Molecular and genetic basis of azole antifungal resistance in the opportunistic pathogenic fungus Candida albicans

Abstract

Candida albicans is an opportunistic yeast and the major human fungal pathogen in the USA, as well as in many other regions of the world. Infections with C. albicans can range from superficial mucosal and dermatological infections to life-threatening infections of the bloodstream and vital organs. The azole antifungals remain an important mainstay treatment of candidiasis and therefore the investigation and understanding of the evolution, frequency and mechanisms of azole resistance are vital to improving treatment strategies against this organism. Here the organism C. albicans and the genetic changes and molecular bases underlying the currently known resistance mechanisms to the azole antifungal class are reviewed, including up-regulated expression of efflux pumps, changes in the expression and amino acid composition of the azole target Erg11 and alterations to the organism’s typical sterol biosynthesis pathways. Additionally, we update what is known about activating mutations in the zinc cluster transcription factor (ZCF) genes regulating many of these resistance mechanisms and review azole import as a potential contributor to azole resistance. Lastly, investigations of azole tolerance in C. albicans and its implicated clinical significance are reviewed.

Introduction

Manifestations of Candida infections range from superficial mucosal and dermal infections such as oral thrush and vaginal yeast infections to life-threatening disseminated bloodstream infections that are associated with mortality rates upwards of 40%–60%.^1–7  Candida infections require extreme attention in immunocompromised individuals, such as AIDS patients, transplant recipients and patients undergoing chemotherapy or immunosuppression therapies, in whom they cause life-threatening invasive infections.^8–10 Studies have suggested that healthy individuals with implanted medical devices, those who have experienced major trauma and patients requiring extended stays in ICUs are at equal risk of acquiring Candida infection.¹¹ Global estimates suggest that invasive candidiasis occurs in ∼700 000 cases annually.¹²

In the USA, Candida species remain the fourth leading cause of nosocomial bloodstream infections and the number three cause of bloodstream infections in ICUs.¹³  Candida species are also the third leading cause of central-line-associated bloodstream infections and the second leading cause of catheter-associated urinary tract infections in the USA.^{14  ,  15} Of all Candida species, Candida albicans dominates almost all patient groups and disease manifestations in terms of incidence, followed by Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei.¹⁶ It is pertinent to mention here that there has been considerable change in species distribution in recent years, dependent upon geographical location and patient population, with a decrease in the proportion of C. albicans and an increase in C. glabrata, C. parapsilosis and other non-albicans Candida species.¹⁶  C. glabrata is the second most common species in the USA and North-Western Europe in the non-outbreak setting.^{17  ,  18} However, C. parapsilosis and/or C. tropicalis are much more frequently encountered than C. glabrata in Latin America, Southern Europe, India and Pakistan.^{17  ,  19–24} Of the five major Candida species, C. krusei is the least common and it is most often found among patients with underlying haematological malignancies with prior antifungal exposure.^{25  ,  26} Another worrisome MDR species that is emerging globally is Candida auris.²⁷

C. albicans is a member of native human microbiota and, as a commensal organism, colonizes the gastrointestinal (GI) tract, reproductive tract, oral cavity and skin of most humans.²⁸ Failure to identify a possible environmental reservoir for C. albicans suggests that this species is exquisitely adapted to healthy mammalian hosts. However, this benign commensal colonization can become pathogenic due to shifts in pH and oxygen levels, alterations in host microbiota (e.g. from antibiotic usage) or changes in the host immune response (e.g. during stress, infection by another microbe or immunosuppressant therapy).²⁹ The increase in the number of Candida infections may be attributed to the availability of modern medical treatments including excessive usage of broad-spectrum antibiotics, anticancer therapy, solid organ transplantation, the presence of indwelling catheters, lack of awareness among multiple medical specialties and poor performance of routine microbiological tests.

Fluconazole is often either the drug of choice or recommended alternative to the echinocandin class for the treatment of most invasive Candida infections.³⁰ Although varying rates of fluconazole resistance have been observed for C. albicans in particular types of infection, resistance rates remain low in general.^{9  ,  31  ,  32} Similarly, C. parapsilosis is usually susceptible to azoles but recent reports suggest the emergence of resistance in invasive infections.^33–36  C. glabrata tends to display high fluconazole MICs, while retaining amphotericin B and echinocandin susceptibility; Candida lusitaniae, on the other hand, is resistant, either intrinsically or acquired, to amphotericin B and the emerging fungal threat C. auris is often MDR.^{17  ,  27  ,  36  ,  37} The susceptibility pattern of various Candida species may also vary depending upon prior antifungal drug exposure, health status of the patient affected, site of isolation and geographical location.

While antifungal resistance in C. albicans is relatively uncommon as compared with species like C. glabrata, gradual use of fluconazole for long-term prophylaxis or treatment may lead to selective pressure resulting in emergence of secondary resistance in otherwise susceptible strains. This has occurred with greatest frequency in the setting of patients suffering from HIV/AIDS during treatment for oropharyngeal candidiasis, where the prevalence of fluconazole-resistant C. albicans isolates in North American oral candidiasis (HIV/AIDS-positive) patients was found to be between 12% and 22%.^38–42

With respect to CLSI clinical breakpoints, fluconazole resistance, defined as MIC values ≥8 mg/L for C. albicans, has been reported overall to be as high as 3.5%.^43–48 Although azole resistance of naive C. albicans isolates is rare, a steady increase in reports of azole resistance, resulting in therapeutic failures, has been a matter of serious clinical concern. In the current review, we aim to address the magnitude of azole resistance in C. albicans and the underlying mechanisms involved in azole resistance development.

