altered ergosterol biosynthetic pathway - an alternate ... · altered ergosterol biosynthetic...

Altered Ergosterol biosynthetic pathway - an alternate multidrug resistance mechanism independent of drug efflux pump in human pathogenic fungi C. albicans

Tulika Prasad*, Sunesh Sethumadhavan and Zeeshan Fatima

Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi-110067, India. * Corresponding author: e-mail: [email protected]; [email protected]

Dimorphic opportunistic Candida albicans has emerged as major human pathogen, causing life threatening diseases in immunocompromised individuals. Their treatment is limited by high toxicity, narrow spectrum of the existing drugs and emergence of multi drug resistance (MDR). Major mechanisms of multi-factorial MDR have been substantially characterized but strategies adopted by clinical resistant Candida isolates suggest that MDR may also be contributed by yet unknown mechanisms. In the efforts to establish newer therapeutic strategies for MDR reversal, levels of EFG1 (a major morphogenic and metabolic regulator) and cellular iron were found to be novel determinants of drug resistance in Candida albicans. Iron known to be critical for host cell invasion; its deprivation resulted in hypersensitivity to different classes of drugs tested and showed upregulation of EFG1. Disruption of EFG1 resulted in selective sensitivity on solid media to drugs targeting ergosterol biosynthetic pathway. Both EFG1 disruption and iron deprivation resulted in enhanced membrane fluidity and therein increased passive drug diffusion. This increase in membrane fluidity correlated with lowered ergosterol levels due to down regulation of ERG11 and increased oleic acid levels due to upregulation of OLE1. Azole inhibited ergosterol pathways are known to result in the accumulation of toxic sterol intermediate and upregulation of ERG3 in both the abovementioned conditions might have resulted in the synergistic diol accumulation. Thus, convergence of EFG1 and cellular iron status with MDR pathways propose an additional mechanism independent of known drug efflux mechanisms mediated by efflux pump proteins.

Keywords Candida albicans; multidrug resistance; ergosterol; potential drug targets

1. Candidiasis and prevalence of MDR in Candida:

Since more than three decades, life threatening superficial and invasive mycoses caused by opportunistic fungal pathogens have emerged as a major concern in immunocompromised individuals. Dimorphic, opportunistic but otherwise commensal organisms like Candida spp. turn pathogenic to cause serious infections resulting in high rates of morbidity and mortality in the hospitalized patients with critical medical conditions and undergoing long term chemotherapy. Candida spp. are the fourth most prevalent microorganisms in nosocomial bloodstream infections [1, 2]. Most candidemias are caused by Candida albicans followed by other non-albicans spp. Pathogenicity among yeasts is extremely variable- the most virulent being Candida albicans [1, 2]. 80% of the human population carries Candida albicans as natural flora in various body parts which shows clinical manifestation ranging from acute, sub-acute, chronic and episodic depending upon the extent of the lowered immune status of the individual [1, 2]. Predisposing factors for candidiasis include AIDS, burn patients, young individual, pregnancy, oral birth control, high fruit diets, steroids, organ transplant, prolonged antibiotic therapy, immunosuppressants, cancer treatments, heart surgery, genetic deficiency, endocrine deficiency diabetes, tuberculosis infections use of catheters, use of dirty needles etc [1, 2]. Developments in various fields of medicine like transplantlogy, indwelling central venous catheters, cancer chemotherapy, use of immune suppressive agents, corticosteroid therapy, advances in surgery etc. [1, 2] are associated with enhanced risk of severe systemic fungal infections. Limitations of therapeutic options and availability of fewer broad spectrum antifungals with minimum side effects poses a serious medical issue to be addressed in the treatment of systemic fungal infections. Furthermore, emergence of drug resistant strains in the biofilms-associated infections, in particular is becoming significant and increasing threat to antifungal therapies [1, 2]. Azole resistant clinical isolates of C. albicans acquire cross-resistance to many unrelated drugs, a phenomenon termed as Multi-Drug Resistance (MDR) [3-5]. MDR is a manifestation of multifactorial phenomenon with various mechanisms known to contribute towards the development of MDR, some of which include the following: 1) Overexpression of the drug efflux pumps belonging to the ABC (ATP-Binding Cassette) Superfamily of transporters e.g. CDR1 and CDR2 [4-7] and MFS (Major Facilitator Superfamily) transporters e.g. MDR1, FLU1, BENr [8-10]. 2) Molecular Alterations of the lanosterol 14-α demethylase, the target enzyme of the azoles. The gene encoding this enzyme is designated as ERG11 in all fungal species, although it has been previously referred to as ERG16 and CYP51A1 in C. albicans. The genetic alterations identified in ERG11 in C. albicans include overexpression of the gene, point mutations in the coding region, gene amplification (leading to overexpression), and gene conversion or mitotic recombination [10, 11]. An azole resistant clinical isolate was reported to have an ERG11 mutation of arginine to lysine at position 467, near the cysteine residue which coordinates with the fifth position of the iron atom in the heme cofactor

