Mutations in TAC1 and ERG11 are major drivers of triazole antifungal resistance in clinical isolates of Candida parapsilosis.

OBJECTIVES: The aim of this study was to determine how mutations in CpERG11 and CpTAC1 contribute to fluconazole resistance in a collection of clinical isolates. METHODS: Sequences of CpERG11 and CpTAC1 were determined for 35 resistant Candida parapsilosis clinical isolates. A plasmid-based CRISPR-Cas9 system was used to introduce mutations leading to amino acid substitution in CpTac1 and CpErg11. Triazole susceptibility was determined by broth microdilution and E-test. Differential expression of genes mediated by CpTAC1 mutation was determined by RNA sequencing, and relative expression of individual transporter genes was assessed with RT-qPCR. RESULTS: Six isolates carried a mutation in CpTAC1 in combination with the CpERG11 mutation, leading to the CpErg11Y132F substitution. When introduced into susceptible isolates, this CpERG11 mutation led to a 4- to 8-fold increase in fluconazole minimum inhibitory concentrations (MIC; 0.125 µg/mL vs. 0.5 µg/mL, 0.125 µg/mL vs. 0.5 µg/mL, and 0.5 µg/mL vs. 4 µg/mL). When introduced into a susceptible isolate, the CpTAC1 mutation leading to the G650E substitution resulted in an 8-fold increase in fluconazole MIC (0.25 µg/mL vs. 2 µg/mL), whereas correction of this mutation in resistant isolates led to a 16-fold reduction in MIC (32 µg/mL vs. 2 µg/mL). CpCDR1, CpCDR1B, and CpCDR1C were overexpressed in the presence CpTac1G650E. Disruption of these genes in combination resulted in a 4-fold reduction in fluconazole MIC (32 µg/mL vs. 8 µg/mL). DISCUSSION: These results define the specific contribution made by the Y132F substitution in CpERG11 and demonstrate a role for activating mutations in CpTAC1 in triazole resistance in C. parapsilosis.

In vivo emergence of high-level resistance during treatment reveals the first identified mechanism of amphotericin B resistance in Candida auris.

OBJECTIVE: Candida auris has emerged as a health-care-associated and multidrug-resistant fungal pathogen of great clinical concern. As many as 50% of C. auris clinical isolates are reported to be resistant to amphotericin B, but no mechanisms contributing to this resistance have been identified. Here we describe a clinical case in which high-level amphotericin B resistance was acquired in vivo during therapy and undertake molecular and genetic studies to identify and characterize the genetic determinant of resistance. METHODS: Whole-genome sequencing was performed on four C. auris isolates obtained from a single patient case. Cas9-mediated genetic manipulations were then used to generate mutant strains harbouring mutations of interest, and these strains were subsequently subjected to amphotericin B susceptibility testing and comprehensive sterol profiling. RESULTS: A novel mutation in the C. auris sterol-methyltransferase gene ERG6 was found to be associated with amphotericin B resistance, and this mutation alone conferred a >32-fold increase in amphotericin B resistance. Comprehensive sterol profiling revealed an abrogation of ergosterol biosynthesis and a corresponding accumulation of cholesta-type sterols in isolates and strains harbouring the clinically derived ERG6 mutation. CONCLUSIONS: Together these findings definitively demonstrate mutations in C. auris ERG6 as the first identified mechanism of clinical amphotericin B resistance in C. auris and represent a significant step forward in the understanding of antifungal resistance in this emerging public health threat.

Delineation of the Direct Contribution of Candida auris ERG11 Mutations to Clinical Triazole Resistance.

