Keywords

14.1 Introduction

Fungal infections have emerged as one of the major causes of human disease, especially in immunocompromised individuals (Shapiro et al. 2011). Fungal infections which are generally superficial can also turn into systemic infections as the disease incidence prolongs (Cannon et al. 2009; Brown et al. 2012). Among the different mycotic infections caused by these opportunistic fungi, candidiasis, an infection caused by Candida, is the most threatening due to severity of the disease and higher worldwide occurrence. Other fungal diseases like cryptococcal meningitis and invasive aspergillosis are also life threatening (López-Martínez 2010). The pathogenicity of fungal infections proceeds in well-organized steps. For example, Candida cell surface adhesion factors first promote its adherence to host surface, followed by an invasion and damage of the host tissues due to release of various virulence factors (Gow and Hube 2012). Eukaryotic fungal pathogens pose an additional therapeutic challenge since they show close evolutionary relationship with the human hosts, thus minimizing the choice of novel drug targets that can be exploited to selectively kill the pathogen (Heitman 2011; Shapiro et al. 2011). Nonetheless, there are many drug categories currently in use against fungal infections which exploit exclusive novel fungal targets. For example, most antifungals are directed against ergosterol which is a typical sterol of fungal cells. The sterol component of cell membranes of fungi is targeted either by blocking the enzymes important for its synthesis or by directly depleting the ergosterol from the plasma membrane (PM) (White et al. 1998). In addition, several other drugs, now in use, also target unique components of cell wall (CW) in fungal cells (White et al. 1998). The fact that the increase in incidence of worldwide fungal infections and emergence of antifungal drug resistance which outcompetes the development of novel antifungal compounds, it becomes important to understand the various facets of infections and to understand the basic mechanisms that govern the development of resistance. This enforces the widening of the hunt for the development of new antifungals targeting novel pathways. This chapter focuses on some of the aspects of antifungals, their mechanisms of action and development of resistance against them.

14.2 Antifungal Drugs

The limited availability of antifungals is a major impediment for the effective treatment of fungal infections (Vandeputte et al. 2012). This is further compounded by the fact that the generation of newer antifungals has lagged behind when compared to the pace of emergence of fungal infections. The components of the fungal CW such as mannans, glucans and chitins; and a few of the enzymes of the ergosterol biosynthetic pathways which are unique to fungal cells are commonly targeted for the development of antifungal agents (St Georgiev 2000; Munro et al. 2001). Among the enzymes of the ergosterol biosynthetic pathway, squalene epoxidase (ERG1), 14α-lanosterol demethylase or CYP51 (ERG11), Δ14–reductase (ERG24) and Δ8-Δ7-isomerase (ERG2) have been the targets of most antifungal agents (Fig. 14.1) (Sanglard et al. 2003). Some of the commonly used antifungal drugs and their mechanisms of action are discussed below:

Fig. 14.1
figure 1

Ergosterol biosynthesis pathway showing specific point of action of select antifungal drugs. Different classes of antifungal drugs are shown on left against the steps of their action in pathway with corresponding enzymes catalyzing the reaction steps shown on right

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14.2.1 Azoles

The fungistatic azoles primarily act on ergosterol biosynthesis by targeting 14α-lanosterol demethylase encoded by ERG11 gene resulting in the inhibition of cytochrome P450-dependent conversion of lanosterol to ergosterol (Fig. 14.1). The resulting ergosterol depletion interferes with the bulk functions of ergosterol as a membrane component, but more importantly, severe ergosterol depletion may also interfere with the “sparking” functions of ergosterol, affecting cell growth and proliferation (White et al. 1998; Sanglard et al. 2009; Shapiro et al. 2011). The blocking of 14α-demethylase results in the accumulation of toxic methylated sterols leading to the membrane stress (Shapiro et al. 2011). In case of yeast Cryptococcus neoformans, azoles such as fluconazole (FLC) and itraconazole also result in the accumulation of obtusifolione in the ergosterol biosynthetic pathway, mainly due to the inhibition of NADPH-dependent 3-ketosteroid reductase (ERG 27), catalyzing the last C-4 demethylation step in ergosterol biosynthesis (Vanden Bossche et al. 1993; Ghannoum et al. 1994).

