Antifungal Drug Resistance: Molecular Mechanisms in Candida albicans and Beyond

Abstract

Fungal infections are a major contributor to infectious disease-related deaths across the globe. Candida species are among the most common causes of invasive mycotic disease, with Candida albicans reigning as the leading cause of invasive candidiasis. Given that fungi are eukaryotes like their human host, the number of unique molecular targets that can be exploited for antifungal development remains limited. Currently, there are only three major classes of drugs approved for the treatment of invasive mycoses, and the efficacy of these agents is compromised by the development of drug resistance in pathogen populations. Notably, the emergence of additional drug-resistant species, such as Candida auris and Candida glabrata, further threatens the limited armamentarium of antifungals available to treat these serious infections. Here, we describe our current arsenal of antifungals and elaborate on the resistance mechanisms Candida species possess that render them recalcitrant to therapeutic intervention. Finally, we highlight some of the most promising therapeutic strategies that may help combat antifungal resistance, including combination therapy, targeting fungal-virulence traits, and modulating host immunity. Overall, a thorough understanding of the mechanistic principles governing antifungal drug resistance is fundamental for the development of novel therapeutics to combat current and emerging fungal threats.

Figure 1.

Antifungal mechanisms of action and structures. (A) Polyenes, such as amphotericin B, act as a fungicidal “sterol sponge” by forming extramembranous aggregates that extract ergosterol from lipid bilayers. (B) Azoles exert fungistatic activity by inhibiting lanosterol 14-α-demethylase (encoded by ERG11), which leads to a block in ergosterol synthesis and the accumulation of toxic sterol intermediates, including 14-α-methyl-3,6-diol produced by Erg3. (C) Fungal cell walls are composed of (1,3)-β-d-glucan covalently linked to (1,6)-β-d-glucan, as well as chitin and mannan. Echinocandins prevent the synthesis of (1,3)-β-d-glucan by inhibiting the (1,3)-β-d-glucan synthase (encoded by FKS1 in C. albicans and C. auris and by both FKS1 and FKS2 in C. glabrata); this results in a loss of cell wall integrity and severe cell wall stress. The structure of a representative antifungal from each class is depicted to the right of each mechanism. Adapted with permission from ref . Copyright 2008 Springer Nature.

Figure 2.

Molecular mechanisms of antifungal drug resistance. (A) Resistance to polyenes is primarily mediated by the depletion of the target ergosterol through loss-of-function mutations in ergosterol biosynthesis genes. This leads to the production of alternate sterols, which do not effectively interact with polyenes and therefore are not extracted from the fungal cell membrane. (B) Resistance to azoles can occur through substitutions to the azole target, Erg11, which leads to lower drug-binding affinity for the lanosterol demethylase enzyme (left panel). Overexpression of the drug target can occur through gain-of-function mutations in the transcriptional activator, UPC2, or through the formation of aneuploidies, such as [i(5L)], which directly increase the copy number of ERG11 (middle panel). Azole resistance is also acquired through the upregulation of ABC transporters (yellow), including Cdr1 and Cdr2, by activating mutations in specific transcription factors (TAC1 in C. albicans and C. auris and PDR1 in C. glabrata). Additionally, overexpression of the MF transporter (pink), Mdr1, through activating mutations in the transcriptional factor, MRR1, confers azole resistance (right panel). Efflux pumps can also be overexpressed through aneuploidy formation. (C) Resistance to echinocandins primarily involves mutations in FKS genes that encode the catalytic subunit of the drug target (1,3)-β-d-glucan synthase. For C. albicans and C. auris, mutations conferring echinocandin resistance occur in FKS1, while for C. glabrata, mutations occur in both FKS1 and its paralogue FKS2.

Figure 3.

Cellular stress responses important for mediating cell wall and cell membrane integrity. A global cellular regulator governing antifungal tolerance and resistance is the molecular chaperone, Hsp90. In C. albicans, Hsp90 is post-translationally regulated by the protein kinase complex CK2 as well as lysine deacetylases (KDACs). Key client proteins of Hsp90 important for mediating cell wall and cell membrane stress responses include the protein phosphatase calcineurin and several components of the PKC cell wall integrity pathway (Pkc1, Bck1, Mkk2, and Mkc1). Additional cellular factors important for mediating tolerance and resistance to the echinocandins include the transcription factor Cas5, the eukaryotic chaperonin containing TCP-1 (CCT) complex, and the protein kinase Yck2. Notably, many other cellular factors contribute to these cellular stress responses, with many more enigmatic regulators that remain to be identified. Perturbation of any of these signaling pathways in combination with the cell wall or cell membrane stress elicited by the echinocandins and azoles, respectively, culminates in fungal growth inhibition.

Figure 4.

Mechanisms of drug potentiation. (A) Two drugs can potentiate the activity of one another when one drug’s action (drug B) increases the bioavailability of another (drug A) within the target cell. For example, in C. glabrata, the small-molecule iKIX1 (structure shown on right) synergizes with the azoles and resensitizes resistant isolates to fluconazole. iKIX1 disrupts the interaction between the KIX domain within the Gal11/Med15 mediator complex (dark purple) and Pdr1, which prevents upregulation of genes encoding efflux pumps (such as PDR5) in response to the azoles. (B) Two drugs targeting proteins of parallel pathways that converge on a single, essential process or function may also display potentiation activity. Pharmacological inhibition of the nonessential stress kinase Yck2 with the novel 2,3-aryl-pyrazolopyridine compound, GW, potentiates echinocandin efficacy in C. albicans and exacerbates echinocandin-mediated cell wall disruption. (C) Fungal stress responses play a major role in basal antifungal tolerance as well as resistance. Hsp90 is a global regulator of stress-response circuitry, promoting tolerance and resistance to drug-induced stress. Key regulators of cellular stress responses (light blue) are stabilized by Hsp90 in response to antifungal treatment, enabling signal transduction that is necessary to survive in the presence of a drug-induced stress. Pharmacological compromise of Hsp90 using the fungal-selective inhibitor CMLD013075 blocks these signaling networks, thereby preventing the evolution of resistance and abrogating resistance once it has evolved.

Figure 5.

Targeting fungal virulence and the host immune response as alternative antifungal strategies. (A) Thwarting fungal infection can be achieved through manipulation of key fungal virulence traits. The C. albicans morphogenetic transition between yeast and filamentous states enables it to adhere to, damage, invade, and disseminate through host tissues, as well as to form biofilms. The small-molecule filastatin inhibits C. albicans filamentation, surface adhesion, and biofilm formation. (B) Vaccination is a potential therapeutic approach to combat fungal disease, particularly in vulnerable populations. Development of the novel anti-Candida vaccine NDV-3A employs the highly immunogenic adhesin and invasin Als3 to elicit an adaptive immune response mediated by antibody and T-cell responses. Antibody-facilitated opsonization for phagocytosis, T-cell mediated proliferation of proinflammatory cytokines IL-17 and IFN-γ, and enhancement of C. albicans neutrophil killing are characteristic of immunization and facilitate protective immunity in the host.