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[10, 11]. One of the common mechanisms of resistance in various clinical isolates is ERG11 overexpression [10, 11]. Gene amplification by increase in the number of gene copies also results in resistance by overexpression of the target enzyme. No clinical isolates of Candida spp. have been identified to have a completely non-functional ERG11, although some point mutations leading to altered drug resistance have been reported in C. albicans. Certain amino acid substitutions F72S, F145I and G227D in Erg11p showed the phenotype of cross-resistance to azoles possibly because of their special location in Erg11p [12]. 3) Appearance of aneuploidy, particularly due to the formation of an isochromosome [i(5L)] by gene amplification of ERG11 and TAC1. A recent study on the rapid acquisition of fluconazole resistance in the same individual revealed that the resistance was conferred by the presence of two extra copies of Chr5L, on the isochromosome [13]. Importantly, ERG11 and a hyperactive allele of TAC1 (encoding a transcriptional regulator of drug efflux pumps) make independent, additive contributions to fluconazole resistance in a gene copy number-dependent manner that is not different from the contributions of the entire Chr5L arm. 4) Alterations in other ERG genes of the ergosterol biosynthetic pathway. Analyses of the sterols help to correlate the alterations in the ergosterol biosynthetic pathway with the acquisition of drug resistance. Azole inhibited pathway or a defective lanosterol demethylase (the predominant azole target enzyme) results in the accumulation of toxic sterol internediates, especially 14α methyl fecosterol and 14α methyl-ergosta-8,24(28)-dien-3β,6α-diol [14]. The accumulation of diol has been associated with growth arrest, altered membrane functions and fluidity in azole treated C. albicans [14]. To prevent the accumulation of toxic diol, the pathogen adapts by having defective sterol Δ5,6-desaturase which is encoded by the gene, ERG3. Fluconazole resistance occurs in > 10% of cases of candidosis during the late stages of AIDS. Clinical studies on such patients revealed that after fluconazole treatment, the toxic sterol, 14α-methylergosta-8,24(28)-dien-3β,6α-diol was altered to 14α-methylfecosterol which is capable of supporting growth [14]. A consequence of this mechanism of azole resistance is that an absence of ergosterol causes cross-resistance to amphotericin B, another major antifungal agent. 5) Altered drug accumulation inside the cells by: either changes in the membrane permeability constraints or altered facilitated drug diffusion. Defects in drug import are common mechanisms of drug resistance. Drug import may be affected by many mechanisms including the sterol composition of the plasma membrane. Several studies have demonstrated that when the ergosterol component of the membrane is eliminated or reduced in favor of other sterol components such as 14a-methyl sterols, there are concomitant permeability changes in the plasma membrane and a lack of fluidity [6, 14]. These changes may lower the capacity of azole drugs to enter the cell. Alteration in the membrane ergosterol and sphingolipid interactions, iron depletion, disruption of genes of ergosterol or sphingolipid biosynthetic pathways, morphogenetic transcription factors which include EFG1, SSK1, etc. results in the alteration of permeability constraints leading to altered drug susceptibilities [15-18]. A recent study has been able to show that import of fluconzaole and other azoles proceeds via facilitated diffusion through a transporter rather than passive diffusion [19]. Analysis of related azoles indicates that competition for azole import depends on an aromatic ring and an imidazole or triazole ring together in one molecule. The mechanism of fluconazole import by facilitated diffusion is conserved among fungal species, including Cryptococcus neoformans, Saccharomyces cerevisiae, and Candida krusei. Fluconazole import was shown to vary among Candida albicans resistant clinical isolates, suggesting that altered drug import may be a previously uncharacterized mechanism of resistance to azole drugs. 6) Molecular alterations in Glucan synthase gene FKS1. FKS1 activity has been linked to triazole resistance in Candida biofilms. Reduction of FKS1 expression render biofilms more susceptible to amphotericin B, anidulafungin, and flucytosine and its increased expression in biofilms leads to enhanced resistance to anidulafungin and amphotericin B. These findings suggest that Candida biofilm glucan sequestration is a multidrug resistance mechanism [20]. Resistance to echinocandin drugs among clinical isolates is associated with amino acid substitutions in two "hot-spot" regions of FKS1 [21]. The mutations, yielding highly elevated MIC values, are genetically dominant and confer cross-resistance to all echinocandin drugs [20]. The FKS1-mediated resistance mechanism is conserved in a wide variety of Candida spp. and accounts for intrinsic resistance of certain species [21]. 7) Molecular alterations in uracil phosphoribosyltransferase gene (FUR1). A recent study represents the first description of the genetic mutation responsible for 5-Fluorocytosine (5FC) resistance [22]. A single nucleotide change from cytosine to thymine at position 301 in the uracil phosphoribosyltransferase gene (FUR1) of C. albicans is responsible for 5FC resistance [22]. Candida albicans is a commensal fungal inhabitant of the normal human microflora that can become opportunistic pathogens of mucosal tissues and invade almost all body sites and organs in response to both host-mediated and fungus-mediated mechanisms. Upto 90% of HIV+ persons show symptomatic episode of oropharyngeal candidiasis (OPC) during progression to AIDS, later turning recurrent. The prevalence of opportunistic fungal infections has increased dramatically among the aged population in recent years and aged mice population show an altered innate and adaptive immune response to C. albicans and are more susceptible to systemic primary candidiasis [23]. One of the toughest challenges faced by modern biology is combating drug resistance in fungal pathogens including Candida albicans [3-5]. Therefore, it is of utmost importance to find a novel therapeutic strategy to reverse MDR in such opportunistic pathogens. Treatment of invasive Candida infections is often complicated by high toxicity, low tolerance or narrow spectrum of activity of the current antifungal drugs and the increasing incidence of azole-resistant strains. The increasing incidence of these problems leads to the emerging need for complete understanding of all the possible mechanisms of drug resistance, in order to predict novel drug target(s) and then develop novel therapeutic

