**1. Introduction**

*Candida albicans* is the most important opportunistic commensal yeast that asymptomatically colonizes the skin, oral cavity, gastrointestinal and genitourinary tracts in healthy people. However, it can cause superficial and invasive infections, especially in immunocompromised individuals [1–3]. Actually, invasive infections due to *Candida* species are considered among the main causes of morbidity and mortality in hospitalized patients. Although there are at least 15 *Candida* species related to human disease, more than 90% of the invasive diseases are related to *C. albicans*, *Candida glabrata*, *Candida tropicalis*, *Candida parapsilosis*, and *Candida krusei* [4–6]. *C. albicans* infections is considered the fourth most common

#### **Figure 1.**

*Mechanisms of action of main antifungals families in the fungal cell. (a) Azoles disrupt the ergosterol synthesis by inhibiting the enzyme 14-α-lanosterol demethylase (ERG11) involved in the transformation of lanosterol into ergosterol in the endoplasmic reticulum. (b) Polyenes disrupt the cell membrane by binding to ergosterol resulting in pore formation. (c) Echinocandins inhibit 1,3-*β*-d-glucan synthase (FKS ½) which causes disruption of the cell wall.*

opportunistic infection in hospitals. Invasive candidiasis (IC) is fatal in about 42% of the reported cases, despite the use of antifungal therapies [7, 8].

Nowadays, the most widely used antifungal drugs for IC include: A) azoles, for instance fluconazole (FLZ), itraconazole (ITC), voriconazole, posaconazole, isavuconazole; B) polyenes such as amphotericin B (AMB); C) echinocandins like caspofungin, micafungin, and anidulafungin [9–11].

These antifungal compounds act on different parts of the fungal cell (**Figure 1**). Azoles interrupt the ergosterol biosynthesis, the main component of the fungal membranes [10, 12, 13]. Polyenes such as AMB interact with ergosterol making pores in the cell membrane [10, 12–14]; while echinocandins act blocking the synthesis of β-d-glucan located in the fungal cell wall [13, 15]. The gradual risk increment for Candida infection and the greater use of antifungal agents has increased resistance towards *Candida* spp. Pharmacological failures in *Candida* spp. treatments have drawn attention to the problem of resistance to antifungals and their molecular mechanisms. *C. albicans* inherently is susceptible to azoles, polyenes, and echinocandins. Mono-resistance to azoles or echinocandins has been reported, as well as combined resistance to azoles and amphotericin, but resistance to multiple compounds that covers all three drug classes is a rare phenomenon and few cases have been reported in *C. albicans* [10, 12, 16].

The following chapter offers an overview of the main genetic mechanisms contributing to the antifungal resistance in *C. albicans*, besides giving an approach for alternative-compounds proposed against their infection.

### **2. Molecular mechanisms of antifungal resistance**

#### **2.1 Azoles**

Fungi cell membrane is mainly integrated by ergosterol, a sterol contributing to several cellular functions, besides modulating membrane fluidity and the structure and function of membrane proteins. The azoles mechanism of action is to inhibit

#### *Molecular Mechanisms of Resistance to Antifungals in* Candida albicans *DOI: http://dx.doi.org/10.5772/intechopen.96346*

14α-lanosterol demethylase, encoded by the *ERG11* gene, which converts lanosterol to ergosterol in the cell membrane (**Figure 1**). This enzyme contains an iron protoporphyrin unit in its active site. Azoles bind to iron causing the blockage of the ergosterol biosynthetic pathway [17–19]. The interruption of ergosterol synthesis allows the accumulation of 14α-methyl sterols, which alters the membrane's stability, permeability, and the action of the enzymes bound to it [20].

The evolution of antimicrobial agent's resistance is common, as there are many microbes able to develop strategies against drugs action. The incremented azoles resistance is mainly a result of their fungistatic rather than fungicidal nature [17–19]. The mechanisms of resistance to azole antifungal agents have been elucidated in *Candida* spp. species and can be classified mainly as: 1) changes in cell wall or in plasma membrane, leading to poor drug absorption; 2) alterations in the affinity of the target drug (i.e. *ERG11* gene), due to a site mutation or its overexpression; 3) drug efflux mediated by membrane transporter proteins belonging to the transporters of the ATP-binding cassette (ABC), namely CDR1 and CDR2 or the transporter of the major facilitator superfamily (MFS), CaMDR1; 4) biofilm formation [18–21]. Although the resistance described in *C. albicans* strains is usually a combination of the mechanisms mentioned above (**Figure 2**) [16].

## *2.1.1 Mutations of the ERG11 target enzyme*

Mutations in the *C. albicans ERG11* gene reduce the affinity for fluconazole and have a moderate effect on posaconazole [17–19]. Several point mutations have been identified in the *ERG11* gene. In resistant strains, there are more than 140 substitutions reported, most of them have a functional additive effect. Two of the most common alterations in *C. albicans* (R467K and G464S), are located near the hemebinding site [20]. Other substitutions related to resistance are A114S, Y132H, Y132F, K143R, Y257H, and K143Q, which contribute to a significant increased resistance (more than four times) to fluconazole and voriconazole [22].