Resistance to fluconazole is one of clinical characteristics most frequently challenging the treatment of invasive Candida auris infections, and is observed among >90% of all characterized clinical isolates. In this work, the native C. auris ERG11 allele in a previously characterized fluconazole-susceptible clinical isolate was replaced with the ERG11 alleles from three highly fluconazole-resistant clinical isolates (MIC ≥256 mg/L), encoding the amino acid substitutions VF125AL, Y132F, and K143R, using Cas9-ribonucleoprotein (RNP) mediated transformation system. Reciprocally, the ERG11WT allele from the same fluconazole-susceptible clinical isolate, lacking any resistance-associated mutation, was introduced into a previously characterized fluconazole-resistant clinical isolate, replacing the native ERG11K143R allele, using the same methods. The resulting collection of strains was subjected to comprehensive triazole susceptibility testing, and the direct impact each of these clinically-derived ERG11 mutations on triazole MIC was determined. Introduction of each of the three mutant ERG11 alleles was observed to increase fluconazole and voriconazole MIC by 8- to 16-fold. The MIC for the other clinically available triazoles were not significantly impacted by any ERG11 mutation. In the fluconazole-resistant clinical isolate background, correction of the K143R encoding mutation led to a similar 16-fold decrease in fluconazole MIC, and 8-fold decrease in voriconazole MIC, while the MIC of other triazoles were minimally changed. Taken together, these findings demonstrate that mutations in C. auris ERG11 significantly contribute to fluconazole and voriconazole resistance, but alone cannot explain the substantially elevated MIC observed among clinical isolates of C. auris. IMPORTANCE Candida auris is an emerging multidrug-resistant and health care-associated pathogen of urgent clinical concern. The triazoles are the most widely prescribed antifungal agents worldwide and are commonly utilized for the treatment of invasive Candida infections. Greater than 90% of all C. auris clinical isolates are observed to be resistant to fluconazole, and nearly all fluconazole-resistant isolates of C. auris are found to have one of three mutations (encoding VF125AL, Y132F, or K143R) in the gene encoding the target of the triazoles, ERG11. However, the direct contribution of these mutations in ERG11 to fluconazole resistance and the impact these mutations may have the susceptibility of the other triazoles remains unknown. The present study seeks to address this knowledge gap and potentially inform the future application the triazole antifungals for the treatment of infections caused by C. auris.

A variant ECE1 allele contributes to reduced pathogenicity of Candida albicans during vulvovaginal candidiasis.

Vulvovaginal candidiasis (VVC), caused primarily by the human fungal pathogen Candida albicans, results in significant quality-of-life issues for women worldwide. Candidalysin, a toxin derived from a polypeptide (Ece1p) encoded by the ECE1 gene, plays a crucial role in driving immunopathology at the vaginal mucosa. This study aimed to determine if expression and/or processing of Ece1p differs across C. albicans isolates and whether this partly underlies differential pathogenicity observed clinically. Using a targeted sequencing approach, we determined that isolate 529L harbors a similarly expressed, yet distinct Ece1p isoform variant that encodes for a predicted functional candidalysin; this isoform was conserved amongst a collection of clinical isolates. Expression of the ECE1 open reading frame (ORF) from 529L in an SC5314-derived ece1Δ/Δ strain resulted in significantly reduced vaginopathogenicity as compared to an isogenic control expressing a wild-type (WT) ECE1 allele. However, in vitro challenge of vaginal epithelial cells with synthetic candidalysin demonstrated similar toxigenic activity amongst SC5314 and 529L isoforms. Creation of an isogenic panel of chimeric strains harboring swapped Ece1p peptides or HiBiT tags revealed reduced secretion with the ORF from 529L that was associated with reduced virulence. A genetic survey of 78 clinical isolates demonstrated a conserved pattern between Ece1p P2 and P3 sequences, suggesting that substrate specificity around Kex2p-mediated KR cleavage sites involved in protein processing may contribute to differential pathogenicity amongst clinical isolates. Therefore, we present a new mechanism for attenuation of C. albicans virulence at the ECE1 locus.

Mutations in TAC1B: a Novel Genetic Determinant of Clinical Fluconazole Resistance in Candida auris.

Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, and yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro Comparative analyses of comprehensive sterol profiles, [3H]fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and relative expression levels of C. aurisERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene TAC1B and to show a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into fluconazole-susceptible isolate AR0387 was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in vitro and that mutations in TAC1B significantly contribute to clinical fluconazole resistance.IMPORTANCECandida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and to be commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrated that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris.

Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae.

This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. TheA. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine ß-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable.IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance.Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

The Interleukin (IL) 17R/IL-22R Signaling Axis Is Dispensable for Vulvovaginal Candidiasis Regardless of Estrogen Status.