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strategies for MDR reversal. To prevent significant morbidity and mortality and for complete eradication of the disease, restricting the chances for relapse in future, there is constant demand for developing newer antifungal agents and modifiers for biologic response. Although, mechanisms of antifungal resistance and the major contributing factors are fairly established, there are evidences to suggest that MDR is a multi-factorial phenomenon which may be contributed by yet unknown mechanisms. For example, alteration in the intracellular iron and levels of a homolog of bacterial two-component response regulators SSK1 result in enhanced drug sensitivity in Candida cells [18, 24] Additionally, there are azoles resistant clinical isolates of C. albicans where mechanisms of resistance appear to be different than the commonly known strategies adopted by Candida [4]. C. albicans can switch from the unicellular yeast form into either of the two distinct filamentous forms, cells with pseudohyphae or true hyphae in response to various environmental stimuli. This ability to switch is considered as an important virulence trait and is also co-regulated with other virulence factors, which are associated with cellular morphology [25]. C. albicans morphology is directly related to environmental conditions and these cues trigger separate signal-transduction pathways, which regulate common targets required to initiate hyphal growth [25]. The transcription factor Efg1p regulates morphogenesis of C. albicans since it induces the yeast-to-hyphal transition and also regulates phenotypic switching and chlamydospore formation of this pathogen [25]. Interestingly, iron deprivation induced hyphal morphogenesis in C. albicans is also mediated through transcriptional regulator Efg1p [17]. Efg1p is an important regulator of morphogenesis and was found to affect the MDR status of C. albicans cells. Disruption of EFG1 resulted in selective sensitivity to azoles and polyenes as compared to the wild-type (WT) strain when grown on solid agar media and displayed increased membrane fluidity and enhanced passive diffusion of drugs in the homozygous efg1 mutant cells [15]. This established a convergence of EFG1 and MDR pathways and thus proposing an additional new role to this important transcription factor of C. albicans. Interestingly, recent studies suggest that there could be a correlation between intracellular iron concentration and multidrug resistance (MDR) phenomenon in mammalian cells since iron plays a key role in providing natural resistance to infections in humans [24]. Iron depletion in mammalian cells with iron chelators is known to activate hypoxia inducible factor-1 (HIF-1) [26, 27] which, in turn, activates its target gene MDR1 [26]. Studies on the role of iron in recurrent vulvovaginal candidosis (RVVC) revealed that iron is not only important for the normal function of host immunity, but is also important for pathogenic Candida owing to the fact that absence of this metal resulted in reduced virulence and hence reduced yeast invasion into the host epithelium [28]. Availability of iron has been found to play a critical role in different clinical infection and this represents a challenge to investigate the role of iron more closely [28]. Iron is usually present in insoluble ferric form complexed to environmental ligands and cannot be taken up by Candida directly [29, 30]. Ferric form is first solubilized by conversion to the ferrous form using the cell surface ferric reductase encoded by CaCFL1 [31]. The ferrous iron is taken up by an iron uptake system comprising of the iron transporter FTR2. But, in low iron availability, Candida uses the high affinity iron uptake system, comprising of a membrane permease-CaFTR1 [32] and a multi-copper oxidase-CaFET3 [33]. The reductive iron uptake in Candida also depends on copper availability, since CaFET3 has an essential requirement for copper, which is mediated by intracellular copper transporter CCC2 [34]. Siderophore transporter (SIT1) also exists in Candida as an additional mechanism for iron uptake and makes use of the low molecular mass organic molecule siderophore to bind extracellular iron [35]. The investigation on whether the availability of iron could have an impact on the susceptibility of Candida to antifungal drugs established iron deprivation as a mechanism to enhance drug susceptibility in Candida cells. Iron depletion was found to introduce an increase in membrane fluidity, which in turn leads to enhanced passive diffusion of drugs thereby resulting in increased drug susceptibility [24]. This could also link changes in membrane fluidity to lowered ergosterol levels due to down regulation of ERG11 in iron deprived Candida cells. Therefore, both EFG1 disruption and iron deprivation resulted in enhanced membrane fluidity and therein increased passive drug diffusion. This increase in membrane fluidity correlated with lowered ergosterol levels due to ERG11 down regulation and increased oleic acid levels due to OLE1 upregulation. Azole inhibited ergosterol biosynthesis is known to lead to the accumulation of toxic sterol intermediate. Simultaneous ERG3 upregulation in both these cases might have synergistically added to the increase in the intracellular toxic diol concentration in the presence of azoles. No change was observed in the transcript levels of the genes encoding the drug efflux pumps. Thus, convergence of EFG1 and cellular iron status with MDR pathways propose an additional mechanism independent of known drug efflux mechanisms mediated by efflux pump proteins.