Current antifungal treatment of C. albicans infections

Currently, antifungal therapy revolves around three main classes of antifungal drugs: the azole antifungals, echinocandins and polyene antifungals such as amphotericin B. The antimetabolite pyrimidine analogue flucytosine is also used in the treatment of certain invasive Candida infections and cryptococcal meningitis, but is limited to combination therapies with amphotericin B.³⁰ The azole antifungals are among the most common therapies for treatment and prophylaxis of both superficial and invasive candidiasis. Through inhibition of the ERG11-encoded protein 14α-lanosterol demethylase, the production of ergosterol normally required for membrane integrity is halted. In C. albicans, the inability to demethylate lanosterol via Erg11 shunts sterol biosynthesis production to other alternative biosynthesis pathways, resulting in the accumulation of 14α-methylergosta-8,24(28)-dien-3β,6α-diol.⁴⁹ The accumulation of this compound is toxic to the cell and results in the fungistatic effect seen with the azole antifungals.⁵⁰

While there are dozens of azole antifungal drugs sold commercially worldwide (ranging from research use to topical antifungal use in humans, to agriculture) only a select few triazoles are typically used for treating invasive candidiasis in the USA. While recent updates to clinical practice guidelines now recommend the echinocandin class of antifungals in many invasive Candida infections, fluconazole remains a viable first-line alternative in many cases, such as in non-neutropenic patients with Candida bloodstream infection.³⁰ Voriconazole is another triazole considered as an alternative or step-down therapy in many instances where fluconazole is also used and the triazoles itraconazole and posaconazole have niche uses in invasive candidiasis, such as alternatives for oropharyngeal or oesophageal candidiasis in fluconazole-refractory disease. Additionally, as no oral echinocandin is currently available commercially, fluconazole and the other triazoles are important for outpatient and step-down therapies for prophylaxis and treatment. The newly approved triazole prodrug isavuconazonium sulphate, while effective against Candida, including azole-resistant Candida species, does not currently have a place in the treatment of invasive candidiasis and is instead reserved for the serious mould infections such as aspergillosis and mucormycosis.^{51  ,  52}

Notably, a new subclass of azole antifungal compounds is in the drug pipeline. The tetrazole compounds, VT-1129, VT-1161 and VT-1598, so named because of an additional nitrogen on the five-membered azole ring that defines the class, are currently awaiting or undergoing clinical trials to treat a wide variety of fungal infections.^{53  ,  54} VT-1161, more specifically, is currently undergoing clinical trials as a treatment for recurrent vaginal candidiasis and may be a plausible future therapy against some azole-resistant C. albicans infections. These compounds have been found to possess potent activity against C. albicans and non-C. albicans species and Cryptococcus species.^54–59 A recent study evaluating the in vitro activities of VT-1161 and VT-1598 compared with other triazoles against azole-resistant C. albicans clinical isolates with well-characterized resistance mechanisms found that VT-1598 retained antifungal activity against azole-resistant isolates with known resistance mechanisms, while resistant isolates with increased efflux pump expression and azole target mutations had increased VT-1161 MICs, though overall VT-1161 possessed good activity against fluconazole-resistant clinical isolates.⁵⁴

There are many barriers to effective treatment with the azole antifungals in invasive candidiasis. Improper dosing and inadequate drug delivery to the source of infection are obvious clinical malpractices to avoid; however, C. albicans and other Candida species have a variety of methods to survive even proper treatment practices.

Epidemiology of azole resistance in C. albicans

In vitro susceptibility testing of yeasts and moulds helps in not only selecting the most clinically active antifungal agent but also in detecting resistance rates. CLSI and EUCAST have developed reference methods to detect in vitro resistance of yeasts and moulds against various antifungal agents. Additionally, both organizations have established defined clinical breakpoints aimed to help predict outcomes for a given antifungal agent and organism.^{60  ,  61} Resistance can be classified as microbiological or clinical. Microbiological resistance occurs when the concentration of antifungal agent required to inhibit the growth of the pathogen is higher than the range seen for WT strains due to the presence of evolved or innate mutational resistance mechanisms. Clinical resistance, on the other hand, is denoted by therapeutic failure in spite of administering appropriate doses of the antifungal agent.

Emergence of azole resistance in Candida species, especially those originating from patients on prolonged azole therapy, have become a worldwide problem thereby stressing the need for antifungal stewardship. Decreased in vitro susceptibility of C. albicans to azoles such as miconazole, econazole, ketoconazole and clotrimazole has been recognized since the late 1970s.^{62  ,  63} Most of the data related to the prevalence of triazole resistance in Candida species originates primarily from studies carried out in the USA and Europe. For example, an azole resistance rate of about 2.3% in C. albicans bloodstream isolates was reported in a 2012 US study, where C. albicans was the most common species isolated, followed by C. glabrata, C. parapsilosis and C. tropicalis.⁶⁴ More broadly, surveillance data analysing 1514 Candida species collected from Europe (41.0%), the Asia Pacific region (24.5%), North America (23.5%) and Latin America (11.0%) during 2013 determined azole antifungal resistance rates of about 0.4% and 0.3% in C. albicans against fluconazole and voriconazole, respectively.¹⁷ Another large population-based surveillance programme, the ARTEMIS DISK Global Antifungal Surveillance Study suggested C. albicans (65.3%) to be the most common species isolated, followed by C. glabrata, C. tropicalis and C. parapsilosis. About 1.4% of C. albicans isolates were resistant to fluconazole, of which 63.6% were cross-resistant to voriconazole.⁴³

