Curr Genet (2013) 59:243–250 DOI 10.1007/s00294-013-0402-1

RESEARCH ARTICLE

Lipids of Candida albicans and their role in multidrug resistance Rajendra Prasad • Ashutosh Singh

Received: 12 June 2013 / Revised: 26 July 2013 / Accepted: 30 July 2013 / Published online: 24 August 2013 Ó Springer-Verlag Berlin Heidelberg 2013

Abstract Over the years, lipids of non-pathogenic yeast such as Saccharomyces cerevisiae have been characterized to some details; however, a comparable situation does not exist for the human pathogenic fungi. This review is an attempt to bring in recent advances made in lipid research by employing high throughput lipidomic methods in terms of lipid analysis of pathogenic yeasts. Several pathogenic fungi exhibit multidrug resistance (MDR) which they acquire during the course of a treatment. Among the several causal factors, lipids by far have emerged as one of the critical contributors in the MDR acquisition in human pathogenic Candida. In this article, we have particularly focused on the role of lipids involved in cross talks between different cellular circuits that impact the acquisition of MDR in Candida. Keywords Lipids
Azole susceptible
Azole resistant
Mitochondria
Cell wall
Candida

Introduction There are 4–5 million diverse species of fungi in nature. While most of them are non-pathogenic, there exist a large number of fungi belonging to almost each fungal phylum that are pathogenic to humans, animals and plants. Human Special Issue: Yeast membranes and cell wall: From basics to applications Communicated by P. Griac. R. Prasad (&)
A. Singh Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India e-mail: [email protected]; [email protected]

fungal pathogens are mostly opportunistic, implying that the success level of their infections depends upon the immune defense system of the host. Due to advancement in preventive treatments such as immune suppression during organ transplantations, and life-threatening diseases like AIDS, these opportunistic fungi find suitable hosts to thrive in (Richardson 2005; Tuite and Lacey 2013). These common opportunistic fungal species are either Candida species like Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida dubliniensis, Candida guilliermondii, Candida krusei, Candida lusitaniae or non-Candida forms like Aspergillus fumigatus, Cryptococcus neoformans, Histoplasma capsulatum, etc. Among these fungal species, C. albicans is well adapted to thrive in most of the organs and niches that the human body can provide, making it the most successful human fungal pathogen (Pfaller and Diekema 2007; Heitman 2011; Pfaller 2012). Not surprisingly, it also contributes to 50–60 % of human infections, followed by non-albicans species, which mostly include C. glabrata, C. parapsilosis, C. tropicalis and C. krusei.

Antifungal resistance Opportunistic fungal pathogens pose an additional clinical situation, as they frequently develop tolerance to commonly used antifungals. Of four main classes of antifungals that are used in the clinic, the triazole drugs (fluconazole, voriconazole, itraconazole and penconazole) are widely used and well tolerated. However, these drugs are susceptible to both target- and efflux-mediated resistance. Azole-resistant strains also show cross resistance to many other drugs and compounds of diverse structure and target, leading to multidrug resistance (MDR) phenomena

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(Morschha¨user 2010; Niimi et al. 2010; Pfaller 2012). Instances of azole resistance have been documented for both C. albicans and non-albicans species (Prasad and Kapoor 2005). Resistance to antifungals could be visualized as a gradual evolving process, wherein different mechanisms of resistance may appear during the course of chemotherapy. The main mechanisms of antifungal resistance include alterations in ergosterol biosynthetic pathway by an overexpression of ERG11 gene, which encodes 14-a demethylase which is a target of commonly used azoles; or by an alteration in target enzymes (point mutations) which leads to reduced affinity to azoles (Prasad and Kapoor 2005; Noe¨l 2012). Reduced intracellular accumulation of drugs is another prominent mechanism of MDR in fungi (Prasad and Kapoor 2005). In C. albicans, for example, this is achieved by increasing the efflux of drugs from the cells by ‘overproducing the plasma membrane (PM) pump proteins’. An induction in the expression levels of genes encoding efflux pump proteins, particularly ABC (ATP Binding Cassette) multidrug transporter proteins Cdr1 or Cdr2 or MFS (Major Facilitator Superfamily) efflux pump protein Mdr1, have been commonly observed in azoleresistant clinical isolates of C. albicans (Prasad and Kapoor 2005). Invariably, MDR Candida cells, which show enhanced expression of efflux pumps 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). Recently, permeability constrains imposed by Candida cells are also proposed to contribute towards the development of MDR. In one instance, altered facilitated diffusion (FD) of azoles has been proposed as a mechanism of resistance. However, identification of membrane transporter protein involved in FD remains elusive (Mansfield et al. 2010). Interestingly, drug inactivation, which is a very common mechanism in bacteria, has not been observed in Candida cells. Of note recent gene profiling and RNA-Seq suggest new regulatory circuitry, which also impact antifungal resistance (Dhamgaye et al. 2012).