Candida albicans, a ubiquitous commensal fungus that colonizes human mucosal tissues and skin, can become pathogenic, clinically manifesting most commonly as oropharyngeal candidiasis and vulvovaginal candidiasis (VVC). Studies in mice and humans convincingly show that T-helper 17 (Th17)/interleukin 17 (IL-17)-driven immunity is essential to control oral and dermal candidiasis. However, the role of the IL-17 pathway during VVC remains controversial, with conflicting reports from human data and mouse models. Like others, we observed induction of a strong IL-17-related gene signature in the vagina during estrogen-dependent murine VVC. As estrogen increases susceptibility to vaginal colonization and resulting immunopathology, we asked whether estrogen use in the standard VVC model masks a role for the Th17/IL-17 axis. We demonstrate that mice lacking IL-17RA, Act1, or interleukin 22 showed no evidence for altered VVC susceptibility or immunopathology, regardless of estrogen administration. Hence, these data support the emerging consensus that Th17/IL-17 axis signaling is dispensable for the immunopathogenesis of VVC.

Abrogation of Triazole Resistance upon Deletion of CDR1 in a Clinical Isolate of Candida auris.

Candida auris has rapidly emerged as a health care-associated and multidrug-resistant pathogen of global concern. In this work, we examined the relative expression of the four C. auris genes with the highest degree of homology to Candida albicansCDR1 and MDR1 among three triazole-resistant clinical isolates as compared to the triazole-susceptible genome reference clinical isolate. We subsequently utilized a novel Cas9-mediated system for genetic manipulations to delete C. aurisCDR1 and MDR1 in both a triazole-resistant clinical isolate and a susceptible reference strain and observed that MICs for all clinically available triazoles decreased as much as 128-fold in the CDR1 deletion strains. The findings of this work reveal for the first time that C. aurisCDR1 and MDR1 are more highly expressed among triazole-resistant clinical isolates of C. auris and that the overexpression of CDR1 is a significant contributor to clinical triazole resistance.

Comparative Analysis of the Capacity of the Candida Species To Elicit Vaginal Immunopathology.

The human fungal pathogen Candida albicans is the major etiological agent of vulvovaginal candidiasis (VVC). Despite this fact, other non-albicans Candida (NAC) species have frequently been reported, as well. Despite their presence in the vaginal environment, little is known about their capacities to elicit immune responses classically associated with C. albicans-mediated immunopathology, including neutrophil recruitment and proinflammatory cytokine signaling. Therefore, using a combination of in vitro and in vivo approaches, we undertook a comparative analysis to determine whether a representative panel of NAC species could colonize, induce immunopathological markers, or cause damage at the vaginal mucosa. Using a murine model of VVC, C. albicans was found to induce robust immunopathology (neutrophils and interleukin 1ß [IL-1ß]) and elicit mucosal damage. However, all the NAC species tested (including C. dubliniensis, C. tropicalis, C. parapsilosis, C. krusei, C. glabrata, and C. auris) induced significantly less damage and neutrophil recruitment than C. albicans, despite achieving similar early colonization levels. These results largely correlated with a notable lack of ability by the NAC species (including C. dubliniensis and C. tropicalis) to form hyphae both in vitro and in vivo Furthermore, both C. dubliniensis and C. tropicalis induced significantly less expression of the ECE1 gene encoding candidalysin, a key fungal virulence determinant driving VVC immunopathology. In order to determine the relative capacities of these species to elicit inflammasome-dependent IL-1ß release, both wild-type and NLRP3-/- THP-1 cells were challenged in vitro While most species tested elicited only modest amounts of IL-1ß, challenge with C. albicans led to significantly elevated levels that were largely NLRP3 dependent. Collectively, our findings demonstrate that although NAC species are increasingly reported as causative agents of VVC, C. albicans appears to be exceedingly vaginopathogenic, exhibiting robust immunopathology, hypha formation, and candidalysin expression. Thus, this study provides mechanistic insight into why C. albicans is overwhelmingly the major pathogen reported during VVC.

Loss of Upc2p-Inducible ERG3 Transcription Is Sufficient To Confer Niche-Specific Azole Resistance without Compromising Candida albicans Pathogenicity.