A surveillance study in Denmark evaluated 303 episodes of fungaemia and reported the predominance of C. albicans, followed by C. glabrata, C. tropicalis and C. parapsilosis.⁶⁵ Of all the C. albicans isolates tested, 3% (n = 6) were resistant to fluconazole. Another Danish study reported a fluconazole resistance rate (defined in this study by an MIC of >4 mg/L) of about 0.6% (7/1183 isolates).⁶⁶ Similarly, in a study from Spain with C. albicans (45.4%) being the predominant species, about 1% of C. albicans strains were fluconazole resistant.⁶⁷ About 3.9% (n = 7) of C. albicans (n = 179) isolates were found be fluconazole resistant in a multicentre study from Belgium during 2013–14.⁶⁸ A 22 year Norwegian national surveillance study of candidaemia found 98.8% of C. albicans isolates to be susceptible to fluconazole; however, only 0.25% of C. albicans isolates were fluconazole resistant during 2004–12.⁶⁹ In a 2015 study conducted in ICU centres across India, 5.2% of C. albicans strains sourced from ICU-acquired candidaemia were identified as fluconazole resistant.⁷⁰ However, low azole resistance rates of about 0.9% and ∼2% have been reported from Australia and South Africa from 2014 to 2015.⁷¹

Overall, the global data suggest that C. albicans remains the predominant infective organism identified in Candida-based infections. While reported resistance rates vary from study to study, collected surveillance data suggest that fluconazole resistance rates and azole resistance rates in general for C. albicans remain relatively low. However, superficial mucosal C. albicans infections pose a considerable threat in immunocompromised populations and the high morbidity and mortality associated with serious invasive Candida infections continues to burden our healthcare systems.^72–74 As such we must not overlook C. albicans as a serious antimicrobial resistance threat. The next section details the specifics of the known azole resistance mechanisms in C. albicans.

Azole resistance mechanisms in C. albicans

The mechanisms of azole resistance in Candida species have been extensively studied in C. albicans. These mechanisms, including the up-regulation of drug transporters, overexpression or alteration of the drug target (Erg11) and mutations in the ERG11 gene resulting in decreased azole inhibition, are summarized in Figure 1 and described in the following section.

Figure 1.

Schematic of known azole resistance mechanisms in C. albicans. The yeast cell wall is not shown in this image. (a) Overexpression of the azole target sterol demethylase (Erg11) via up-regulation of ERG11 may counteract target inhibition via the azoles. (b) Amino acid changes in sterol demethylase resulting from (c) mutations in the ERG11 gene can result in decreased azole inhibition of sterol demethylase. (d) Aneuploidy can result in increases in expression of efflux genes and ERG11, as well as increased genetic diversity of C. albicans isolates in response to drug exposure. Intracellular concentrations of azole antifungal drug can be reduced through up-regulated expression of (e) ABC and (g) MFS transporters. (f) Alternative usage of sterols through defective C5-sterol desaturase (Erg3) can lead to alternative sterol biosynthesis pathways that ignore sterol demethylase inhibition. ER, endoplasmic reticulum. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Efflux pumps

Cdr1 and Cdr2

Like many infecting organisms, C. albicans is able to utilize efflux systems to transport drugs extracellularly, thereby reducing intracellular drug concentrations (Figure 1e). As such, the overexpression of azole-targeting efflux pumps is a key mechanism driving azole resistance in C. albicans. The ATP-binding cassette (ABC) transporters Cdr1 and Cdr2 have both been well documented as drivers of azole resistance and were first implicated in clinical azole resistance in isolates in HIV-infected patients with oropharyngeal candidiasis.^75–77 In two separate studies, it was found that isolates of C. albicans taken from HIV-infected individuals had increased mRNA levels of CDR1, which has been shown to confer resistance to the azole antifungals.^77–79

Importantly, the zinc cluster transcription factor (ZCF) Tac1 was discovered to regulate the expression of both CDR1 and CDR2 and that a co-dominant mutation in the putative C-terminal activation domain conferred hyperactivity of the Tac1 transcription factor, leading to constitutive increased expression of CDR1 and CDR2.^{80  ,  81} In a comparison of azole-susceptible versus azole-resistant matched isolates from the same patient, constitutive high expression of these transporters was suggested to be a result of both increased transcription and increased mRNA stability, the latter of which is tied to mRNA hyperadenylation and loss of heterozygosity at the poly(A) polymerase 1 locus.^{82  ,  83} It was further demonstrated that deletion of CDR1 in an azole-resistant clinical isolate containing a Tac1 gain-of-function mutation was directly responsible for reducing resistance to fluconazole, ketoconazole and itraconazole.⁸⁴ While CDR2 deletion also reduced resistance to fluconazole and ketoconazole, the effect was much weaker compared with CDR1 deletion, suggesting Cdr1 is the major contributor towards Tac1-mediated azole resistance. Since then, multiple gain-of-function mutations in Tac1 (Figure 2a) leading to increased expression of the Cdr1 and Cdr2 efflux transporters have been described.^{81  ,  85–87}

Figure 2.

Known and suspected gain-of-function mutations found in clinical isolates along the zinc cluster transcription factor genes (a) TAC1, (b) MRR1 and (c) UPC2. Numbers along the bottom of the gene denote the amino acid position. Light spotted regions at the N-terminus indicate the putative DNA-binding domains of each gene, previously reported.^{80  ,  107  ,  196  ,  197} The black region at the N-terminus of each gene represents the conserved Zn(II)Cys(6) zinc finger domain. Known gain-of-function mutations are denoted above the gene by solid lines.^{81  ,  87  ,  103  ,  104  ,  109  ,  112–114  ,  198–201} Suspected (unconfirmed) mutations found in azole-resistant clinical isolates that may contribute to decreased azole susceptibility are denoted above the gene by dashed lines.^{85  ,  202} ID, inhibitory domain; *, nonsense (stop) mutation.

