*2.1.4 Efflux pumps*

A mechanism to decrease the azoles intracellular concentration is increasing their output. This class of resistance is mediated by the activity of transport systems such as the pleiotropic drug resistance (PDR) class of ATP-binding cassette transporters (ABC) and major facilitators superfamily (MFS) transporters [17]. These membrane proteins translocate compounds across cell membranes actively using different energy sources. ABC proteins are primary transporters that use ATP hydrolysis. MFS pumps are secondary transporters that use the motive force of the proton across the plasma membrane. Both types of transporters contain distinctive protein domains that confer substrate specificity: nucleotide-binding domains (NBD) in ABC pumps and transmembrane domains (TMD) in ABC and MFS pumps. Fungal PDR proteins appear to share common features on both sides of the two TMDs that separate the cytosolic from the outer cytosolic space [18, 26]. This probably reflects the fact that the cytosolic part is the motor that drives the transport of a variety of substrates through the lipid bilayer through the core of the protein into the outer cytosolic space or the outer layer of the lipid bilayer [26].

*C. albicans* contains 28 ABC proteins and 96 potential MFS transporters [18]. In this species, the main transporters, related to resistance, of the ABC proteins are CDR1 and CDR2 (resistance drugs to Candida 1 and 2) [21], while for MFS it is MDR1 (Multidrug Resistance 1). CDR1 and CDR2 overexpression improves drug output and reduces its accumulation in cells [23]. Positive regulation of MDR1 results in increased azole output [17]. Several cis-acting regulatory elements responsible for the regulation of the *CDR1* and *CDR2* genes have been identified. Promoter deletion studies have revealed five different regulatory elements in the CDR1 promoter, including one BEE (basal expression element), one DRE (drugsensitive element), two SRE (steroid sensitive element), and one NRE (negative regulatory element). Internal deletions of the BEE and DRE motifs in the CDR1 promoter affect baseline CDR1 expression and drug-induced expression, respectively. SRE1 and SRE2 are involved in steroid hormone responses: SRE1 responds only to progesterone and SRE2 to progesterone and β-estradiol. Finally, the deletion of the NRE motif leads to an increase in the baseline expression of CDR1. In contrast to CDR1, the CDR2 promoter contains only one DRE motif. Among these diverse cis-acting elements, DRE is the only element involved in constitutive high expression and transient up-regulation of CDR1 and CDR2. In *C. albicans*, CDR1 is the main contributor to azole resistance among ABC transporters [23, 26].

In *C. albicans* a gene encoding a CaNdt80p protein similar to the *Saccharomyces cerevisiae* meiosis-specific transcription factor Ndt80p has been identified. Alteration of CaNdt80 affects the basal expression of CDR1 and reduces its ability for upregulation in the presence of miconazole. More recently, Ndt80p was involved in the global effect of azole resistance through its regulon, including several genes implicated in ergosterol metabolism [23]. Additionally, MDR1 is the only MFS transporter involved in the azole resistance of clinical isolates. MDR1 usually does not express detectable levels in fluconazole-susceptible isolates but is constitutively up-regulated in some fluconazole-resistant strains. A region called BRE (benomyl response

element) or MDRE (*MDR1* drug resistance element), respectively, was identified. This region is responsible for the constitutively high expression of *MDR1* in fluconazole-resistant isolates. Hyperactive alleles confer a constitutive overexpression of *MDR1* and therefore, resistance to fluconazole [23]. *MDR1* expression in *C. albicans* cells is enhanced by benomyl, methotrexate, and several other unrelated drugs, and found to be more pronounced in some of the azole-resistant clinical isolates [21].

The up-regulation of ABC and MFS transporters is mediated by specific regulations in resistant fungal pathogens. Point mutations defined as gain-of-function (GOF) mutations in these regulators confer an inherently high level of expression of the transporters in drug-resistant strains. GOF mutations in the transcription factor Upc2p led to increased resistance to fluconazole in *C. albicans* [17]. GOF mutations in the transcription factors TAC1 and MRR1 lead to upregulation of the CDR1/CDR2 and MDR1 drug efflux pumps, respectively [16, 18]. An important question related to strategies to overcome efflux-mediated antifungal resistance is the relative contribution of each efflux pump protein to clinically significant antifungal resistance in *C. albicans*. It is now clear that the CDR1, CDR2, and MDR1 transporters are the main efflux pumps that mediate resistance of *C. albicans* to azole drugs. However, MDR1 is relatively specific for fluconazole, while many azole drugs can act as substrates for CDR1 and CDR2. Interestingly, several fluconazole-resistant *C. albicans* isolates overexpress only CDR1 and CDR2, but not MDR1, while other strains overexpress only MDR1, reflecting the existence of at least two different transcriptional pathways that are responsible for the upregulation of these genes in azoles [26].