2. Functional relevance of lipids in Candida:

Lipids are vital structural components of biomembranes and play a significant role in cellular signaling, membrane microdomain organization and dynamics, membrane trafficking and energy storage utilizing cellular lipid droplets and plasma lipoproteins [36].

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Among various classes of yeast lipids, membrane sterol, which is also the target of azoles, is one of the important constituents; it is responsible mainly for rigidity, stability, and resistance to physical stresses [16]. Loss of sterol generally results in destabilization of the membrane, leading to increased membrane permeability and altered drug susceptibilities in yeast cells [37]. Recent reports show the existence of discrete membrane microdomains, known as lipid rafts which are composed predominantly of sphingolipid and sterol, within lipid bilayers [38]. Yeast sphingolipids are another important membrane lipid components differing from those of mammalian cells in being structurally less complex and containing phosphatidylinositol as part of their polar head groups [39]. Drug-resistant mammalian cells have shown up-regulation of lipids and proteins that constitute lipid rafts and the caveolar membrane [40] ABC drug transporter Cdr1p of C. albicans and its homologue, human P glycoprotein/ multidrug resistance protein (Pgp/MDR1) have been demonstrated to be localized predominantly in respective sterol-enriched membrane domains and respective depletion of ergosterol and cholesterol have resulted in impaired drug transport mediated by these transporters [40, 41]. To summarize, sphingolipids and sterol as individual components as well as their mutual interactions are significant for the proper functioning of the ABC drug efflux pump proteins. Recent reports also suggest the involvement of sterol- and sphingolipid-enriched microdomains in hyphal morphogenesis in C. albicans, wherein membrane lipid polarization appears to contribute to the ability of this pathogen to grow in a highly polarized manner to form hyphae [39, 42]. Additionally, ergosterol and sphingolipids as individual components are also important determinants of hyphal morphogenesis in Candida albicans [16, 39] Sterol-rich membrane domains play a significant role in cell polarity and cytokinesis in Schizosaccharomyces pombe [43]. Lipid raft domains enriched in sphingolipids and sterols are involved in virulence and pathogenesis, by mediating the partitioning of specific proteins into lipid rafts, such as the acylated proteins, pleckstrin homology (PH) domain containing proteins and GPI-anchored proteins which include important virulence factors [44]. These GPI-anchored proteins include members of the adhesion protein family (Hwp1p and Als1p), epithelial adhesion protein-family (Eap1p, Dfg5p and Phr1p) and secreted aspartyl protease family (Sap9p and Sap10p) [42]. In all cases, membrane sphingolipids gain importance for the formation of signalling regulators. The different morphogical forms of Candida albicans appear to have different roles for host-cell interaction and virulence and host conditions trigger virulence attributes of this fungal pathogen [25]. It has been proposed that changes in the physical state of membranes are directly sensed and transmitted by specific signalling pathways triggering protective stress responses [45]. Specific inducers and elevated temperatures lead to hyphal development or regulate chlamydospore development. The fluidity of cellular membranes is determined to a large extent by their lipid composition and by ambient temperatures. High levels of unsaturated fatty acids, low amounts of sterols in eukaryotic membranes and high temperatures increase fluidity [45]. Levels of oleic acid show direct effect on specific components of the hyphal induction machinery [45]. Since, both aerobic morphogenetic signaling and oleic acid biosynthesis require oxygen, therefore, oleic acid acts as a sensor activating specific morphogenetic pathways in normoxic conditions. One major player in lipid signaling pathways of pathogenic fungi is Farnesol (FA), a 15-carbon oxygenated lipid made up of isoprene moieties, which is the first quorum-sensing molecule identified in eukaryotes [46]. FA is known to inhibit the hyphal formation through alteration of gene expression in Candida involving Ras1-adenylate cyclase and MAP kinase pathways [46]. FA exposure in Aspergillus alters the production of ergosterol and induces apoptosis-like changes [46]. All oxygenated lipids are collectively known as oxylipins. Fungal oxylipins are derived from oleic acid (18:1), linoleic (18:2) and linolenic acid (18:3), after the addition of an Oxygen molecule to polyunsaturated fatty acids. Eicosanoids like prostaglandins (PG), prostaglandin-like molecules, leucotriens and thromboxanes are oxygenated lipids shown to be produced in C. albicans and C. neoformans [47], even though these yeasts do not have cyclooxygenases. Many pathogenic fungi are known to produce eicosonoid by utilising the host which enables them to manipulate the host immune response [46]. Candida albicans produces endogenous eicosanoids from exogenous arachidonic acid, suggesting that host arachidonic acid can be used to synthesize fungal prostaglandin (PG) [47]. Fungal oxylipins compete with the host PG, such as PGE2, thus suggesting that fungal PG could interfere with the modulation of the host PG regulated immune responses [47, 48]. The eicosanoids/ oxylipins pathway in C. albicans also plays a central role in the control of morphogenesis and biofilm formation and, thus, these lipids are also viewed as regulators of Candida virulence [49]. The enzyme(s) responsible for PG synthesis in C. albicans is (are) yet not known. The innate effector phase or protective TH1 (T Helper cells 1) response to fungal infection is downregulated by Fungal PGE2 [48]. Sphingolipids are crucial determinants for normal hyphal growth in C. albicans [39, 42] which is an important attribute for virulent trait. Other sphingolipids such as glucosylceramide (GlcCer) are vital in inducing hyphal elongation [50]. Glycosphingolipid glucosylceramide synthase 1 (GCS1) in C. albicans is reported to be required for virulence [51]. Diacylglycerol, an important component of the sphingolipid signaling pathway activates melanin production through laccase and transcription of antiphagocytic protein, both of which are involved in virulence [46]. Therefore, most infectious disease pathogens are found to often adopt ingenious strategies for the evolution of better ways of cell survival, drug resistance, virulence and pathogenicity by altering lipid homeostasis in host cells and exploiting of the complexity of the lipidome.