MDR transporters in yeast The genome of C. albicans possesses 28 ABC and 95 MFS proteins; however, only ABC transporters Cdr1 and Cdr2 proteins, and MFS transporter Mdr1 protein are known to be multidrug transporters which play major role in drug extrusion from the resistant strains (Gaur et al. 2005, 2008). ABC proteins are generally made up of two transmembrane domains (TMDs), each consisting of six transmembrane segments (TMS) and two cytoplasmically located

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nucleotide binding domains (NBDs) which precede each TMD (Prasad et al. 1995; Prasad and Goffeau 2012). While it appears that several TMSs associate together to form the substrate binding site(s), this alone is probably not sufficient for substrate transport across the membrane bilayer. Vectorial transport of these substrates requires energy from the hydrolysis of ATP carried out at the NBDs (Fig. 1a). MFS transporters to which Mdr1 protein belongs are proton-antiporters which employ electrochemical gradient of protons to power drug efflux (Fig. 1b). The full transport protein has 12 TMSs and unlike the ABC proteins, lacks NBDs (Prasad and Goffeau 2012). Since ABC and MFS transporters are among the major players that contribute to azole resistance in clinical isolates of Candida, there is a spurt in research on all aspects of these genes and their encoded proteins. Therefore, considerable attention is also being paid to the structural and functional aspects of these proteins, which in turn could lead to better strategies for designing modulators/ inhibitors of these pumps (Maurya et al. 2013). In this review, we highlight overwhelming instances of membrane lipids as a new player that affects MDR status of Candida cells. We begin with a discussion on the evidence that suggests possible role of lipids in growth and survival of Candida cells in general, and in particular, in MDR. The later part of review deals with the lipidomics of clinical MDR isolates of Candida and talks about how imbalances in lipid composition could affect drug tolerance in Candida cells.

Evidence for involvement of lipids in MDR Ergosterol and sphingolipids levels affect drug susceptibility Recently, it has been shown that any imbalance in ergosterol or sphingolipids (SLs) composition results in decreased susceptibility of yeast cells to antifungals. Mutants of ergosterol or SL biosynthetic pathways have revealed a close link between their levels and MDR in C. albicans as well as in S. cerevisiae (Mukhopadhyay et al. 2004; Pasrija et al. 2005a, b, 2008). Thus, the ERG gene disruptants were hypersusceptible to various drugs and similar was the case observed for SLs pathway mutants (Prasad et al. 2005).

ABC MDR transporter proteins are preferentially localized within microdomains of membrane The microdomains, also called ‘Lipid rafts’ in various organisms, play an important role in cell signaling, protein

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Fig. 1 Schematic representation of the two major classes of multidrug transporters in C. albicans. a Putative topology of an ABC transporter. Structurally, ABC transporters comprise of two homologous halves and each half comprises a hydrophilic nucleotide binding domain (NBD) followed by a hydrophobic transmembrane domain (TMD) containing six transmembrane helices (TMHs). ABC transporters export various structurally unrelated drug molecules using energy from ATP hydrolysis (Prasad and Goffeau 2012). b Putative topology of an MFS transporter. A typical MFS transporter comprises of two TMDs, each comprising of six TMHs linked by cytoplasmic loops and do not possess NBDs. MFS transporter effluxes drugs against proton (H?) gradient (Prasad and Goffeau 2012)