Azoles mainly include two subclasses based on the number of nitrogen atoms in a ring; The first class includes imidazoles which consist of miconazole, oxiconazole, econazole, ketoconazole, tioconazole, and clotrimazole with two nitrogen atoms in an azole ring, while another class includes triazoles such as FLC, posaconazole, itraconazole, terconazole, and voriconazole which contain three nitrogen atoms in a cyclic ring (Fig. 14.2). Imidazoles are mainly used for the mucosal fungal infections while triazoles are administered both for the systemic as well as for the mucosal infections (Sanglard et al. 2009; Vandeputte et al. 2012). Depletion of membrane ergosterol due to the use of azoles are also shown to disrupt vacuolar ATPase functions resulting in an impairment of the vacuolar acidification and ion homeostasis (Zhang et al. 2010). Since azoles are fungistatic, their prolonged use poses greater threat of emergence of drug resistance among the surviving fungal population (Shapiro et al. 2011).

Fig. 14.2
figure 2

Structure of various azole antifungal compounds. These include imidazoles with two nitrogen atoms in a ring (i) Clotrimazole, (ii) Econazole, (iii) Ketoconazole (iv) Miconazole, (v) Oxiconazole, (vi) Tioconazole or triazoles containing three nitrogen atoms in a ring, (vii) Itraconazole (viii) Fluconazole (ix) Voriconazole (x) Posaconazole

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14.2.2 Polyenes

Polyenes are the amphipathic organic natural molecules called macrolides and are generally produced by Streptomyces (Vandeputte et al. 2012). Polyenes directly bind to ergosterol of fungal cell membranes leading to the formation of pores in membrane, resulting in the loss of ionic balance, membrane integrity and cell death (Sanglard et al. 2009) (Fig. 14.1). Polyenes mainly include amphotericin B (AmpB), natamycin and nystatin (Fig. 14.3). AmpB is mostly effective in systemic invasive fungal infections and is used generally against Cryptococcus, Candida and Aspergillus species (Lemke et al. 2005; Sanglard et al. 2009) while nystatin and natamycin are preferred for topical infections due to their low absorption (Vandeputte et al. 2012). Although polyenes are fungicidal in nature and have been in use for a long time but they show many side effects in humans which limits their use. However, lipid formulations of AmpB are less toxic and are relatively better for the treatment of fungal infections (Shapiro et al. 2011).

Fig. 14.3
figure 3

Structure of various antifungal compounds. These include polyenes (i) AmpB (ii) Natamycin (iii) Nystatin; pyrimidine analogs (i) 5 Fluorocytosine (ii) 5 Fluorouracil and Allylamine, thiocarbamates and morpholines antifungals as (i) Fenpropimorph (ii) Terbinafine (iii) Amorolfine (iv) Tolnaftate

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14.2.3 Pyrimidine Analogs

Pyrimidine analogs which include 5-fluorocytosine (5-FC) and 5-fluorouracil (5-FU) are the synthetic structural analogs of nucleotide cytosine (Fig. 14.3). Pyrimidine analog 5-FC is converted to 5-FU by cytosine deaminase which after conversion to downstream products gets incorporated into DNA and RNA during the synthesis of these biomolecules where it inhibits cellular functioning by blocking protein synthesis or inhibiting DNA replication. These drug analogs show activity against different Candida and Cryptococcus species (Lemke et al. 2005; Sanglard et al. 2009). 5-FC is rapidly absorbed and thus gives good bioavailability, however; it also shows many side effects (Lemke et al. 2005; Vandeputte et al. 2012). 5-FC is comparatively less effective antifungal drug because the fungal cells frequently develop tolerance to it. For this reason, it is generally preferred in combination therapy (Sanglard et al. 2009).

14.2.4 Allylamine, Thiocarbamates and Morpholines

Allylamines and thiocarbamates inhibit the ERG1 gene of ergosterol biosynthesis while morpholines which include fenpropimorph and amorolfine (Fig. 14.3) inhibit the ERG24 and ERG2 genes of ergosterol biosynthesis (Fig. 14.1). Allylamines include terbinafine while thiocarbamates include tolnaftate (Fig. 14.3). All of these drugs are mostly used for the control of dermatophyte fungal infections (Gubbins and Anaissie 2006; Sanglard et al. 2009).