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3. Lipids are key players to mediate MDR in Candida albicans:

Evidences suggest that impaired import of an antifungal agent involve membrane alterations through changes in the sterol and/ or lipid content and is an important attribute of azole resistance [16, 37]. The involvement of membrane lipid phase in drug resistance is now well documented and ABC drug transporter Cdr1p of C. albicans and its homologue, human P glycoprotein/ multidrug resistance protein (Pgp/MDR1) have been found to be sensitive to the nature and the physical state of the lipid matrix [37, 52]. The emergence of drug resistance in serial isolates of C. albicans from patients undergoing azole treatment is in most cases from a previously more susceptible strain. The step-wise acquisition of resistance has been associated with the overexpression of certain MDR genes, such as CDR1, CDR2, CaMDR1, and ERG11 [53]. But overexpression of known MDR genes does not directly correlate with azole resistance in all instances, implying the contribution of yet other unknown mechanisms of resistance. Therefore, it appears that azole resistance in C. albicans is a multifactorial phenomenon. In vitro acquired drug resistance exhibited by a fluconazole-adapted series of isolates has correlated well with observed in vivo clinical scenario wherein a stepwise increase in acquisition of MDR traits is observed [6]. In the sequentially fluconazole-adapted isolates of C. albicans, the gradual onset of resistance mechanisms is associated with changes in membrane lipid fluidity and asymmetry. The adapted resistant strains were found to exhibit overexpression of drug extrusion pump-encoding genes, particularly CDR1 and CDR2 and the gene for the azole target enzyme, ERG11 and changes in the membrane order due to the lowered ergosterol content and altered membrane lipid asymmetry [6]. The sequential rise in fluconazole MICs in these isolates was also accompanied by cross-resistance to other azoles and terbinafine. The asymmetrical distribution of phosphatidylethanolamine (PE) between two monolayers of plasma membrane was changed; PE being more exposed to the outer monolayer in the resistant strain [6]. Therefore, the interplay between the physical state of membrane lipids and azole resistance mechanisms in C. albicans demonstrates their close association. Some of the clinical azole-resistant C. albicans isolates has been shown to exhibit altered membrane phospholipid and sterol compositions [54]. Recent study on the isogenic erg mutants of Saccharomyces cerevisiae could demonstrate that the drug resistance is closely associated with status of membrane lipids which appears to be a cumulative effect of drug diffusion, extrusion pumps and the membrane lipid environment [37]. Cdr1p, when expressed in erg mutants of S. cerevisiae, has been found to interact more with membrane lipids, particularly with ergosterol and sphingolipid, and thus is more severely affected by lipid perturbations than the MFS pump, CaMdr1p. A close interaction exists between membrane ergosterol and sphingolipids [16]. Reduction in the content of either of these two components either genetically or by using chemical inhibitor has been shown to result in destabilization of this interaction showing deleterious effects on the drug susceptibilities of C. albicans cells [16]. Ergosterol and sphingolipids as individual components as well as the interaction between them is a critical determinant of the functioning of the ABC drug efflux pump, Cdr1p [16, 37, 39, 55, 56]. Disruption of the interactions between ergosterol and sphingolipids lead to the trafficking problems of Cdr1p resulting in impaired drug efflux. Cdr1p like its homologue, human Pgp, may be preferentially associated with membrane microdomains rich in sterol and sphingolipids. Therefore, it appears that not only there exists a close interaction between membrane lipid constituents and the drug susceptibilities of C. albicans cells but there is also a well-coordinated control of their synthesis [16, 37, 39, 55, 56].

