Fig 8C). In four of the five cases, Hst 5 resistance was fully or largely mediated by the hyperactive Mrr1, because the percent killing was elevated to the levels of the matched susceptible isolates when MRR1 was deleted. Isolate 6692 was an exception, because the mrr1Δ mutants derived from it retained the increased Hst 5 resistance. This strain apparently possesses other mechanisms of Hst 5 resistance that override the contribution of the hyperactive Mrr1. Collectively, these results demonstrate that fluconazole-resistant C. albicans strains with Mrr1 GOF mutations have simultaneously acquired increased Hst 5 resistance, because the overexpression of MDR1 and other Mrr1 target genes not only mediates fluconazole resistance but also increased resistance to the antimicrobial peptide.

Discussion
The acquisition of GOF mutations in zinc cluster transcription factors is a frequent cause of fluconazole resistance in clinical C. albicans strains. GOF mutations in Mrr1, Tac1, and Upc2 enable the cells to continue to grow in the presence of the drug and outcompete wild-type cells in the population. However, the hyperactive transcription factors also decrease the fitness of the cells in the absence of drug selection, at least under some environmental conditions [20,44,46]. As Mrr1, Tac1, and Upc2 regulate different sets of genes [31,45,47], the fitness defect must have a different basis in each case; consequently, it is aggravated in highly resistant strains containing several hyperactive transcription factors [20]. The deregulated gene expression probably causes an unnecessary waste of energy and reduces the ability of the cells to appropriately adapt to specific host niches. Here, we have uncovered a previously unrecognized effect of hyperactive forms of Mrr1 in addition to their ability to mediate fluconazole resistance. The overexpression of the multidrug efflux pump MDR1 (and possibly to some extent also FLU1) and other Mrr1 target genes confers increased resistance to the antifungal peptide Hst 5, which is present in the saliva of humans. Fluconazole-resistant strains with GOF mutations in Mrr1 have mainly been isolated from the oral cavity of HIV-infected patients suffering from oropharyngeal candidiasis [13,14,16,42,44,[48][49][50][51][52][53][54][55]. Increased Hst 5 resistance may therefore represent an additional advantage that counterbalances the fitness costs of Mrr1 hyperactivity and helps the mutants to establish themselves in this host niche. Nevertheless, Mrr1 mutations have been found only after fluconazole treatment of oropharyngeal candidiasis and have not been detected in pretreatment isolates from the same patients. This suggests that exposure to Hst 5 is not sufficient to select for Mrr1 GOF mutations when C. albicans colonizes the oral cavity as a harmless commensal without causing disease symptoms and does not have to cope with antimycotic therapy.
It is difficult to estimate how strongly a hyperactive Mrr1 contributes to the survival and competitive fitness of C. albicans in the oral cavity of humans. After short-term exposure to Hst 5, as in our in vitro experiments, strains with a hyperactive Mrr1 exhibited significantly reduced killing compared to wild-type controls. In the natural habitat, Hst 5 is constantly produced, resulting in continued exposure of C. albicans to the antifungal peptide, and the ability to avoid its toxic effects may present an even more significant advantage. The fitness gain due to enhanced Hst 5 resistance will likely also depend on the Hst 5 concentration, which varies among individuals and is generally lower in HIV-infected persons [56]. As Hst 5 is produced only by humans and other primates, the relevance of increased Hst 5 resistance cannot be meaningfully studied in mouse models of experimental candidiasis.
Although we initially searched for regulators of FLU1 expression in order to identify transcription factors that might confer Hst 5 resistance, our subsequent experiments revealed that Flu1 contributed little, if at all, to Hst 5 resistance in derivatives of strain SC5314 carrying hyperactive forms of MRR1. Instead, we unexpectedly found that overexpression of the efflux pump MDR1 was one mechanism by which such strains became less susceptible to killing by the antimicrobial peptide. MDR1 overexpression, therefore, confers increased resistance not only to fluconazole but also to Hst 5, which is a previously unrecognized function of this efflux pump that is of potential clinical relevance. It is well possible that FLU1 makes a more important contribution in some clinical isolates with MRR1 GOF mutations, because all such isolates that were tested concomitantly overexpressed both MDR1 and FLU1, and some of them may contain FLU1 alleles that encode more efficient Hst 5 transporters. Allelic differences that affect the efficiency of drug transport have been previously reported for the drug efflux pump CDR2 [57]. Nevertheless, a hyperactive Mrr1 still conferred increased Hst 5 resistance, albeit less efficiently, even in strains lacking both MDR1 and FLU1 as well as TPO2, encoding a related putative transporter, indicating that additional Mrr1 target genes are involved in this phenotype. This is similar to Mrr1-mediated fluconazole resistance, which is only partially mediated by Mdr1 and involves other Mrr1 target genes that remain to be identified [45]. The effect of specific GOF mutations on the expression of individual Mrr1 target genes varies. For example, the T360I and T896I mutations caused comparable MDR1 expression levels, but only the T360I mutation also resulted in strong TPO2 overexpression (see Fig 6B). Therefore, the degree of resistance to fluconazole and Hst 5 conferred by a particular Mrr1 GOF mutation will depend not only on the expression levels of MDR1 but also on those of the other target genes that contribute to these phenotypes.
The successful establishment and expansion of genetically altered variants within a population require that the beneficial effects of a mutation outweigh its negative consequences. As noted above, the increased Hst 5 resistance may not sufficiently offset the fitness costs caused by a hyperactive Mrr1 to select for MRR1 GOF mutations in strains colonizing the oral cavity of healthy persons. In contrast, the increased fluconazole resistance of such strains supports their emergence in the presence of the drug. It is intriguing that antimycotic therapy promotes the evolution of strains which, as a consequence of a drug resistance mutation, have acquired increased resistance to an innate host defense mechanism and are better adapted to some stressful conditions encountered in the host even in the absence of antifungal drug treatment.