14.2.5 Echinocandins

The lipopeptide echinocandins which include caspofungin, micafungin and anidulafungin (Fig. 14.4) are comparatively recent class of antifungal drugs which target the synthesis of CW components by acting as non-competitive inhibitors of β-1,3 glucan synthase required for β-glucan synthesis (Fig. 14.5) (Perlin 2011; Shapiro et al. 2011). Defects in the synthesis of CW components affect the integrity of fungal cells resulting in CW stress. As a result, echinocandin treated cells become osmotically sensitive, form pseudohyphae, show separation defects, reduced sterol contents and thickened CW. Echinocandins are generally non-toxic to mammalian cells because they act on specific CW synthesis pathway unique to fungal cells (Sanglard et al. 2009; Perlin 2011; Shapiro et al. 2011).

Fig. 14.4
figure 4

Structure of various echinocandin antifungal compounds targeting unique components in fungal CW. These include (i) Caspofungin (ii) Anidulafungin and (iii) Micafungin

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Fig. 14.5
figure 5

Different mechanisms of multidrug resistance adopted by fungal cells. The commonly observed mechanisms of drug resistance particularly against azoles, polyenes and echinocandins include (1) changes in membrane property/ lipid composition affecting normal drug import (2) over expression of drug efflux proteins leading to rapid drug extrusion (3) an alteration of the drug target (genes encoding ergosterol biosynthetic pathway enzymes or glucan synthases) leading to poor binding of toxic drugs to its target (4) overproduction of genes synthesizing the drug target proteins. (5) Echinocandins block the activity of glucan synthase important for synthesis of CW components affecting CW integrity leading to cell stress

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14.2.6 Emerging Novel Antifungals

Keeping in view the facts that there are limited antifungal strategies and paucity of effective drugs, there is continuous hunt for the development of novel and effective antifungals to combat the fungal infections. Many different drug categories which show promising antifungal activity are at various stages of the development and some of them are listed in Table 14.1 and few potential antifungal strategies are discussed in the following section.

Table 14.1 Novel antifungal agents showing promise for antifungal drug development either against particular fungal species or showing broad range antifungal effects
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14.2.7 Emerging Antifungal Strategies

As mentioned above, the development of novel drug candidates have not kept pace with the frequency of the development of tolerance to available drugs, it is imperative to search for the different strategies to combat fungal infections. For instance, the transcription factor Upc2 which regulates the expression of ERG genes has been exploited as a potential target for the development of antifungal drugs. Many small molecules screened from the commercially available compounds collection library have already been shown to inhibit the azole mediated up regulation of Upc2 and its target genes in S. cerevisiae and C. glabrata. However, the full potential of Upc2 remains to be explored before it could be successfully employed to improve antifungal strategies (Gallo-Ebert et al. 2014). Several new targets which include glucan synthesis, 26S proteasome, cAMP homeostasis, microtubule dynamics, and translational elongation also show great promise as antifungal targets (Roemer and Krysan 2014).

Combination therapy wherein different drugs are given in combination to treat fungal infections is among most favorite strategies. Some of the benefits of combination therapies include broad spectrum of treatment, synergy of effects between different drugs, lower doses of drug usage and lesser chances of the development of drug resistance. Synergistic effect shown by the drugs mainly occurs due to the additive effects on both CW and cell membrane components of fungal cell. For instance, the damaged CW due to one of the antifungal components potentiates effective action of drugs directed against cell membrane components. The compromised CW integrity could also facilitate permeability of drugs across cell membranes to intracellular targets. Combination of azoles and allylamines displays synergistic effects due to the inhibition of the same pathway at different steps (Tobudic et al. 2010a; Rodrigues et al. 2014). Notably, combination therapy requires critical evaluation of the possible antagonistic and agonist property of different drugs when administered in combinations (Lewis and Kontoyiannis 2001). Table 14.2 summarizes some of the drugs which show better efficacy when given in combination.