sorting and virulence (van Meer et al. 2008; Simons and Sampaio 2011). Lipid rafts are highly enriched in SLs and cholesterol (in mammalian cells) or ergosterol (in yeasts), and are characterized by their resistance to detergent solubility (Simons and Sampaio 2011). Although debated among different groups, these lipid rafts are well characterized in C. albicans as detergent resistant fractions (DRM) of the PM (Pasrija et al. 2008). When analyzed, these DRMs were found quite enriched in SL and sterols. Both SL and sterols are rigid structures, and probably provide hydrophobic surface to stabilize the interaction with large hydrophobic proteins. As discussed above, yeast cells susceptibility to drugs is influenced by the imbalances in membrane raft constituents (Pasrija et al. 2005b). Our results have shown that the ABC efflux proteins like Cdr1, Cdr2 and Mdr1 belonging to MFS superfamily show different propensities towards lipid. While, Cdr1 protein is preferentially localized within the ‘lipid rafts’, no such preference to lipids or domains is evident in Mdr1 protein. Disruption of SL biosynthetic genes like IPT1 (inositol phosphoryl transferase), FEN1, SUR4 or FEN12 (the fatty acid elongase) resulted in improper localization of Cdr1 protein. Similarly, deletion of ergosterol biosynthetic pathway genes led to mislocalization of Cdr1 protein, thereby affecting its functionality. In contrast, the imbalances of SL or ergosterol levels did not affect the localization and functionality of sister MDR transporter, Mdr1 protein; which remained properly localized within the PM (Pasrija et al. 2008).

MDR proteins as lipid translocators In most cell types in various organisms, membrane phospholipids are asymmetrically distributed across the PM (Ikeda et al. 2006). Amino-phosphoglycerides such as phosphatidylethanolamine (PE) and phosphatidylserine (PS) are predominantly located on the inner leaflet of the lipid bilayer, whereas the phosphatidylcholine (PC) and other lipids are localized on both the outer and inner leaflets of bilayer (Pomorski and Menon 2006). The asymmetrical distribution of membrane lipids is very specific, and its loss results in various physiological consequences in Candida (Smriti et al. 2002; Shukla et al. 2006). The lipid asymmetry is maintained by membrane bound phospholipid translocators divided into three classes, namely (a) bi-directional energy dependent scramblase, energy-dependent translocator that moves lipids (b) towards the cytoplasmic surface of the PM (flippase) or (c) away (floppase) from the cytoplasmic surface of the membrane (van Helvoort et al. 1996; Smriti et al. 2002; Holthuis and Levine 2005; Ikeda et al. 2006). It has been observed that yeast cells including Candida also possess asymmetric PM, and some of the ABC drug extrusion pumps can mediate phospholipid translocation (Daleke 2007; Sebastian et al. 2012). In Candida, both Cdr1 and Cdr2 proteins mediate an energy-dependent phospholipid translocation between the two leaflets of lipid bilayer (Smriti et al. 2002). Because of common binding sites of Cdr1 protein, the floppase activity can be competitively

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inhibited by drug substrates (Smriti et al. 2002; Shukla et al. 2006). Lipids in hyphae and biofilms The ability of C. albicans to switch from yeast to hyphae form or to assemble biofilms has been strongly linked to its pathogenesis and in evading host immune response (Mukherjee et al. 2003; Chandra et al. 2007). Studies involving the expression profiling of C. albicans biofilms revealed differential regulation of lipid biosynthesis genes (Yeater et al. 2007; Lattif et al. 2008). Lipidomics of the planktonic and biofilm forms of C. albicans confirmed a severe remodeling of glycerophospholipid (GPL) and SL composition within the two forms. Further the study showed that treatment with either myriocin or aureobasidin A (SL biosynthesis inhibitors), and disruption of IPT1 gene, abolished biofilm formation in C. albicans. This study has also demonstrated that SL enriched lipid rafts are critical for biofilm formation in C. albicans (Lattif et al. 2011). Earlier studies have also demonstrated that the mutants of both SL and sterol biosynthetic pathway genes are defective in hyphae formation (Pasrija et al. 2005a; Prasad et al. 2005). Together these reports strongly suggest that lipids are crucial for the development of hyphae and modulation of biofilms in C. albicans. Lipids as virulence factors Recently, a screening of homozygous deletion mutant library showed that glucosylceramide biosynthesis is required for virulence in C. albicans (Noble et al. 2010). Mutants of HSX11 (glucosyltransferase), Orf19.260 (or Sld1, sphingolipid desaturase) and Orf19.4831 (SL methyltransferases/cyclopropane synthases), which are involved in biosynthesis of glucosylceramide and HET1 (SL transfer protein), show attenuated virulence and proliferation. The glucosylceramide pathway is Candida specific and is not found in other fungi like S. cerevisiae and Schizosaccharomyces pombe. Interestingly, the virulence effecter glucosylceramide acts independent of morphogenetic switching in C. albicans (Noble et al. 2010). In a separate study, CHO1 (PS synthase) and PSD1/PSD2 (PS decarboxylases) gene mutants, which are defective in PS and PE biosynthesis, also show attenuated virulence of C. albicans in a mouse model (Chen et al. 2010). Ergosterol levels affect vacuolar functions and MDR A genetic screen of S. cerevisiae haploid disruption mutants against anti-arrhythmic drug amiodarone displayed a hypersusceptible phenotype for some VMA and ERG genes (Zhang et al. 2010). Results presented in that study