Inactivation of sterol Δ5,6-desaturase (Erg3p) in the prevalent fungal pathogen Candida albicans is one of several mechanisms that can confer resistance to the azole antifungal drugs. However, loss of Erg3p activity is also associated with deficiencies in stress tolerance, invasive hyphal growth, and attenuated virulence in a mouse model of disseminated infection. This may explain why relatively few erg3-deficient strains have been reported among azole-resistant clinical isolates. In this study, we examined the consequences of Erg3p inactivation upon C. albicans pathogenicity and azole susceptibility in mouse models of mucosal and disseminated infection. While a C. albicanserg3Δ/Δ mutant was unable to cause lethality in the disseminated model, it induced pathology in a mouse model of vaginal infection. The erg3Δ/Δ mutant was also more resistant to fluconazole treatment than the wild type in both models of infection. Thus, complete loss of Erg3p activity confers azole resistance but also niche-specific virulence deficiencies. Serendipitously, we discovered that loss of azole-inducible ERG3 transcription (rather than complete inactivation) is sufficient to confer in vitro fluconazole resistance, without compromising C. albicans stress tolerance, hyphal growth, or pathogenicity in either mouse model. It is also sufficient to confer fluconazole resistance in the mouse vaginal model, but not in the disseminated model of infection, and thus confers niche-specific azole resistance without compromising C. albicans pathogenicity at either site. Collectively, these results establish that modulating Erg3p expression or activity can have niche-specific consequences on both C. albicans pathogenicity and azole resistance.IMPORTANCE While conferring resistance to the azole antifungals in vitro, loss of sterol Δ5,6-desaturase (Erg3p) activity has also been shown to reduce C. albicans pathogenicity. Accordingly, it has been presumed that this mechanism may not be significant in the clinical setting. The results presented here challenge this assumption, revealing a more complex relationship between Erg3p activity, azole resistance, C. albicans pathogenicity, and the specific site of infection. Most importantly, we have shown that even modest changes in ERG3 transcription are sufficient to confer azole resistance without compromising C. albicans fitness or pathogenicity. Given that previous efforts to assess the importance of ERG3 as a determinant of clinical azole resistance have focused almost exclusively on detecting null mutants, its role may have been grossly underestimated. On the basis of our results, a more thorough investigation of the contribution of the ERG3 gene to azole resistance in the clinical setting is warranted.

Candidalysin Drives Epithelial Signaling, Neutrophil Recruitment, and Immunopathology at the Vaginal Mucosa.

Unlike other forms of candidiasis, vulvovaginal candidiasis, caused primarily by the fungal pathogen Candida albicans, is a disease of immunocompetent and otherwise healthy women. Despite its prevalence, the fungal factors responsible for initiating symptomatic infection remain poorly understood. One of the hallmarks of vaginal candidiasis is the robust recruitment of neutrophils to the site of infection, which seemingly do not clear the fungus, but rather exacerbate disease symptomatology. Candidalysin, a newly discovered peptide toxin secreted by C. albicans hyphae during invasion, drives epithelial damage, immune activation, and phagocyte attraction. Therefore, we hypothesized that Candidalysin is crucial for vulvovaginal candidiasis immunopathology. Anti-Candida immune responses are anatomical-site specific, as effective gastrointestinal, oral, and vaginal immunities are uniquely compartmentalized. Thus, we aimed to identify the immunopathologic role of Candidalysin and downstream signaling events at the vaginal mucosa. Microarray analysis of C. albicans-infected human vaginal epithelium in vitro revealed signaling pathways involved in epithelial damage responses, barrier repair, and leukocyte activation. Moreover, treatment of A431 vaginal epithelial cells with Candidalysin induced dose-dependent proinflammatory cytokine responses (including interleukin 1α [IL-1α], IL-1ß, and IL-8), damage, and activation of c-Fos and mitogen-activated protein kinase (MAPK) signaling, consistent with fungal challenge. Mice intravaginally challenged with C. albicans strains deficient in Candidalysin exhibited no differences in colonization compared to isogenic controls. However, significant decreases in neutrophil recruitment, damage, and proinflammatory cytokine expression were observed with these strains. Our findings demonstrate that Candidalysin is a key hypha-associated virulence determinant responsible for the immunopathogenesis of C. albicans vaginitis.

Overexpression of Candida albicans Secreted Aspartyl Proteinase 2 or 5 Is Not Sufficient for Exacerbation of Immunopathology in a Murine Model of Vaginitis.