Additionally, it was shown that increased CDR1 expression could be induced in the presence of agents such as oestradiol or fluphenazine.⁸⁸ While it was previously shown that CDR1 expression could be induced in response to fluconazole in a Cdr1-GFP fusion protein construct, a more recent study found that no increase in CDR1 or CDR2 expression as measured by quantitative reverse transcription PCR (RT–qPCR) could be observed in the presence of fluconazole in a C. albicans strain with 6His3Flag-tagged Tac1.^{89  ,  90} Both the constitutive and transient expression of CDR1 and CDR2 genes seem to be regulated via interactions at multiple cis-acting elements within the CDR1 and CDR2 promoters.⁹¹ The presence of drug-responsive elements (DREs) within each gene’s respective promoter regions, for example, has been shown to contribute to the induced and constitutive expression of CDR1 and CDR2, while a separate basal expression element (BEE) contributes to CDR1 baseline expression.^{80  ,  88  ,  92}

In a genome-wide location analysis of Tac1 binding sites using ChIP-chip, Tac1 was shown to bind to the DRE of both CDR1 and CDR2 under non-activating conditions, indicating that post-binding mechanisms such as repressor interaction loss or co-activator recruitment may occur during Tac1 activation.⁹² More recently, components of the tail module of the mediator complex have been identified as required for Tac1-activated CDR1 expression. A study examining the co-activator requirements for hyperactive Tac1 regulation described decreased fluconazole MICs in TAC1 gain-of-function mutant strains lacking either Med3 or Med15, both of which are part of the mediator tail module.⁹⁰ Furthermore, not only was Tac1-driven transcription of CDR1 decreased in gain-of-function Tac1 mutant strains lacking either Med3 or Med15, but deletion of MED3 also diminished xenobiotic-induced expression levels of CDR1, CDR2 and RTA3 with fluphenazine and oestradiol. ChIP assays revealed that occupancy of the mediator tail module at the CDR1 DRE increases during Tac1 hyperactivation, as well as with TAC1 overexpression; however, higher occupancy at the DRE did not result in increased fluconazole MICs, unless Tac1 was activated. Importantly, the authors also delineated a C-terminal region of Tac1 (amino acids 856 to 981), i.e. the transcription activation domain (TAD), whose interaction with an as-yet-uncharacterized inhibitory middle domain may be crucial to Tac1 hyperactivation and have posited that gain-of-function mutations in Tac1 may work to antagonize the normally inhibitory interaction between these two regions.⁹⁰

Mdr1

Mdr1, part of the major facilitator superfamily (MFS) of transporters (Figure 1g), has also been shown to play a role in fluconazole and voriconazole resistance, though it does not effect large MIC changes with other azole antifungals such as itraconazole, isavuconazole and posaconazole.^93–95 The gene encoding the Mdr1 efflux pump in C. albicans was originally discovered by its ability to confer resistance to several structurally unrelated compounds such as the anti-mitotic drug benomyl, methotrexate, cycloheximide, sulfometuron methyl, 4-nitroquinoline-N-oxide and bentriazoles in Saccharomyces cerevisiae expressing the C. albicans MDR1 gene, which at the time was known as BEN^r.^{96  ,  97} It was not until serial isolates taken from HIV-infected patients with oropharyngeal candidiasis were analysed for ERG11 and MDR1 mRNA expression levels that a link was established between the MDR1 gene and fluconazole resistance.⁹⁸

A study assessing sequential isolates of C. albicans in patients with recurrent oropharyngeal candidiasis treated with fluconazole found that increased MDR1 mRNA, as well as increased CDR1/2 and ERG11 gene expression, correlated with increasing fluconazole drug resistance, indicating that MDR1 plays a role in the multifaceted development of resistance in C. albicans.⁹⁹ When the MDR1 gene was disrupted in two fluconazole-resistant clinical C. albicans isolates, susceptibility to fluconazole increased compared with the resistant isolates with intact MDR1, demonstrating a direct relationship between MDR1 and fluconazole resistance in clinical isolates.¹⁰⁰

Importantly, fluconazole-susceptible and fluconazole-resistant matched pairs (serial isolates taken from the same patient in which fluconazole resistance developed over time) were shown to lack major polymorphic differences in the nucleotide sequence of their respective MDR1 promoter regions, despite high expression of MDR1 mRNA in the fluconazole-resistant isolate.¹⁰¹ Subsequent expression of GFP from the MDR1 promoter in the fluconazole-resistant isolate but not the fluconazole-susceptible isolate indicated that MDR1 expression was likely guided by a trans-regulatory factor. Induction of MDR1 mRNA expression through benomyl and tert-butyl hydrogen peroxide identified several cis-acting elements in the MDR1 promoter involved in induction with these agents, though fluconazole itself failed to induce the MDR1 promoter.¹⁰²

It was not until more recently that the zinc cluster transcription factor Mrr1 was discovered to regulate the Mdr1 efflux pump. Transcriptional profiling of three MDR1-overexpressing clinical isolates via DNA microarray analysis revealed 21 genes coordinately up-regulated with MDR1 and 7 genes down-regulated in all three isolates.¹⁰³ Among these, the then-putative ZCF, Zcf36, later known as the MDR regulator Mrr1, was hypothesized to control MDR1 expression. Deletion of MRR1 in MDR1-overexpressing isolates F5 and G5 managed to abolish MDR1 promoter activity in a P_MDR1-GFP reporter fusion, implicating Mrr1 in the constitutive expression of MDR1 in these fluconazole-resistant clinical isolates. Interestingly, deletion of MRR1 in each MDR1-overexpressing isolate did not yield identical MICs to strains lacking MDR1 in the same background. Deletion of MDR1 alone in both cases resulted in a higher MIC in the resistant clinical isolate background compared with deletion of its transcriptional regulator Mrr1, providing evidence that Mrr1 may regulate other determinants of fluconazole resistance apart from Mdr1.