Strains and growth conditions
Candida albicans strains used in this study are listed in S1 Table. The complete library of C. albicans strains containing artificially activated zinc cluster transcription factors has been described previously [34]. All strains were stored as frozen stocks with 17.2% glycerol at -80˚C and subcultured on YPD agar plates (10 g yeast extract, 20 g peptone, 20 g glucose, 15 g agar per litre) at 30˚C. Strains were routinely grown in YPD liquid medium at 30˚C in a shaking incubator. For selection of nourseothricin-resistant transformants, 200 μg/ml nourseothricin (Werner Bioagents, Jena, Germany) was added to YPD agar plates. To obtain nourseothricinsensitive derivatives in which the SAT1 flipper cassette was excised by FLP-mediated recombination, transformants were grown overnight in YCB-BSA-YE medium (23.4 g yeast carbon base, 4 g bovine serum albumin, 2 g yeast extract per litre, pH 4.0) without selective pressure to induce the SAP2 promoter controlling caFLP expression. Alternatively, strains containing a SAT1 flipper cassette in which the caFLP gene is expressed from the MAL2 promoter (as in plasmids pMRR1R4 to pMRR1R10) were grown overnight in YPM medium (10 g yeast extract, 20 g peptone, 20 g maltose per liter) instead of YCB-BSA-YE to induce the MAL2 promoter. Appropriate dilutions of the cultures were plated on YPD agar plates and grown for 2 days at 30˚C to obtain single colonies. Nourseothricin-sensitive clones were identified by restreaking on YPD plates and on YPD plates containing 100 μg/ml nourseothricin.

Plasmid constructions
A FLU1 deletion construct was obtained by amplification of the FLU1 upstream and downstream regions from genomic DNA of strain SC5314 with the primer pairs FLU1.01/FLU1.02 and FLU1.03/FLU1.04, respectively (all oligonucleotide primers used in this study are listed in S2  Table). The PCR products were digested with SacI/SacII and XhoI/ApaI, respectively, and cloned on both sides of the modified SAT1 flipper cassette contained in plasmid pSFS5 [58] to result in pFLU1M1. To reintroduce an intact FLU1 copy into flu1Δ mutants, the FLU1 coding region plus upstream and downstream sequences was amplified with primers FLU1.01 and FLU1_compleR. The PCR product was digested with SacI/SacII and substituted for the FLU1 upstream region of plasmid pFLU1M1 to generate pFLU1K1. A P FLU1 -GFP reporter fusion was constructed as follows. The FLU1 upstream region was amplified with primers FLU1.01 and FLU1rev_upstream, and a GFP-T ACT1 fragment was amplified from plasmid pNIM1 [59] with primers FLU1forw_GFP and ACT19. The gel-purified PCR products were then used as templates for a fusion PCR with primers FLU1.01 and ACT19. The PCR product was digested with SacI/SacII and inserted instead of the FLU1 upstream region in plasmid pFLU1M1 to generate pFLU1G1.
A WAR1 deletion construct was generated by amplifying the WAR1 flanking sequences with the primer pairs WAR1.01/WAR1.02 and WAR1.03/WAR1.04, digesting the PCR products with SacI/SacII and XhoI/ApaI, respectively, and inserting the fragments on both sides of the SAT1 flipper cassette of pSFS5, generating pWAR1M1. Similarly, a ZCF35 deletion cassette was obtained by amplifying the ZCF35 flanking sequences with the primer pairs ZCF35-7/ ZCF35-8 and ZCF35-9/ZCF35-10 and cloning the SacI/SacII-and XhoI/ApaI-digested PCR products in pSFS5 to generate pZCF35M3. A TPO2 deletion cassette was generated by amplifying the TPO2 flanking sequences with the primer pairs TPO2.01/TPO2.02 and TPO2.03/ TPO2.06 and cloning the SacI/SacII-and XhoI/KpnI-digested PCR products in pSFS5 to generate pTPO2M2. A new MDR1 deletion cassette was generated by substituting the SAT1 flipper cassette from pSFS5 for the old SAT1 flipper cassette in the previously described plasmid pMDR1M2 [45], yielding pMDR1M3.