The secreted aspartyl proteinases of Candida albicans have long been implicated in virulence at the mucosal surface, including contributions to colonization and immunopathogenesis during vulvovaginal candidiasis. In an effort to disentangle hypha-associated virulence factor regulation from morphological transition, the purpose of this study was to determine if overexpression of SAP2 or SAP5 in an efg1Δ/Δ cph1Δ/Δ mutant could restore the capacity to cause immunopathology during murine vaginitis to this avirulent hypofilamentous strain. Two similar yet distinct genetic approaches were used to construct expression vectors to achieve SAP overexpression, and both genetic and functional assays confirmed elevated SAP activity in transformed strains. Similar to previous findings, intravaginal challenge of C57BL/6 mice with hypha-defective strains attained high levels of mucosal colonization but failed to induce robust vaginal immunopathology (neutrophil recruitment, interleukin-1ß [IL-1ß] secretion, and lactate dehydrogenase release) compared to that with the hypha-competent control. Moreover, constitutive expression of SAP2 or SAP5 in two distinct sets of such strains did not elicit immunopathological markers at levels above those observed during challenge with isogenic empty vector controls. Therefore, these results suggest that the physiological contributions of SAPs to vaginal immunopathology require hypha formation, other hypha-associated factors, or genetic interaction with EFG1 and/or CPH1 to cause symptomatic infection. Additionally, the outlined expression strategy and strain sets will be useful for decoupling other downstream morphogenetic factors from hyphal growth.

Loss of C-5 Sterol Desaturase Activity Results in Increased Resistance to Azole and Echinocandin Antifungals in a Clinical Isolate of Candida parapsilosis.

Among emerging non-albicans Candida species, Candida parapsilosis is of particular concern as a cause of nosocomial bloodstream infections in neonatal and intensive care unit patients. While fluconazole and echinocandins are considered effective treatments for such infections, recent reports of fluconazole and echinocandin resistance in C. parapsilosis indicate a growing problem. The present study describes a novel mechanism of antifungal resistance in this organism affecting susceptibility to azole and echinocandin antifungals in a clinical isolate obtained from a patient with prosthetic valve endocarditis. Transcriptome analysis indicated differential expression of several genes in the resistant isolate, including upregulation of ergosterol biosynthesis pathway genes ERG2, ERG5, ERG6, ERG11, ERG24, ERG25, and UPC2 Whole-genome sequencing revealed that the resistant isolate possessed an ERG3 mutation resulting in a G111R amino acid substitution. Sterol profiles indicated a reduction in sterol desaturase activity as a result of this mutation. Replacement of both mutant alleles in the resistant isolate with the susceptible isolate's allele restored wild-type susceptibility to all azoles and echinocandins tested. Disruption of ERG3 in the susceptible and resistant isolates resulted in a loss of sterol desaturase activity, high-level azole resistance, and an echinocandin-intermediate to -resistant phenotype. While disruption of ERG3 in C. albicans resulted in azole resistance, echinocandin MICs, while elevated, remained within the susceptible range. This work demonstrates that the G111R substitution in Erg3 is wholly responsible for the altered azole and echinocandin susceptibilities observed in this C. parapsilosis isolate and is the first report of an ERG3 mutation influencing susceptibility to the echinocandins.

The RTA3 Gene, Encoding a Putative Lipid Translocase, Influences the Susceptibility of Candida albicans to Fluconazole.

The RTA3 gene, coding for a member of the Rta1p-like lipid-translocating exporter family, is coordinately upregulated with the ATP-binding cassette transporter genes CDR1 and CDR2 in azole-resistant clinical isolates of Candida albicans that carry activating mutations in the transcription factor Tac1p. We show here that deleting RTA3 in an azole-resistant clinical isolate carrying a Tac1p-activating mutation lowered fluconazole resistance by 2-fold, while overexpressing RTA3 in an azole-susceptible clinical isolate resulted in enhanced fluconazole tolerance associated with trailing growth in a liquid microtiter plate assay. We also demonstrate that an Rta3p-green fluorescent protein (GFP) fusion protein localizes predominantly to the plasma membrane, consistent with a putative function for Rta3p as a lipid translocase.

Azole Antifungal Resistance in Candida albicans and Emerging Non-albicans Candida Species.