Some clinical isolates share common mutations with environmental azoleresistant strains, suggesting that some azole-resistant clinical isolates could have their origin in the environment [23]. This resistance appears to be driven by the

#### **Figure 2.**

*Schematic overview of the main mechanisms of drug resistance against azoles, AMB, and echinocandins adopted by* Candida albicans*. (a) Alteration of the enzyme target (azoles and echinocandins), (b) overexpression of drug efflux proteins (azoles), (c) Reduction of sterols in the plasma membrane (AMB), (d) increased stress tolerance and altered the fungal cell wall (echinocandins and AMB).*

agricultural use of azoles. In patients without azoles treatment, resistance has been identified derived from the environment. These cases involved a Cyp51A substitution at position 98 (from leucine to histidine), and a 34 base tandem repeat (TR) in the cyp51A promoter, leading to overexpression. Both changes are necessary to confer resistance. In particular, these resistant isolates can be crossed with susceptible strains, suggesting that resistance could be transferred through the sexual cycle. Strains with these alterations have emerged throughout Europe and beyond. Additionally, a new environmentally selected resistance mutation (TR46, Y121F, T289A) was reported among patients in the Netherlands [20].

#### *2.1.2 Dysregulation of the target enzyme ERG11*

One way to decrease the drug effective concentration is the overexpression of *ERG11* [17]. This overexpression is common among azole-resistant *C. albicans* clinical isolates. This contributes directly to resistance, since an increase in the target requires more drug for inhibition, reducing susceptibility [19]. *ERG11* overexpression arises either from genetic dosing through gene duplication or from positive regulation of the gene by trans-acting factors [23]. Multiple mechanisms explain the constitutive overexpression of *ERG11* in azole-resistant clinical strains. First, amplification of the *ERG11* gene can occur by the formation of an isochromosome with two copies of the left arm of chromosome 5 [i (5 L)], in which *ERG11* resides, or by duplication of the entire chromosome. Second, the activation of mutations in the gene encoding the transcription factor Upc2 positively regulates most of the ergosterol biosynthesis genes [18, 20].

### *2.1.3 Alteration of the ergosterol biosynthesis pathway (point mutations in ERG genes)*

Brief exposures of two to three hours to azoles cause transient upregulation of the *ERG* gene family in *C. albicans.* These data suggest a common regulation of ergosterol biosynthetic pathway in the presence of inhibitors. Longer *in vitro* exposures to azoles (minimum 24 h) leads to constitutive up-regulation of the *ERG* genes decreasing drug susceptibility [23].

Modification of the metabolic pathway can be effective at different points, as example, alteration of the last steps of biosynthesis through the inactivation of the *ERG3* gene results in no toxic methylated sterols production, leading to azole crossresistance. Furthermore, mutations in non-essential genes of this pathway (*ERG3, ERG6, ERG24,* and *ERG2*) also lead to a decrease, or even a total absence, of ergosterol in the plasma membrane [17]. Lanosterol demethylase inactivity or defectiveness due to azoles induce ergosterol depletion and toxic 14α-methyl-3,6-diol sterols accumulation. The presence of 14α-methyl sterols can modify the function and fluidity of the plasma membrane [21]. The additive mutation in the *ERG3* gene prevents the formation of this toxic product from 14α-methylfecosterol and leads to the accumulation of non-toxic sterols (Mishra, 2007; Shukla, 2016). Although this mechanism is not the most frequent one, it has been identified in several clinical isolates of *C. albicans* [23]. Mutations in *ERG3* are sufficient to induce azole resistance in *Candida* spp., but they are rarely associated with high resistance [20].

Four clinically isolated *C. albicans* erg3 mutants (CA12, CA488, CA490, and CA108) were reported as resistant to fluconazole, voriconazole, itraconazole, ketoconazole, and clotrimazole under CLSI test conditions. Importantly, CA12 and CA108 retained an azole-resistant phenotype even when tested in the presence of FK506, a multi-drug flux inhibitor. In contrast, CA488, CA490, along with three isolates (CA6, CA14, and CA177, in which ergosterol comprised more than 80% of the total sterol

#### *Molecular Mechanisms of Resistance to Antifungals in* Candida albicans *DOI: http://dx.doi.org/10.5772/intechopen.96346*

fraction and ergosta 7,22-dienol was undetectable) exhibited azole sensitive phenotypes in the inhibitor FK50 presence. CA108 mutant strain contains multiple amino acid substitutions in ERG3, but only a single conserved polymorphism (E266D) in sterol 14α-demethylase (ERG11). CA12 contains a substitution (W332R) in ERG3 and no residue changes in ERG11. Furthermore, CA488 and CA490 were found to harbour multiple residue changes in both ERG3 and ERG11 [24]. Furthermore, the residue 193 in ERG3 was found to play an important role in azole resistance [25].