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clearly demonstrated that both ERG and VMA gene disruptants failed to acidify the vacuoles, suggesting a tight link between the ergosterol levels and vacuolar function. Also, treatment with fluconazole, lead to alkalinity of vacuolar pH in a dose-dependent manner, and aggravated cytosolic acidification in C. albicans. Together, this study showed that ergosterol is essential for proper functionality of V-ATPases, which in turn are required to maintain the vacuolar pH and intracellular ion homeostasis. This study established a new mechanism for azole induced killing of C. albicans. Also, both VMA and ERG gene mutants showed defects while growing on non-fermentable carbon source (indicating mitochondrial respiration defects), susceptibility to calcofluor white (in chitin-mediated cell wall stress) and defective virulence in murine models of Candidiasis (Zhang et al. 2010). These data suggest a possibility of a link between the ergosterol, vacuole, cell wall and mitochondria, and change in the overall integrity of these results in altered susceptibility to azoles (discussed later). The above discussions strongly suggest that Candida lipids not only are physiologically relevant, but also affect its MDR status. These aspects have prompted recent investigations towards a comprehensive lipid analysis by way of performing high throughput lipidomics of Candida to discover new roles, which could be exploited as therapeutic targets. Coinciding with the spurt in research, there has been some revolutionary technological advancement towards analyzing the lipid molecules (Shevchenko and Simons 2010). Classical tools to study lipids in yeasts have been radioactive labeling, biochemical assays, thin layer chromatography, gas chromatography and high performance liquid chromatography (Wenk 2005). However, in the last decade a massive advancement has taken place in analyzing various lipids using techniques like mass spectrometry (MS) and nuclear magnetic resonance (Wenk 2005). One initial lipidomics study on non-pathogenic Saccharomyces cerevisiae accelerated our ability to analyze yeast lipid metabolism and signaling, and the factors that regulate them in a high throughput manner using MS (Ejsing et al. 2009).

Lipids of Candida Over time, lipids have continuously evolved and diversified into thousands of different structures to meet the physiological demands of evolving fungi like C. albicans. This flexibility in lipids is achieved by the availability of lipid biosynthetic gene pool resource, which is able to utilize fatty acids (FAs) and different backbones to form an array of structures (Shevchenko and Simons 2010). Further,

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depending upon the FA chain length, position of FA, number and position of double bonds (unsaturation), the lipids can be sub-categorized into several ‘molecular species’ (Shevchenko and Simons 2010; Singh et al. 2010) (Fig. 2a). This artillery of available lipids allows fungi like C. albicans the flexibility and leverage to modulate their structure, function and homeostatic environment to adapt to any condition. By employing electrospray ionization tandem mass spectrometry (ESI–MS/MS), the phospholipidomes of eight hemiascomycetous human pathogenic Candida species, namely C. albicans, C. glabrata, C. parapsilosis, C. kefyr, C. tropicalis, C. dubliniensis, C. krusei and C. utilis, have recently been characterized (Singh et al. 2010). Nine major GPL classes namely PC, PE, phosphatidyl serine (PS), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), phosphatidic acid (PA), lysoPG, lysoPE, lysoPC (depicted in Table 1); four major groups of SL, ceramides, inositol phosphorylceramide (IPC), mannosyl inositol phosphorylceramide (MIPC) and M(IP)2C; neutral lipids like sterols, diacylglycerol (DAG) and triacylglycerol (TAG), were identified. Notably, the study could also characterize over 200 molecular species of GPLs and SLs (Fig. 2b). Interestingly, although no major differences among Candida species in GPL class composition were detected; distinct differences in phospholipid species could be identified. In contrast, differences in SL class composition as well as in molecular species were quite evident.