Within the limited antifungal armamentarium, the azole antifungals are the most frequent class used to treat Candida infections. Azole antifungals such as fluconazole are often preferred treatment for many Candida infections as they are inexpensive, exhibit limited toxicity, and are available for oral administration. There is, however, extensive documentation of intrinsic and developed resistance to azole antifungals among several Candida species. As the frequency of azole resistant Candida isolates in the clinical setting increases, it is essential to elucidate the mechanisms of such resistance in order to both preserve and improve upon the azole class of antifungals for the treatment of Candida infections. This review examines azole resistance in infections caused by C. albicans as well as the emerging non-albicans Candida species C. parapsilosis, C. tropicalis, C. krusei, and C. glabrata and in particular, describes the current understanding of molecular basis of azole resistance in these fungal species.

Multidrug Transporters and Alterations in Sterol Biosynthesis Contribute to Azole Antifungal Resistance in Candida parapsilosis.

While much is known concerning azole resistance in Candida albicans, considerably less is understood about Candida parapsilosis, an emerging species of Candida with clinical relevance. We conducted a comprehensive analysis of azole resistance in a collection of resistant C. parapsilosis clinical isolates in order to determine which genes might play a role in this process within this species. We examined the relative expression of the putative drug transporter genes CDR1 and MDR1 and that of ERG11. In isolates overexpressing these genes, we sequenced the genes encoding their presumed transcriptional regulators, TAC1, MRR1, and UPC2, respectively. We also sequenced the sterol biosynthesis genes ERG3 and ERG11 in these isolates to find mutations that might contribute to this phenotype in this Candida species. Our findings demonstrate that the putative drug transporters Cdr1 and Mdr1 contribute directly to azole resistance and suggest that their overexpression is due to activating mutations in the genes encoding their transcriptional regulators. We also observed that the Y132F substitution in ERG11 is the only substitution occurring exclusively among azole-resistant isolates, and we correlated this with specific changes in sterol biosynthesis. Finally, sterol analysis of these isolates suggests that other changes in sterol biosynthesis may contribute to azole resistance in C. parapsilosis.

Disruption of the transcriptional regulator Cas5 results in enhanced killing of Candida albicans by Fluconazole.

Azole antifungal agents such as fluconazole exhibit fungistatic activity against Candida albicans. Strategies to enhance azole antifungal activity would be therapeutically appealing. In an effort to identify transcriptional pathways that influence the killing activity of fluconazole, we sought to identify transcription factors (TFs) involved in this process. From a collection of C. albicans strains disrupted for genes encoding TFs (O. R. Homann, J. Dea, S. M. Noble, and A. D. Johnson, PLoS Genet. 5:e1000783, 2009, http://dx.doi.org/10.1371/journal.pgen.1000783), four strains exhibited marked reductions in minimum fungicidal concentration (MFCs) in both RPMI and yeast extract-peptone-dextrose (YPD) media. One of these genes, UPC2, was previously characterized with regard to its role in azole susceptibility. Of mutants representing the three remaining TF genes of interest, one (CAS5) was unable to recover from fluconazole exposure at concentrations as low as 2 µg/ml after 72 h in YPD medium. This mutant also showed reduced susceptibility and a clear zone of inhibition by Etest, was unable to grow on solid medium containing 10 µg/ml fluconazole, and exhibited increased susceptibility by time-kill analysis. CAS5 disruption in highly azole-resistant clinical isolates exhibiting multiple resistance mechanisms did not alter susceptibility. However, CAS5 disruption in strains with specific resistance mutations resulted in moderate reductions in MICs and MFCs. Genome-wide transcriptional analysis was performed in the presence of fluconazole and was consistent with the suggested role of CAS5 in cell wall organization while also suggesting a role in iron transport and homeostasis. These findings suggest that Cas5 regulates a transcriptional network that influences the response of C. albicans to fluconazole. Further delineation of this transcriptional network may identify targets for potential cotherapeutic strategies to enhance the activity of the azole class of antifungals.

UPC2A is required for high-level azole antifungal resistance in Candida glabrata.