#### **2.2 Polyenes**

The potent fungicidal activity of polyenes derives from their ability to selectively bind sterol at the fungal cell membrane (**Figure 1**). Four models have been proposed as the mode of action for polyenes: 1) the pore formation model, 2) the surface adsorption model, 3) the sterol sponge model, and 4) the oxidative damage model [14]. The pore formation model is the most studied mechanism, where polyenes are directly intercalated with the ergosterol membrane forming ion channels that permeabilize and kill yeast cells [14, 27]. Additionally, indirect mechanisms of fungal cells damage have been identified due to the effect of polyene compounds, such as those mediated by reactive oxygen species (ROS) and by the secretion of interleukin-1β (IL-1β) by host cells [28, 29].

The polyene AMB is a broad-spectrum drug and is one of the main antifungals used for ICs [10, 14]. AMB is heptane isolated from *Streptomyces nodosus* producing high toxicity. Hence, a liposomal AMB (Ambisome R) has been developed to minimize side effects and increase treatment efficacy [10, 14, 30, 31]; however, the high costs of this drug limited its use. Resistance to AMB is rare, despite 50 years of clinical use as monotherapy, although resistant *C. albicans* strains have been found in different studies [32–35]. The alterations in the composition of the sterols and phospholipids of the membrane, the regulation of oxidative stress, and alterations of the fungal cell are the more frequent resistance mechanisms described for AMB in fungi [10, 12, 14]. In *C. albicans*, resistance to AMB is associated with ergosterol replacement by a precursor molecule or by sterols reduction at the plasma membrane (**Figure 2**) [10, 12, 14].

#### *2.2.1 Alteration in the composition of sterols in the cell membrane (mutations in ERG genes)*

The most common mechanism for acquired resistance to AMB in *C. albicans* is attributed to alterations in the composition of sterols of the fungal cell membrane [10, 12, 14, 36]. Different mutations in *ERG* genes (*ERG11, ERG3, ERG2,* and

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

*ERG6*) have been associated with this mechanism in *Candida* spp. [14, 37, 38]. Loss of function of the *ERG11* and *ERG3* genes (lanosterol 14α-demethylase and C-5 sterol desaturase, respectively), leads to the exchange of ergosterol for alternative sterols such as lanosterol, eburicol, and 4,14-dimethyl-zymosterol in the membrane of *C. albicans*, [14, 36, 39]. Resistance to AMB in *C. albicans* is also associated with an aminoacidic substitution in *ERG11* and with *ERG5* (sterol desaturase C-22) disfunction, again associated with an alternative membrane sterol composition [14, 39, 40]. In other *Candida* spp., the inactivation of *ERG6* [11, 14, 37] and *ERG2* had a similar effect [11, 14]. Resistance to AMB is rarely found in combination with resistance to other antifungal drugs, although certain mutations that induce resistance to polyenes can lead to cross-resistance to azoles [14, 36, 41].

#### *2.2.2 Response to oxidative stress and alterations in the cell wall*

Fungal resistance mechanisms are also related to oxidative stress regulation, allowing the cell to tolerate exposure to AMB [14, 30]. In *C. albicans*, one of the described mechanisms of stress tolerance to AMB includes the heat shock protein 90 (Hsp90) molecular chaperone, which regulates a large number of proteins involved in several fungal cellular processes [42, 43]. In addition to alterations in the composition of sterols in the plasma membrane and the regulation of oxidative stress, studies in fungi have correlated resistance to AMB with fungal cell wall alterations [14, 44, 45]. In AMB resistant *C. tropicalis* strains, an enlargement of the cell wall has been observed with increased levels of 1,3-β-glucans [14, 44], suggesting an affectation in the penetration of AMB through the cell wall [14, 45].