Table 14.2 Various regimes of combinatorial antifungal therapy. Various antifungal and/or non-antifungal drug combinations which show better efficacy in combination compared to the effects shown by independent drugs
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14.3 Resistance Against Antifungal Drugs

Fungal cells have developed several strategies to deal with the antifungals. They have learnt to modify the antifungal drug targets or most commonly increase the efflux of the incoming drugs. Some of the known mechanisms of MDR are depicted in Fig. 14.5 and are briefly discussed below.

14.3.1 Azole Target Protein (Erg11p) Is Modified in Resistant Isolates of Candida

The modification of the target protein represents one of the commonest mechanisms of MDR where the target protein of azoles, Erg11p, is modified by the chromosomal mutations leading to the replacement of native amino acids. This is evident from the fact that several point mutations in ERG11 gene which encodes Erg11p have been identified in clinical drug resistant isolates of Candida. Interestingly, these mutations appear to be predominantly restricted to certain hot spot regions of Erg11p. The exact placement of all the identified mutations in a 3D model of the protein confirms that these mutations are not randomly distributed but rather are clustered in select hot spot regions (Marichal et al. 1999; Wang et al. 2009). These point mutations either individually or in combination, invariably prevent normal binding of FLC to target protein by reducing the affinity of the drug towards Erg11p (Wang et al. 2009; Morio et al. 2010).

14.3.2 Azole Resistance Leads to an Overexpression of ERG11

Apart from spontaneous point mutations in ERG11 (discussed above), in many FLC resistant clinical isolates, very often an over expression of ERG11 is also observed (Hoot et al. 2011; Flowers et al. 2012; Sasse et al. 2012). The zinc cluster transcription factor Upc2p regulates the expression of ERG11 and other genes involved in ergosterol biosynthesis (White and Silver 2005). Several studies have confirmed that an overexpression of UPC2 increases resistance of Candida cells towards azole drugs, while its disruption results in cells hypersusceptibility to azoles. A comparison of sequence of UPC2 between genetically matched pair azole susceptible and resistant isolates led to the identification of point mutations in the encoded protein. These gain of function (GOF) mutations results in an over expression of ERG11 and hyper-resistance to azoles (Heilmann et al. 2010; Hoot et al. 2011; Flowers et al. 2012).

14.3.3 Azole Resistance and Ergosterol Biosynthetic Pathway

In clinical azole resistance, several mutations have also been detected in other ERG genes. For example, the point mutations in ERG3 also occur which could be present either alone or in combination with ERG11 mutations, resulting in a change in the ratios of various cell sterol biosynthetic intermediates, and increased tolerance to azoles and polyenes. The cytochrome P450 spectral studies performed in a system reconstituted with purified ERG5 (Δ22-desaturase or CYP61) of C. glabrata revealed an interactions between azoles and the heme-protein, implying that ERG5 could also be a target of azoles and may contribute in the development of antifungal resistance (Lamb et al. 1999). Indeed, C. albicans drug resistant clinical isolate with a combination of a single mutation in the ERG5 gene along with a stretch of amino acid duplication in the ERG11 gene has been identified (Martel et al. 2010). This mutant was not only resistant to azoles but also displayed collateral resistance to AmpB due to the depletion of membrane ergosterol (Martel et al. 2010). ERG6 in C. glabrata is involved in azole resistance due to various base pair alterations leading to missense mutations (Vandeputte et al. 2007). Similarly, an erg6 disruptant strain of C. lusitaniae was susceptible to AmpB due to decreased membrane ergosterol levels. Coinciding with this, several clinical isolates of C. lusitaniae show increased expression of ERG6 along with a decrease in ERG3 expression and enhanced resistance to AmpB (Young et al. 2003).

Together, the azole-induced upregulation of ERG11, along with other genes of the ergosterol biosynthetic pathway, suggests the existence of a common mechanism of upregulation in C. albicans (Henry et al. 2000). Transcript profiling of azole treated Candida show that almost 15 % of genes differentially expressed upon drug treatment fall under the category of sterol metabolism in a wild type strain of C. albicans (Liu et al. 2005). Notably, while a global regulation of ERG genes was evident from the transcript profiling, several genes of diverse functions as well as of unknown functions were also differentially regulated by the drug treatment (De Backer et al. 2001; Liu et al. 2005). This reinforces that azole resistance could be the result of many factors which remains to be identified. The dissection of the mechanisms mediating these phenotypes could provide newer insights into the phenomenon of MDR.