Candida glabrata, the second most common cause of Candida infections, is associated with high rates of mortality and often exhibits resistance to the azole class of antifungal agents. Upc2 and Ecm22 in Saccharomyces cerevisiae and Upc2 in Candida albicans are the transcriptional regulators of ERG11, the gene encoding the target of azoles in the ergosterol biosynthesis pathway. Recently two homologs for these transcription factors, UPC2A and UPC2B, were identified in C. glabrata. One of these, UPC2A, was shown to influence azole susceptibility. We hypothesized that due to the global role for Upc2 in sterol biosynthesis in S. cerevisiae and C. albicans, disruption of UPC2A would enhance the activity of fluconazole in both azole-susceptible dose-dependent (SDD) and -resistant C. glabrata clinical isolates. To test this hypothesis, we constructed mutants with disruptions in UPC2A and UPC2B alone and in combination in a matched pair of clinical azole-SDD and -resistant isolates. Disruption of UPC2A in both the SDD and resistant isolates resulted in increased susceptibility to sterol biosynthesis inhibitors, including a reduction in fluconazole MIC and minimum fungicidal concentration, enhanced azole activity by time-kill analysis, a decrease in ergosterol content, and downregulation of baseline and inducible expression of several sterol biosynthesis genes. Our results indicate that Upc2A is a key regulator of ergosterol biosynthesis and is essential for resistance to sterol biosynthesis inhibitors in C. glabrata. Therefore, the UPC2A pathway may represent a potential cotherapeutic target for enhancing azole activity against this organism.

UPC2 is universally essential for azole antifungal resistance in Candida albicans.

In Candida albicans, the transcription factor Upc2 is central to the regulation of ergosterol biosynthesis. UPC2-activating mutations contribute to azole resistance, whereas disruption increases azole susceptibility. In the present study, we investigated the relationship of UPC2 to fluconazole susceptibility, particularly in azole-resistant strains. In addition to the reduced fluconazole MIC previously observed with UPC2 disruption, we observed a lower minimum fungicidal concentration (MFC) for a upc2Δ/Δ mutant than for its azole-susceptible parent, SC5314. Moreover, the upc2Δ/Δ mutant was unable to grow on a solid medium containing 10 µg/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed higher fungistatic activity against the upc2Δ/Δ mutant than against SC5314. UPC2 disruption in strains carrying specific resistance mutations also resulted in reduced MICs and MFCs. UPC2 disruption in a highly azole resistant clinical isolate containing multiple resistance mechanisms likewise resulted in a reduced MIC and MFC. This mutant was unable to grow on a solid medium containing 10 µg/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed increased fungistatic activity against the upc2Δ/Δ mutant in the resistant background. Microarray analysis showed attenuated induction by fluconazole of genes involved in sterol biosynthesis, iron transport, or iron homeostasis in the absence of UPC2. Taken together, these data demonstrate that the UPC2 transcriptional network is universally essential for azole resistance in C. albicans and represents an attractive target for enhancing azole antifungal activity.

Mitochondrial two-component signaling systems in Candida albicans.

Two-component signal transduction pathways are one of the primary means by which microorganisms respond to environmental signals. These signaling cascades originated in prokaryotes and were inherited by eukaryotes via endosymbiotic lateral gene transfer from ancestral cyanobacteria. We report here that the nuclear genome of the pathogenic fungus Candida albicans contains elements of a two-component signaling pathway that seem to be targeted to the mitochondria. The C. albicans two-component response regulator protein Srr1 (stress response regulator 1) contains a mitochondrial targeting sequence at the N terminus, and fluorescence microscopy reveals mitochondrial localization of green fluorescent protein-tagged Srr1. Moreover, phylogenetic analysis indicates that C. albicans Srr1 is more closely related to histidine kinases and response regulators found in marine bacteria than are other two-component proteins present in the fungi. These data suggest conservation of this protein during the evolutionary transition from endosymbiont to a subcellular organelle. We used microarray analysis to determine whether the phenotypes observed with a srr1Δ/Δ mutant could be correlated with gene transcriptional changes. The expression of mitochondrial genes was altered in the srr1Δ/Δ null mutant in comparison to their expression in the wild type. Furthermore, apoptosis increased significantly in the srr1Δ/Δ mutant strain compared to the level of apoptosis in the wild type, suggesting the activation of a mitochondrion-dependent apoptotic cell death pathway in the srr1Δ/Δ mutant. Collectively, this study shows for the first time that a lower eukaryote like C. albicans possesses a two-component response regulator protein that has survived in mitochondria and regulates a subset of genes whose functions are associated with the oxidative stress response and programmed cell death (apoptosis).