Similar to the zinc cluster transcription factor-encoding gene TAC1 in CDR1- and CDR2-overexpressing isolates, mutated MRR1 alleles were present in the MDR1-overexpressing clinical isolates.¹⁰³ Moreover, sequencing of the MRR1 alleles in the MDR1-overexpressing isolates F5 and G5 revealed a loss of heterozygosity and retention of two copies of a mutant MRR1 allele in both clinical isolates. Replacement of a single copy of the mutant MRR1 allele encoding either a P683S or G997V amino acid substitution into SC5314 lacking both native copies of MRR1 resulted in increased resistance to fluconazole and increased MDR1 promoter activity as measured by a P_MDR1-GFP reporter construct, demonstrating the presence of gain-of-function mutations in MRR1 that regulate MDR1 expression in C. albicans.

Indeed, multiple amino acid substitutions in Mrr1 originating from both human clinical isolates and in vitro-generated strains have been shown to be gain-of-function mutations driving constitutive MDR1 expression (Figure 2b).¹⁰⁴ Furthermore, through DNA sequence analysis of the F1–F5 matched-series clinical isolates, it was revealed that gain-of function mutations appear to occur in a single MRR1 allele initially, which provides an intermediate level of resistance compared with the matched isolates collected chronologically later in the series that were homozygous for the MRR1  ^P683S allele. Through analysis of marker polymorphisms along chromosome 3 distal to MRR1 and proximal to the telomere, it was shown that loss of heterozygosity with selection of mutated MRR1 alleles was accomplished through mitotic recombination in clinical isolates, though chromosome loss was also observed as a cause in MDR1-overexpressing strains developed in vitro. This parallels the N977D homozygous mutation discovered in Tac1p along with loss of heterozygosity at chromosome 5 in C. albicans.  ⁸¹

Upc2 and increased ERG11 expression

Upc2 is another Zn(II)-Cys(6) transcription factor involved in sterol biosynthesis and azole resistance. Initially identified as a homologue of the S. cerevisiae (ScUPC2) gene, UPC2 in C. albicans was initially reported to up-regulate the ergosterol biosynthesis genes ERG2 and ERG11 when exposed to fluconazole.¹⁰⁵ The overexpression of ERG11 had been shown to contribute to decreased azole susceptibility through increased production of the azole target 14α-lanosterol demethylase (Figure 1a).^{78  ,  98  ,  106  ,  107} However, not only was the induction of ERG11 abolished when UPC2 was deleted, but the deletion strain was also hypersusceptible to the azole drugs, as well as the squalene epoxidase (Erg1) target drug terbinafine, the Erg2 and Erg24 target drug fenpropimorph and the HMG-CoA reductase inhibitor lovastatin, illustrating UPC2’s important role in sterol biosynthesis regulation.^{105  ,  107}

Importantly, overexpression of Upc2 via expression of the ORF from the MET3 promoter increased resistance to fluconazole and ketoconazole, and it was also demonstrated that the DNA-binding domain of Upc2 bound directly to ergosterol biosynthesis genes, binding to a putative sterol responsive element in ERG2.¹⁰⁷ Furthermore, investigation of the ERG11 promoter through deletion and linker scan mutations concluded that two 7 bp inverted-repeat sequences located −231 and −251 bp upstream of the start codon form an azole-responsive element (ARE) regulated by Upc2 and that this ARE was sufficient to induce ERG11 expression.¹⁰⁸

Upc2, however, was also shown to have a regulatory role in more than just sterol biosynthesis genes. Through genome-wide gene expression profiling, several ergosterol biosynthesis genes, including ERG11, were determined to be coordinately up-regulated with UPC2 in fluconazole-resistant isolates and ChIP-microarray location analysis identified 202 promoters bound by Upc2, including not only ergosterol biosynthesis genes (NCP1, ERG11, ERG1, ERG2, ERG24, ERG4, ERG5, ERG6, ERG9, ERG10, ERG25, ERG251 and UPC2) but also other transcription factors and the drug transporter genes CDR1 and MDR1.^109–111 As with the zinc cluster transcription factors Tac1 and Mrr1, gain-of-function mutations in Upc2 were found to lead to increased gene expression of ERG11 conferring fluconazole resistance in clinical isolates (Figure 2c).^{109  ,  112–114} When UPC2 was disrupted, the Δupc2 mutants had decreased cellular ergosterol content and were more susceptible to fluconazole not only in the azole-susceptible SC5314 strain but also in a highly resistant clinical isolate 12–99, which overexpresses CDR1, MDR1 and ERG11 and possesses a mutation in ERG11, suggesting that UPC2 is required for clinical azole drug resistance.^{114  ,  115}

Other zinc cluster transcription factors

Apart from the ZCFs Tac1, Mrr1 and Upc2, which are well-known determinants of C. albicans azole resistance, there has been significant interest in the role of the remaining ZCFs as well. Laboratory strains exhibiting hyperactive transcription factors Aro80, Mrr2, Stb5, Cta4, Zcf25, Zcf35 and Znc1 have also displayed increased azole MICs comparable to hyperactive Tac1, Mrr1 and Upc2, demonstrating that in theory other ZCFs may play a role in azole resistance in C. albicans.¹¹⁶ Additionally, in the hyperactive ZCF strains, Znc1 and Mrr2 appeared to activate the CDR1 promoter, indicating that these ZCFs may contribute to increased efflux pump expression.