4. Membrane Sterol affects drug susceptibility independent of the drug efflux pumps:

The acquisition of MDR is a gradual process involving an inter play of various interdependent and independent factors. The lipid micro-environment is a critical determinant of the shift from drug susceptible to multi-drug resistant in C. albicans. There are evidences showing that lipid composition of the Candida cells affects the drug susceptibility [16, 37, 39, 55, 56]. This could be reasoned out as the lipid composition is directly associated with the membrane order and functionality of drug efflux pump proteins.

Membrane protein:

Efflux pump proteins of the ABC (ATP-binding cassette) superfamily and MFS (major facilitator superfamily) of transporters are common exporters of structurally unrelated drugs. The ABC transporters use the energy derived from ATP hydrolysis to power the efflux, whereas the MFS transporters make use of proton gradient across the plasma membrane for drug extrusion. It has been suggested that the activities of the yeast efflux pumps, particularly those that belong to the ABC superfamily, are influenced by subtle modifications in membrane lipid composition [16, 37, 39, 55, 56]. Alterations in sterol and sphingolipid levels result in destabilization of the membrane and enhanced drug susceptibilities of C. albicans cells [16, 39]. Existence of discrete microdomains within the lipid bilayer of yeast membranes, which are predominantly composed of sphingolipids and sterols also strongly support the observation that lipids have a major role to play in drug resistance [16, 38, 39]. Moreover, it appears that membrane sphingolipids and sterols, both individually and through their mutual interactions, can critically affect the functioning of drug efflux pump proteins.

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EFG1 Mediated Drug Resistance:

Candida albicans being a dimorphic pathogen, can switch in response to various environmental stimuli from the unicellular yeast form into either of the two distinct filamentous forms: cells with pseudohyphae or true hyphae. This ability to switch is coregulated with other virulence factors and is an important virulence trait [15, 25].The transcription factor Efg1p is a morphogenetic regulator in C. albicans inducing the yeast-to-hyphal transition and also regulates phenotypic switching and chlamydospore formation [15]. Efg1p also mediates the iron deprivation-induced hyphal morphogenesis in C. albicans [17]. Considering the importance of the Efg1p regulator in morphogenesis, it is important to evaluate if defects in morphogenic signaling pathways would also affect the MDR status of C. albicans cells. Homozygous ∆efg1 mutant cells showed selective sensitivity to azoles and polyenes as compared to the wild-type strain when grown on solid agar media (Fig. 1A) [15]. The ∆efg1 null mutant cells displayed increased membrane fluidity, leading to enhanced passive diffusion of drugs (Fig. 2A) [15]. The increased membrane fluidity could be related to reduced ergosterol levels (Fig. 2B) and enhanced oleic acid levels due to considerable upregulation of OLE1 gene – which encodes fatty acid desaturase (steroyl CoA desaturase) and is responsible for synthesizing oleic acid. ∆efg1 mutation resulted in a marked increase in the OLE1 transcription as compared to the WT cells with a significant increase in the oleic acid content of the membrane. However, despite the fact that no major changes in the overall phospholipid content were evident in ∆efg1 mutant cells, the observed increase in membrane fluidity implied a direct link between Ole1p and Efg1p levels. The statistically significant decrease in ergosterol contents (24%) as observed in null ∆efg1 mutant cells [Fig. 2B] could be attributed to the considerable ERG11 downregulation (the rate-limiting step in ergosterol biosynthesis) (Fig. 4). The transcript levels of ERG3 were found to be directly affected by the EFG1 levels [58] and ∆efg1 null mutants showed significant ERG3 upregulation (Fig. 4). Several antifungals have been associated with the increase in the levels of reactive oxygen species (ROS). Interestingly, efg1 mutant cells displayed enhanced levels of endogenous ROS but the enhanced ROS levels which could be reversed by the addition of antioxidants could not reverse drug sensitivity of efg1 mutant cells. Overexpression of drug efflux pumps is one of the well known mechanisms for the development of resistance in C. albicans. Disruption of the morphogenic regulator EFG1 enhanced the drug sensitivity of C. albicans cells by a mechanism that appeared to be independent of the drug efflux pumps as no change was observed in the efflux activity and the transcript levels of the genes encoding the major drug transporters [Fig. 3A]. This could demonstrate the convergence of EFG1 and MDR pathways and established EFG1 as a regulator of morphogenesis, metabolism and drug resistance, thus assigning a new role to this important morphogenic regulator in C. albicans [25].