14.3.4 Drug Import Impacts Tolerance

The hydrophobic nature of drugs facilitates their easy import by passive diffusion. However, the contribution of drug import in the overall scenario of MDR is not well established. Nonetheless, there are a few instances to suggest that passive diffusion of drugs could be an important determinant of MDR. For example, fluctuations in membrane fluidity are shown to affect passive diffusion leading to an increase in susceptibility to drugs. The erg mutants of C. albicans possess high membrane fluidity, which led to an enhanced diffusion and susceptibility to azoles (Kohli et al. 2002; Prasad et al. 2010). In another study, permeability constrains imposed by Candida cells have been reemphasized in the development of MDR. It is shown that azoles can enter in C. albicans, C. kruesi and C. neoformans cells by diffusion (Mansfield et al. 2010). The kinetics of import in de-energized cells suggests that FLC import proceeds via facilitated diffusion (FD) mediated through a transporter rather than by passive diffusion. Other azoles compete for FLC import, suggesting that all the azoles utilize the same FD mechanism. FLC import was also shown to vary among C. albicans resistant clinical isolates, suggesting that altered FD may be a previously uncharacterized mechanism of resistance to azole drugs (Mansfield et al. 2010). However, the identification of a membrane transporter protein involved in FD of azoles remains elusive (Mansfield et al. 2010). Interestingly, drug inactivation which is a common mechanism in bacteria has not been observed in Candida cells.

14.3.5 Drug Efflux as a Common Strategy of Drug Tolerance

Increased efflux, which results in reduced intracellular accumulation of the incoming drugs, is another prominent mechanism of MDR in fungi (Prasad et al. 1995; Prasad and Kapoor 2005). In C. albicans, for example, this is achieved by increasing the efflux of drugs from cells by overproducing the PM efflux pump proteins. An over expression of genes encoding efflux pump proteins, particularly ABC (ATP Binding Cassette) multidrug transporter proteins Cdr1 and Cdr2 or MFS (Major Facilitator Superfamily) efflux pump protein Mdr1, have been commonly observed in azole resistant clinical isolates of C. albicans (White T et al. 2002; Karababa et al. 2004; Kusch et al. 2004; Prasad and Kapoor 2005). Invariably, MDR Candida cells, which show enhanced expression of efflux pump encoding genes, also show simultaneous increase in the efflux of drugs, thus implying a causal relationship between efflux pump encoding gene expression levels and intracellular concentration of the drug (Cannon et al. 2009). A brief description of these transporters is included, however, for more details, the reader is recommended to see the accompanying chapter “Efflux Pump Proteins of Candida in Clinical Drug Resistance”.

14.3.5.1 ABC Transporters

The inventory of ABC transporters of C. albicans revealed that there are twenty-eight putative ABC superfamily members, including twelve half transporters that largely remain uncharacterized (Gaur et al. 2005). ABC transport proteins are classified into nine families (A to I) according to the nomenclature adopted by the Human Genome Organization (HUGO) (Dean et al. 2001; Verrier et al. 2008). Of these, yeast proteins belonging to ABCB (MDR) (Thornewell et al. 1997; Sanguinetti et al. 2006; Lamping et al. 2010), ABCC (MRP) (Decottignies et al. 1998; Pagant et al. 2010) and ABCG (PDR) (Golin et al. 2007; Prasad and Goffeau 2012) transporters are most often associated with the antifungal resistance.

Full ABC proteins are made up of two (or three) transmembrane domains (TMDs) and two cytoplasmic nucleotide-binding domains (NBDs). NBDs are the nucleotide binding sites, which bind and hydrolyze ATP required to power the efflux of substrates bound within TMDs drug binding sites. Each TMD is usually comprised of six transmembrane segments (TMS), which generally are continuous alpha helices arranged to form drug binding sites (Prasad and Goffeau 2012).