In 2015, the first clinical isolate containing putative gain-of-function mutations in the ZCF Mrr2 was described, possessing amino acid substitutions that appeared to increase CDR1 but not CDR2 expression.¹¹⁷ In contrast to this finding, a recent report investigating these mutations in MRR2 when inserted into the native locus, as well as when the gene is overexpressed, failed to identify any changes in fluconazole susceptibility or CDR1 expression compared with WT.¹¹⁸ Given the contradictory findings, the clinical impact of other ZCFs outside of Tac1, Mrr1 and Upc2 in azole resistance remains in question.

Mutations in ERG11

Mutations in the ERG11 gene itself are another common azole resistance mechanism in C. albicans (Figure 1b and c), in stark contrast to the haploid organism C. glabrata, in which the frequency of mutations in ERG11 appears relatively low.^{16  ,  119} Most ERG11 mutations are thought to alter the ability of the azoles to bind and inhibit the lanosterol demethylase enzyme, either through steric hindrance in the ligand-binding pocket where the azole antifungals compete with the ergosterol precursor or by altering or reducing important interactions, such as the H-bond interactions with the azole ring and the haem group of the Erg11 protein.

The earliest point mutation resulting in an amino acid substitution (R467K) in the ERG11 gene of a clinical isolate was sequenced and discovered in 1997.¹²⁰ Soon after, multiple additional amino acid substitutions in Erg11 were uncovered and, notably, the effect of these amino acid changes on triazole antifungal MICs was not uniform.¹²¹ This altered susceptibility to the azoles was noted to likely be a result of altered interaction with azole binding and overall reduced fluconazole-binding affinity to the mutant 14α-lanosterol demethylase target enzyme.^{122  ,  123} As the azole antifungals emerged as a common first-line option in the treatment of many superficial and invasive fungal infections, documentation of new ERG11 mutations in azole-resistant clinical isolates continued to appear, though not all documented mutations were definitively shown to be tied to azole resistance.^124–137

The isolated contribution towards azole resistance of several predominant Erg11 amino acid substitutions found in clinical C. albicans isolates was investigated through homozygous replacement of the native ERG11 alleles with a mutant ERG11 ORF encoding either a single or double amino acid substitution in the Erg11 protein.¹³⁸ Single substitutions in Erg11 showed variable responses to fluconazole and other triazoles ranging from no effect to a 16-fold increase in MIC over the fluconazole-susceptible parent and double substitutions in Erg11 further increased the MIC beyond the individual contribution to azole resistance of any single amino acid substitution. Interestingly, while some substitutions, such as Y132F/H, K143R, G307S and S405F, are located on the exposed active-site cavity of the Erg11 protein and presumably directly influence or interfere with azole binding, other substitutions that are not predicted to directly interact with azole binding, such as the G450E substitution found on the fungal CYP51-specific β5-hairpin, may indirectly affect resistance through interaction with cytochrome P450 reductase–Erg11 interactions.¹³⁹ Importantly, the crystal structure of the Erg11 protein of C. albicans has been resolved, leading to further insight into the interactions between azole drugs and their target and the development of 3D models useful for future antifungal discovery.^{139  ,  140}

Recently, substitutions in purified, recombinant C. albicans Erg11 expressed from Escherichia coli revealed that Erg11 variants differ substantially in their baseline catalytic turnover and affinity for azole binding.¹⁴¹ Despite some substitutions showing relatively loose azole binding, their observed contribution to azole resistance in in vitro C. albicans susceptibility testing was significant, making it conceivable that Erg11 amino acid substitutions may contribute to azole resistance through more nuanced ways than simply reducing azole binding affinity.

Mutations in ERG3 and alternative sterol biosynthesis

Alterations in sterol biosynthesis pathways in C. albicans remain an important, albeit rare, clinical mechanism of azole antifungal resistance (Figure 1f). Defective C5-6 desaturase, homologous to the CaErg3 enzyme C-5 sterol desaturase, identified in azole-resistant isolates of S. cerevisiae initially led to the currently hypothesized mode of action of the azole antifungals.

14α-Methylergosta-8,24(28)-dien-3β,6α-diol, a metabolic by-product not normally produced in significant amounts during normal ergosterol biosynthesis, was found to accumulate in C. albicans cells following treatment with fluconazole.⁵⁰ However, isolates with defective C-5 sterol desaturase, which lacked detectable amounts of ergosterol presumably because they no longer possessed the Erg3 enzyme function required for ergosterol production, avoided accumulation of 14α-methylergosta-8,24(28)-dien-3β,6α-diol after treatment with fluconazole. The continued growth of isolates with defective Erg3 in the presence of fluconazole has led to the current belief that 14α-methylergosta-8,24(28)-dien-3β,6α-diol is toxic to the C. albicans cell and its accumulation results in the inhibition of growth. Moreover, mutations and amino acid substitutions that confer defects in Erg3 also confer resistance to the azole antifungals, specifically due to the inability to produce 14α-methylergosta-8,24(28)-dien-3β,6α-diol, which requires Erg3.