Strain constructions
C. albicans strains were transformed by electroporation [43] with the following gel-purified linear DNA fragments. The insert from pFLU1M1 was used to sequentially delete the two wild-type alleles of FLU1 in strain SC5314, and the insert from pFLU1K1 was used to reintroduce an intact FLU1 copy into the homozygous mutants. The insert from pFLU1G1 was used to express the GFP reporter gene under the control of the endogenous FLU1 promoter in various strain backgrounds. The cassettes from plasmids pMRR1GAD1, pZCF34GAD1, pWAR1-GAD1, and pZCF35GAD1 [34] were used to introduce artificially activated forms of MRR1, MRR2, WAR1, and ZCF35, respectively, into P FLU1 -GFP reporter strains and flu1Δ mutants. The deletion cassettes from pMDR1M3, pTPO2M2, pWAR1M1, and pZCF35M3 were used to generate mdr1Δ, tpo2Δ, war1Δ, and zcf35Δ mutants of strain SC5314. The cassettes from pMDR1M3 and pTPO2M2 were also used for the construction of flu1Δ mdr1Δ tpo2Δ triple mutants. The inserts from plasmids pMRR1R4 to pMRR1R10 were used to replace the wildtype MRR1 alleles of strain SC5314 by mutated alleles with different GOF mutations. The insert from pMRR1R4 was also used to introduce the G997V GOF mutation into both MRR1 alleles of flu1Δ, mdr1Δ, and tpo2Δ single mutants and flu1Δ mdr1Δ tpo2Δ triple mutants. The correct integration of each construct as well as recycling of the SAT1 flipper cassette were confirmed by Southern hybridization using the flanking sequences as probes. Introduction of the MRR1 GOF mutations was confirmed by reamplification of the genes from heterozygous and homozygous mutants and sequencing of the PCR products. In each case, two independent series of strains were generated and used for further analysis.

Isolation of genomic DNA and Southern hybridization
Genomic DNA from C. albicans strains was isolated as described previously [60]. The DNA was digested with appropriate restriction enzymes, separated on a 1% agarose gel, transferred by vacuum blotting onto a nylon membrane, and fixed by UV crosslinking. Southern hybridization with enhanced chemiluminescence-labeled probes was performed with the Amersham ECL Direct Nucleic Acid Labelling and Detection System (GE Healthcare UK Limited, Little Chalfont Buckinghamshire, UK) according to the instructions of the manufacturer.

Northern hybridization analysis
Overnight cultures of the strains were diluted 10 −2 in fresh YPD medium and grown for 4 h at 30˚C. Total RNA was extracted by the hot acidic phenol method [61] combined with a purification step with the RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA samples were separated on a 1.2% agarose gel, transferred by capillary blotting onto a nylon membrane, fixed by UV crosslinking, and hybridized with digoxigenin-labeled MDR1 (positions 647 to 1688 in the MDR1 coding sequence, amplified with primers NB-MDR1_FW and NB-MDR1-RV), TPO2 (positions 115 to 1154 in the TPO2 coding sequence, amplified with primers TPO2_forN3 and TPO3_revN3), and ACT1 (positions 1512 to 1677 in the ACT1 coding sequence, amplified with primers ACT_RT and ACT2_RT) probes. The bound probes were detected with a peroxidase-labeled anti-digoxigenin AP-conjugate (Roche, Basel, Switzerland).