#### **2.3 Echinocandins**

Echinocandins are lipo-peptides that inhibit 1,3-β-d-glucan synthetase, which is responsible for the biosynthesis of 1,3-β-d-glucan, one of the main components of the fungal cell wall, causing osmotic instability and therefore the death of fungal cells (**Figure 1**) [10, 13]. This class of drugs has certain advantages attributable to its effects on the fungal cell wall, including a lower risk of side effects since animal cells do not have this structure [10]. Echinocandins have a limited spectrum, but for *Candida* species, they have broad fungicidal activity. The 1,3-β-d-glucan synthetase target comprises a GTP binding protein Rho, which helps regulate the biosynthetic capacity of glucan synthetase, and a catalytic subunit, FKS, which encodes three related genes, *FKS1*, *FKS2*, and *FKS3*. *FKS1* is essential in *C. albicans* and other *Candida* spp. Whereas *FKS1* and *FKS2* are functionally redundant in *C. glabrata*, *FKS3* is very low expressed compared to other genes [46], not being a significant contributor to biosynthetic capacity in general.

Echinocandins are the first major new class of antifungal drugs on the market in decades. Consequently, it is of vital importance to assess the nature of the resistance mechanism to this class of drugs. Mutations that affect the target site are the most likely resistance mechanism that exists (**Figure 2**), since unlike azoles, echinocandins are poor substrates for drug exit through efflux transporters, ruling out this mechanism of resistance [10, 13]. Specific mutations have already been reported in two highly conserved regions of the Fks1 subunit of glucan synthetase, a membrane protein, which can confer resistance *in vitro* in *Candida* isolates to caspofungin, the first echinocandin approved for the treatment of yeast infections [10, 13, 47, 48]. Other ways in which there may be the acquisition of resistance to echinocandins in *C. albicans* is through different response pathways to cellular stress, as well as some clinical factors such as empirical therapy, prophylaxis, gastrointestinal reservoirs, or intra-abdominal infections.

#### *2.3.1 Acquired FKS mutations*

Resistance-associated amino acid substitutions occur in two highly conserved hot-spot (HS) regions of the FKS genes. The residues they encompass are Phe641– Pro649 and Arg1361 in *C. albicans* and other *Candida* spp. Substitutions of amino acids Ser645 and Phe641 cause 75% resistance in *C. albicans* [10, 13]. Pharmacodynamic studies conducted in murine models infected with *C. albicans* demonstrated that mutations in the *FKS1* gene confer resistance to echinocandins [48, 49]. Mutations in *FKS1* lead to a decrease in the virulence of *C. albicans* in murine models of IC. Furthermore, high doses of caspofungin are effective against *C. albicans*, including resistant isolates that presented point mutations in *FKS1* [50, 51]. Several studies have reported that mutations in the *FKS1* gene produce changes in the morphology of the cell wall of *C. albicans*, observing a decrease in 1,3-β-d-glucan levels in contrast to the increased amount of chitin in response to echinocandin exposure [51]. Data suggest that increased chitin in the *C. albicans* cell wall could provide a window of opportunity to acquire mutations in *FKS1*, even without exposure to caspofungin [52].

#### *2.3.2 Adaptive stress responses*

The fungal cell wall is a dynamic structure that changes during growth and development, requires 1,3-β-d-glucan crosslinking, an essential polymer for the survival of the fungal cell. Echinocandins alter the integrity of the cell wall and induce stress in the cell. In response to this, the fungal cell possesses a repertoire of mechanisms to protect the cell against such destabilization. Protection against cell wall weakening is induced through a variety of stress adaptation mechanisms, which involve protein kinase C (PKC), calcineurin, and Hsp90 [10, 13]. Stress signals in the cell wall are transmitted through the Rho GTPase, which mobilizes various effectors. Its activation alters several carbohydrate polymers along with the structure and remodelling of the cell wall. The Hsp90 heat shock protein organizes a cellular stress response circuit that has a major impact on resistance to echinocandins. Also, the genetic or chemical modulation of the Hsp90 protein reduces tolerance to echinocandins [52]. In response to the inhibition of FKS by the action of echinocandins, a greater amount of chitin is produced helping to maintain the integrity of the cell wall as chitin replaces 1,3-β-d-glucan, thus reducing sensitivity to drugs [10, 13, 48].