Fig. 1. Drug resistance profiling of 1. WT, 2. Homozygous Δefg1 mutants, 3. WT and 4. Iron starved cells as determined by spot assays. For spot assays, five microlitres of five fold serial dilutions of each yeast culture (A600 nm 0.1) was spotted on to YPD plates in the absence (control) and presence of drugs. Growth differences were evaluated as described elsewhere [16, 39] using drug free controls following incubation of the plates for 48 hours. Growth was not affected by the presence of solvents used for the drugs. Drugs were used at the following concentrations for WT and Δefg1 mutants: Fluconazole- FLC (1 µg/ml), Itraconazole- ITR (0.25 µg/ml), Ketoconazole- KTC (0.05 µg/ ml), Nystatin- NYS (1.3 µg/ml), Amphotericin B- AMB (0.3 µg/ml), Cycloheximide- CYC (0.1 µg/ml), Methotrexate- MTX (1 µg/ml), Crystal Violet- CV (0.1 µg/ ml), O-Phenanthrolene- O-PHE (2 µg/ml) and Sulfomethyl methuron- SMM (1 µg/ml). Drugs were used at the following concentrations for WT and iron starved cells in the absence and presence of 200 µM BPS: Fluconazole- FLC (0.5 g/ ml). Anisomycin- ANI (5 µg/ ml), Cycloheximide- CYC (300 µg/ ml), Nystatin- NYS (10 µg/ ml) and Amphotericin B- AMB (0.25 µg/ml).

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Iron Deprivation and Pathogenicity:

Recent studies suggest a correlation between intracellular iron concentration and the multidrug resistance (MDR) phenomenon in mammalian cells [26, 27] as iron plays a key role in providing natural resistance to infections in humans [57]. Intracellular iron levels have been shown to affect the drug susceptibilities in Candida cells [24]. Iron depletion in Candida albicans with bathophenanthrolene disulfonic acid and ferrozine as chelators enhanced its sensitivity to different classes of drugs, including fluconazole, anisomycin, cycloheximide, nystatin and amphotericin B (Fig. 1). Iron depletion further introduced an increase in membrane fluidity, which in turn lead to enhanced passive diffusion of drugs, thereby resulting in enhanced drug susceptibility (Fig. 2A). The changes in membrane fluidity could be related to lowered ergosterol levels (Fig. 2B) in iron deprived Candida cells probably because of down regulation of ERG11 (Fig. 3). Interestingly, the efflux of the fluorescent substrate R6G mediated by the Cdr1p and Cdr2p efflux proteins remained unchanged, irrespective of the iron levels in Candida cells. Also deletion of the drug efflux pumps could not further increase the drug sensitivity of the iron starved cells. This could imply that the synergism between antifungals and iron starvation is not directly related to the activity of the efflux pump proteins. Changes in ERG genes under iron deprivation conditions affected ergosterol levels, resulting in higher membrane fluidity, enhanced passive drug diffusion and increased drug susceptibilities. ERG11 was considerably down regulated under iron-restricted conditions, while ERG3 showed the opposite effect under similar conditions [24].

Fig. 2. A. Left panel shows the mean fluorescence polarization “p” values of the cells S.D. of three independent sets of experiments as depicted on X-axis and right panel shows the mean of the OD527 of the supernatant (lower OD implies higher concentration of R6G inside the cells) for monitoring the passive diffusion of R6G S.D. of three independent sets of experiments as depicted on X-axis for 1. WT, 2. Homozygous Δefg1 mutants, 3. WT and 4. Iron starved cells.