Fluconazole susceptibility assays
The fluconazole susceptibilities of the strains were determined by a previously described broth microdilution method [62], with slight modifications. A 2-day-old colony from a YPD agar plate was suspended in 2 ml of a 0.9% NaCl solution, and 4 μl of the suspension was mixed with 2 ml 2x SD-CSM medium (13.4 g yeast nitrogen base without amino acids [YNB; BIO 101, Vista, Calif.], 40 g glucose, 1.54 g complete supplement medium [CSM, BIO101]). A twofold dilution series of fluconazole (Sigma GmbH, Deisenhofen, Germany) was prepared in water, starting from an initial concentration of 512 μg/ml. One hundred microliters of each fluconazole solution was then mixed with 100 μl of the cell suspension in a 96-well microtiter plate and the plates were incubated for 48 h at 37˚C. The MIC of fluconazole was defined as the drug concentration that abolished or drastically reduced visible growth compared to a drug-free control.

Dilution spot assays
Overnight cultures of the strains in SD-CSM medium were diluted to an optical density at 600 nm of 2.0. Ten-fold dilutions from 10 0 to 10 −5 were prepared in a 96-well microtiter plate and ca. 5 μl of the cell suspensions transferred with a replicator onto SD-CSM agar plates without or with 0.5 or 1.0 μg/ml MPA or 5 μg/ml fluconazole. Plates were incubated for 2 days at 30˚C and photographed.

Flow cytometry
Overnight cultures of the GFP reporter and control strains were diluted 10 −2 in fresh YPD medium and grown for 3 h at 30˚C. The cultures were tenfold diluted in 1 ml cold phosphatebuffered saline (PBS) and flow cytometry was performed using the MACSQuantAnalyzer (Miltenyi Biotec; Bergisch Gladbach, Germany) equipped with an argon laser emitting at 488 nm. Fluorescence was detected using the B1 fluorescence channel equipped with a 525 nm band-pass filter (bandwidth 50 nm). Twenty thousand cells were analyzed per sample and counted at a flow rate of approx. 500 cells per second. Fluorescence data were collected by using logarithmic amplifiers. The mean fluorescence (arbitrary values) was determined with MACSQuantify (Version 2.4, Miltenyi Biotec) software.

Hst 5 sensitivity assays
The susceptibility of C. albicans cells to Hst 5 was measured using microdilution plate assays as previously described [63]. Briefly, 10 ml of YPD medium was inoculated with single colonies of each strain. Cells were grown overnight at room temperature. Overnight cultures were diluted to an A 600 of 0.3 to 0.4 and then incubated at 30˚C with shaking (220 rpm) until an A 600 of *1.0 was reached. Cells were washed twice with 10 mM sodium phosphate buffer (NaPB), pH 7.4. The cells (1 × 10 6 ) were then mixed with different concentrations of Hst 5 at 30˚C for 60 min and diluted in 10 mM NaPB. Aliquots of 500 cells were spread onto YPD agar plates and incubated for 24 to 48 h until colonies became visible. The percent killing was calculated as [1 − (number of colonies from Hst 5-treated cells/number of colonies from control cells)] × 100%. Assays were performed in quadruplicate for each strain. The reference strain SC5314 was included in each set of experiments to validate assay conditions.

Statistical analysis
Since the FLU1 expression and Hst 5 sensitivity assays were performed under controlled conditions in vitro, we assumed that the collected data fit a standard normal distribution. The oneway ANOVA test was used when three or more groups were compared with each other, and the two-tailed t-test was used when only two groups were compared, as indicated in the figures. Values for the two independently constructed strains A and B were combined in each case, except for the experiments shown in Fig 4B, which were performed on separate occasions. All statistical tests were conducted using GraphPad Prism version 7.03 software.
Supporting information S1 Fig. MPA sensitivity of flu1Δ mutants. Serial dilutions of the wild type and two independent series of heterozygous and homozygous flu1Δ mutants and complemented strains were spotted onto SD-CSM agar plates without or with 0.5 μg/ml MPA and grown for 2 days at 30˚C. The following strains were used in this experiment: SC5314 (Wild type), SCFLU1M2A and -B (FLU1/flu1Δ), SCFLU1M4A and -B (flu1Δ/flu1Δ), SCFLU1K2A and -B (flu1Δ/flu1Δ + FLU1). Strain S2UI1, which carries a mutated IMH3 allele that confers MPA resistance (MPA R ), was included as an additional control. (PDF) S2 Fig. FLU1 expression in strains carrying a P FLU1 -GFP reporter fusion in the indicated genetic backgrounds. Strains were grown to log phase in YPD medium and the mean fluorescence of the cells was determined by flow cytometry. The results obtained with two independently generated reporter strains are shown in each case (means and standard deviations from three biological replicates). The background fluorescence of otherwise identical strains without GFP is indicated by the black part of each column.