While relatively few in number compared with isolates containing other more common resistance mechanisms, several mutant ERG3 clinical isolates have been identified and reportedly possess resistance to the azole antifungals and cross-resistance to the polyene amphotericin B.^{50  ,  129  ,  142–145} However, some question the clinical significance of ERG3 mutant isolates during infection as isolates with an ERG3 resistance phenotype also displayed hyphal growth defects and attenuated virulence.^146–148 Recently, it has been shown that changes within the ERG3 promoter that affect expression and activity may be sufficient to confer azole resistance in niche-specific instances without affecting C. albicans pathogenicity, which implicates changes in the ERG3 locus as potentially more clinically relevant than previously believed.¹⁴⁹

Aneuploidy and loss of heterozygosity

Aneuploidy plays a role in azole resistance, as well as genetic diversity in C. albicans (Figure 1d). Gain or loss of chromosomes has been tied to azole resistance and attenuated virulence.^{150  ,  151} Loss of heterozygosity at gene loci of isolates serially passaged in azole-containing media, as well as laboratory strains passaged through mice models of haematogenously disseminated disease, both suggest that significant genetic rearrangements take place as a result of host and drug stress.^{152  ,  153} Studies using comparative genome hybridization in various C. albicans laboratory strains observed aneuploidies mainly on chromosomes 1, 2 and 5, and it was later shown that some genomic changes such as isochromosome formation confer azole resistance in C. albicans.^{154  ,  155}

Overrepresentation of efflux pumps, ERG11 or ZCF genes involved in azole resistance as a result of chromosomal non-disjunctions, trisomy or isochromosome formation is very likely to contribute to decreased drug resistance in these cells. Indeed, it has been shown for both TAC1 and MRR1 that loss of heterozygosity was responsible in some clinical isolates for homozygous gain-of-function mutations conferring increased efflux pump expression and azole resistance.^{81  ,  104} Recent analysis of large-scale genomic changes in 43 clinical isolates of C. albicans showed that, while loss-of-heterozygosity events were persistent and often associated with resistance, aneuploidies in clinical isolates were often transient and did not correlate with drug resistance.¹⁵⁶ The discovery of tetraploid cells of C. albicans, containing four homologous sets of chromosomes, and their ability to generate a diverse range of fitness and heterogeneity through increased rates of heterozygosity loss suggests that aneuploidy plays an important role in adaptive fitness to selective pressures under these circumstances.^{157  ,  158} The recent investigations into trisomy of chromosome R and chromosome 4 possibly conferring azole resistance in two different clinical isolates of C. albicans also underscores the importance of aneuploidy in the microevolution of drug-resistant C. albicans.^{117  ,  159}

Azole import

Currently there is limited evidence suggesting that reduced azole uptake has any clinical relevance to azole resistance in C. albicans or any other Candida species. However, it should be mentioned that it is plausible that reduced azole import could be a potential uncharacterized mechanism of drug resistance. In a study examining the in vitro accumulation of fluconazole in the Candida cell, it was observed that fluconazole import displayed saturation kinetics in de-energized cells unable to efflux drug via active transport mechanisms like the ABC transporter Cdr1.¹⁶⁰ Additionally, maximal accumulation of fluconazole did not progress in a temperature-dependent manner, inconsistent with simple passive diffusion. The kinetics of fluconazole import, therefore, suggest that fluconazole is taken up by means of a carrier protein via facilitated diffusion and that this mechanism is likely conserved across many fungal species.^{160  ,  161}

The carrier protein(s) responsible for importing fluconazole and presumably the other azoles, however, has/have not yet been identified despite prolific research. In theory, cells possessing defective carrier protein (via mutation, for example) or protein in low amounts would not accumulate lethal or significantly inhibitory amounts of drug, resulting in resistance.

Hsp90 axis

While not directly involved in azole resistance, Hsp90 is an important molecular chaperone for which there is increasing evidence of its regulatory role in azole resistance development. Hsp90 is involved in the folding of a diverse set of client proteins and the regulation of signal transduction pathways and has been shown to potentiate the evolution of drug resistance traits, including some azole resistance mechanisms.^162–167 In C. albicans, for example, serial clinical isolates taken from the same patient treated with fluconazole over 2 years showed that azole resistance was initially dependent on Hsp90 and its client protein calcineurin, though resistance evolved towards Hsp90-independent resistance mechanisms over time.¹⁶⁴ It is worth noting that by stabilizing the calcineurin protein and the pathway that is essential in the cellular drug-induced stress response, Hsp90 has been shown to affect not only azole resistance but resistance to the echinocandin drug class as well.^{165  ,  167  ,  168} Additionally, Hsp90 has been shown to be involved in the regulation of protein kinase C (PKC) signalling, which is involved in the activation of the calcineurin pathway and subsequent drug-induced stress response to the azoles.¹⁶⁶ More recently, it was discovered that Hsp90 is also involved in azole tolerance as part of the Rim pathway in C. albicans.¹⁶⁹ This, on top of its involvement in C. albicans morphogenesis and virulence trait development, undoubtedly makes Hsp90 an attractive therapeutic target, especially as its role in azole resistance becomes increasingly defined.