The PDR protein subfamily of C. albicans comprises seven full-size members: Cdr1p, Cdr2p, Cdr3p, Cdr4p, Cdr11p, CaSnq2p and Ca4531. The C. albicans Cdr1p and Cdr2p proteins are active multidrug transporters, while Cdr3p and Cdr4p do not efflux drugs and play no apparent role in the development of antifungal resistance (Prasad and Goffeau 2012). Other transporters in related fungi, including CgCDR1 (Sanglard et al. 1999), CgCDR2 (PDH1) (Miyazaki et al. 1998) and SNQ2 (Torelli et al. 2008) in C. glabrata, ABC1 in C. krusei (Katiyar and Edlind 2001) and AFR1 in C. neoformans (Sanguinetti et al. 2006), are multidrug transporters and play a role in the development of MDR in these pathogenic species.

14.3.5.2 MFS Transporters

MFS transporters are the second major superfamily of transporters (Saier et al. 1999). A phylogenetic analysis identified 95 putative MFS transporters in C. albicans (Gaur et al. 2008). Most MFS transporters consist of two domains of six-TMSs within a single polypeptide chain with few exceptions (Stephanie et al. 1998). On the basis of hydropathy and phylogenetic analysis, the drug efflux MFS proteins can be divided into two distinct types; Drug: H+ Antiporter-1 (DHA1), consisting of 12 TMSs and Drug: H+ Antiporter-2 (DHA2) that contains 14 TMSs. MDR1 of DHA1 subfamily is a major multidrug transporter of C. albicans. Homologues of CaMDR1 have been identified from C. dubliniensis and C. glabrata, which are designated as CdMDR1 and CgMDR1, respectively (Moran et al. 1998; Sanglard et al. 1999). It appears that an increased expression of CdMDR1 is one of the main mechanisms of FLC resistance in clinical isolates of C. dubliniensis (Moran et al. 1998). Since CgMDR1 confers specific resistance to FLC, its constitutive expression in C. glabrata may be responsible for the intrinsically low susceptibility of this yeast species to triazoles (Sanglard et al. 1999).

Among all the MFS proteins, only one member, MDR1, has been implicated clinically to be involved in azole resistance in S. cerevisiae. FLU1, a close homologue of MDR1 has also been implicated in FLC resistance in S. cerevisiae. However, an over expression of FLU1 has not been detected in FLC resistant clinical isolates of C. albicans. None of the other 95 members of this superfamily are implicated in MDR (Gaur et al. 2008).

As an important MDR gene of the MFS family, MDR1 of C. albicans has been extensively studied for its role in drug resistance. The functional evaluation of critical amino acid residues of the Mdr1 protein revealed that the residues of TMS5 which harbor antiporter motifs are potentially significant for their functionality and contribute to drug:H+ transport. Independent of the substrate specificity of the antiporter, the antiporter motif in the predicted TMS5 is well conserved in all of the functionally related subgroups in bacteria and plants (Pasrija et al. 2007).

14.3.6 Echinocandin Resistance

Echinocandins inhibit the synthesis of β-1,3-glucans which is one of the major component of fungal CW (Fig. 14.5). Mutation in the FKS genes encoding echinocandin drug target glucan synthase enzyme results in its decreased sensitivity towards drug and development of resistance (Fig. 14.5) (Perlin 2007). Point mutations in FKS genes are the only known mechanism by which fungi develop resistance to echinocandin antifungal drugs (Park et al. 2005; Balashov et al. 2006). Drug resistant mutations developed in FKS genes generally fall in two “hot spots” regions of FKS1, essential for enzyme activity (Perlin 2007). Garcia-Effron et al. has also reported that mutations in Fks1 protein (Fks1p) lower the activity of β-glucan synthase without altering its affinity for the drug as is also the case with azole drug target Erg11p (Garcia-Effron et al. 2009). Fks1p mutations in “hot-spot” regions have been characterized in C. albicans as well as in non-albicans spp. (Perlin 2011). A paralog of FKS1 in C. glabrata, FKS2, is also responsible for echinocandin resistance (Perlin 2007).

14.4 Concluding Remarks

The available arsenals of antifungals targeting mostly sterols or its synthesis machinery or CW components of fungal cells are reasonably successful in combating fungal infections. However, the fungistatic nature of many of these antifungals limits their success. The synergy among different drugs is being also projected as an alternate strategy. The limited availability of antifungals and emergence of clinical drug resistance necessitate search of newer compounds and new targets. The current research does promise for novel targets and better drugs.