B. Relative percentages of ergosterol content as a percentage of the wet weight of the cells S.D. of more than three independent sets of experiments for 1. WT, 2. Homozygous Δefg1 mutants, 3. WT and 4. Iron starved cells. Fluorescence polarization values, passive diffusion of R6G and ergosterol content were measured as described previously [16, 39].

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Fig. 3. A. Northern blot analysis of ERG and ERG11 in lanes, 1. WT, 2. Homozygous Δefg1 mutants, 3. WT and 4. Iron starved cells. The upper panel panels show transcript levels and the lower panel represents loading control for indicating equal gel loading of total RNA for the respective gene transcripts. Northern analysis was performed as described earlier [39].

B. Schematic representation of the ergosterol biosynthetic pathway showing the antagonistic regulation of ERG11 and ERG3 genes.

ERG3 and ERG11 regulation in drug resistance:

The role of ERG3 in azole resistance originates from the observation that treatment of yeasts with azoles resulted in the accumulation of 14α-methylated sterols and 14α-methylergosta-8, 24 (28)-dien-3,6-diol. Formation of the latter sterol metabolite is thought to be catalyzed by sterol 5, 6-desaturase encoded by ERG3; thus, inactivation of ERG3 can suppress toxicity and therefore cause azole resistance [58, 59]. Analysis of the sterol compositions of two separate azole-resistant C. albicans clinical isolates revealed the accumulation of ergosta-7,22-dienol, which is a feature consistent with the absence of sterol 5,6-desaturase activity [59, 60]. EFG1 disruption or iron deprivation of Candida cells has been shown to regulate ergosterol synthesis genes, ERG11 and ERG3 in antagonistic manner. ERG11 downregulation together with ERG3 upregulation resulted in lowering of ergosterol levels and increase in the intracellular concentration of the toxic diol, which is known to cause cell growth arrest (Fig. 3B). The resulting drug susceptibilities to several unrelated drugs and metabolic inhibitors in EFG1 null mutant cells or iron derived cells therefore, might be the result of alterations in ergosterol biosynthetic pathway resulting in altered membrane fluidity and enhanced drug permeability.

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Fig. 4. Upper left panel shows the transcript levels for CDR1, CDR2 and CaMDR1, genes encoding the major drug transporters along with the loading control for indicating equal gel loading of total RNA for the respective gene transcripts. Lower left panel shows no change in the efflux activity for the substrate R6G as measured by the mean of the OD527 of the supernatant (lower OD implies higher concentration of R6G inside the cells) at regular time intervals S.D. of three independent sets of experiments as depicted on X-axis for the WT and homozygous Δefg1 mutants. R6G efflux was performed as described elsewhere [16, 39].

Upper right panel shows the drug susceptibilities of the drug efflux pump mutant with four drug pumps disrupted namely, Δcdr1 Δcdr21 Δcamdr1 Δflu1 as determined by spot assays in the absence and presence of 100 µM BPS. To detect any growth effect of BPS, a low concentration of FLC was used (0.125 µg/ ml) where mutant cells could grow. The lower right panel shows the minimum inhibitory concentration (MIC) of drug efflux pump mutant with fluconazole (FLC) in the absence and presence of BPS and is represented as histogram and the OD of the cells at 600 nm is plotted on Y axis. The clear bars (open) represent the cells grown without BPS and the shaded bars (grey) represent cells grown in the presence of 100 µM BPS. The concentration of FLC for MIC80

was 0.125 µg/ ml for the mutant cells grown with or without BPS.

Conclusive remarks:

Convergence of EFG1 and cellular iron status with MDR pathways propose an additional mechanism involving the altered ergosterol biosynthetic pathway but independent of known drug efflux mechanisms mediated by efflux pump proteins (schematic representation in Fig. 5).

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Fig. 5. Proposed model to show the convergence of EFG1 and cellular iron status with MDR pathways and existence of an additional mechanism involving altered ergosterol biosynthetic pathway, independent of known drug efflux mechanisms mediated by efflux pump proteins

Acknowledgements: The support to TP by Ranbaxy Science foundation in the form of Ranbaxy Science Scholar Award, Department of Biotechnology (DBT) Innovative Young Biotechnologist Award (IYBA), 2008 (BT/BI/12/045/2008), Department of Science and Technology (DST) support under PURSE Program and Council of Scientific and Industrial Research (CSIR) funded Open Source Drug Discovery (OSDD) grant (OSDD/HCP001/11FYP/2010-11/134) is gratefully acknowledged.

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