Biofilms and drug resistance

Biofilms are an active, complex, 3D network of cells that adhere to solid surfaces or are present at liquid–air interfaces. C. albicans forms highly structured biofilms consisting of cells at different stages of growth, i.e. budding yeast-form cells (round), oval pseudohyphal cells and cylindrical hyphal cells surrounded by extracellular matrix. Biofilms are intrinsically resistant to antifungal drugs, in particular the azoles, leading to therapeutic failure. Various studies have described the influence of high fungal cell density, reduction in growth rate, lack of nutrition availability, production of extracellular exopolysaccharide matrix, presence of recalcitrant ‘persister’ cells that exhibit tolerance to antifungal drugs, gene expression alterations and alterations in membrane sterol content on the Candida cell membrane in this setting. Certain efflux pumps in planktonic cells are often up-regulated within the first few hours of surface contact with azole antifungals and remain up-regulated throughout the biofilm development.^170–174 This up-regulation of efflux pumps results in biofilm recalcitrance to treatment with antifungal agents. Biofilm drug resistance may also be attributed to the secreted extracellular matrix, which acts as a physical barrier to drug penetration and thereby directly contributes to the structural integrity of the biofilm.^175–178 Moreover, the biofilm matrix glucan has demonstrated the ability to bind and sequester antifungal drugs, including fluconazole, preventing them from acting intracellularly.¹⁷⁹ The polysaccharide β-1,3-glucan and the pathway involved in its delivery and accumulation in the biofilm matrix is therefore believed to be contributing to drug resistance properties of the biofilms.

Azole tolerance

C. albicans is usually classified as either susceptible (and susceptible dose-dependent) or resistant when referring to clinical antifungal breakpoints, which are used in helping to predict response to therapy against fluconazole or other antifungal agents.^{30  ,  46  ,  60  ,  180  ,  181} Antifungal ‘tolerance’ is an oft-used and loosely defined term that refers generally to the residual growth of cells at or above inhibitory concentrations of drug.

Unlike resistant cells, which may be able to survive drug treatment due to gene-dependent mechanisms directly affecting the drug, drug target or accumulation of the drug in the cell, cells displaying tolerance have been defined as those able to survive transient exposure to drug without an accompanying change in MIC.^182–184 The ‘trailing growth’ phenomenon, describing the residual population of cells that grow at supra-MIC levels observed in broth dilution assays, can be considered related to azole tolerance and has been well documented in C. albicans.^184–187 Recently, RTA3, encoding a putative lipid translocase coordinately up-regulated with CDR1 in clinical isolates with Tac1 gain-of-function mutations, has been associated with increased azole tolerance and trailing growth in C. albicans.¹⁸⁸

The presence and degree of trailing growth has been known to be affected by different parameters such as temperature and pH and can affect the MIC interpretation depending on the time of reading (24 h versus 48 h).^{186  ,  189  ,  190} However, the clinical significance of trailing growth has been dubious at best. Past literature suggests that the C. albicans isolates exhibiting trailing growth phenomena respond to azole treatment similarly to susceptible isolates in murine models and in a small sample of HIV-infected patients with recurrent oropharyngeal candidiasis.^191–193

Importantly, a recent study delineated parameters to describe the antifungal tolerance observed with certain strains. The ‘fraction of growth’ (FoG) measured on antifungal disc diffusion assay and the ‘supra-MIC growth’ (SMG) as measured by broth microdilution assay are objective measurements assessing growth either inside the zone of inhibition or above the MIC for a given strain, respectively.¹⁹⁴ Neither parameter correlated with MIC and instead measured a drug response distinct from MIC. Interestingly, FoG and SMG were independent of inoculum size, cell density or drug concentration and, in contrast to tolerance in bacteria, tolerant C. albicans cells did not have reduced growth rates. Additionally, FoG did not correlate with growth rate or colony size, indicating that high levels of FoG are not due simply to faster cell growth. Instead, C. albicans isolates with high levels of FoG or SMG suggest a larger subpopulation that can respond to high levels of antifungal drug stress better than isolates with low FoG or SMG.

When comparing the drug responses of clinical isolates taken from patients with persistent candidaemia versus patients with candidaemia that was cleared after a single treatment course of fluconazole, significantly higher FoG and SMG levels were observed in clinical isolates originating in patients with persistent candidaemia.¹⁹⁴ This was despite the fact that the tested isolates in both groups all had in vitro MICs considered to indicate fluconazole susceptibility and identified FoG and SMG as potential parameters to guide treatment outcome in patients with candidaemia. This tied with the identification of several adjuvant drugs that could reduce FoG but not MIC in clinical isolates suggests that drug tolerance in C. albicans may be an overlooked issue of significant clinical relevance.

Future directions

As we have discussed, C. albicans has an arsenal of defence mechanisms that it uses, alone and in combination, to overcome inhibition by the azole antifungals. Despite the numerous resistance pathways already discovered, continued investigation is required to fully characterize azole resistance in C. albicans. For example, the azole importer(s) in Candida and other fungal species is/are yet to be identified and its/their discovery would plausibly reshape our approach to overcoming azole resistance.

Ongoing research is also identifying potential novel mechanisms of azole resistance, though the clinical significance of these are still unknown. For example, though there is a report of mutations in other ergosterol biosynthesis genes, such as ERG5, conferring azole resistance in C. albicans, this report is isolated.¹²⁹ Recent literature showed that alteration of sphingolipid synthesis mediated through FEN1 and FEN12 deletion results in fluconazole resistance and these composition changes are similar to what is observed in WT cells treated with fluconazole.¹⁹⁵ Furthermore, transposon-based mutagenesis and disruption of the C. albicans genome in a haploid strain identified a number of potential genes involved in drug stress response through Gene Set Enrichment Analysis, providing further insight into C. albicans acquisition of drug resistance.¹⁹⁵

As more of the machinery driving azole resistance becomes unveiled, we can begin to isolate the cogs critical to its function. Targeting these components with novel drug therapeutic approaches and improved use of our existing armamentarium becomes the immediate goal in addressing the rising problem of azole resistance and antifungal drug resistance in general. Moreover, understanding the molecular and genetic basis by which C. albicans gains resistance provides the foundational knowledge needed to predict and possibly prevent azole resistance through improved treatment practices and rapid diagnostics in the coming years.

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