Table 1 Interspecies comparison of GPL classes in Candida species

Fig. 2 Lipid species diversity in Candida. a Lipid species can exist as isobars and isomers. For example, PC 32:1 comprising of 32 carbon atoms and 1 double bond might exist as isobars (change in FA chain lengths) or isomers (change in FA position form sn-1 to sn2); these different forms are referred as ‘molecular lipid species’. Recent advancements in MS-based methods have allowed us to distinguish and detect these molecular lipid species with similar masses with high

accuracy (Shevchenko and Simons 2010). b Phospholipid species detected in Candida. Direct MS analysis allows us to detect the various lipid species as the total number of carbons and the double bonds. The pie diagram depicts the lipid species detected for each phospholipid class in Candida (Singh et al. 2010). However, each lipid species can be categorized further into several molecular lipid species (Shevchenko and Simons 2010)

Lipid class

Mode

Detection

Scan

Amount (%)

PC

Positive ion

[M ? H]?

Pre 184.1

34–44

PE

Positive ion

[M ? H]?

NL 141.0

21–32

PI

Positive ion

[M ? NH4]?

NL 277.0

13–25

PS

Positive ion

[M ? NH4]

?

NL 185.0

4–9

PG

Positive ion

[M ? NH4]?

NL 189.0

0.8–3

PA

Positive ion

[M ? NH4]?

NL 115.0

1–4

LysoPC

Positive ion

[M ? H]?

Pre 184.1

1–5

LysoPE

Positive ion

[M ? H]?

NL 141.0

0.5–2

LysoPG

Negative ion

[M - H]-

Pre 152.9 \0.04

The table depicts the nine major GPL classes that are routinely detected in Candida using MS (Singh et al. 2010). In MS, the detection is based on sequential precursor (Pre) or neutral loss (NL) scans performed either in positive or negative mode that generates a specific fragment characteristic to each GPL class. In Candida, PC and PE are the most abundant GPL classes and possess maximum enrichment in terms of lipid species, which LysoPG is the least abundant class. Overall, the lipid diversity and complexity of Candida lipids is remarkable. Also each lipid class comprises of several molecular species which can be distinguished on the basis of number and position of double bonds as well as the FA chain lengths (Fig. 2b)

For instance, the abundance of many SL species is distinct in C. albicans compared to non-albicans strain like C. glabrata or non-pathogenic yeast like S. cerevisiae (Ejsing et al. 2009; Singh et al. 2010). Particularly, lipid species IPC (42:0;3, 42:0;4, 44:0;3 and 44:0;4) MIPC (42:0;3, 42:0;4, 44:0;3 and 44:0;4) and M(IP)2C (42:0;3

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and 44:0;3) are most abundant in C. albicans. In contrast, S. cerevisiae are quite enriched in overall M(IP)2C content. Further, the ceramides like the 42:0;3 species are also abundant in C. albicans compared to the other yeasts. However, the physiological consequences of these changes are not completely understood. These GPL profiles could be further discriminated and statistically validated by the principal component analysis. The comparative lipidomics of Candida species revealed that there is no typical lipid marker which separates them, rather each Candida strain has a typical lipid imprint (Singh et al. 2010). Lipidomics of azole-susceptible (AS) and -resistant (AR) clinical isolates The adaptation of C. albicans to tolerate antifungals is accompanied with many specific and global changes in lipid homeostasis. Although many studies have been done earlier to address the lipid composition of AS and AR isolates of C. albicans, these have provided only limited information due to technical limitations and randomness in the background of the isolates (Hitchcock et al. 1986; Lo¨ffler et al. 2000). To address this problem, recently, the detailed lipidomics of several genetically matched (isogenic) as well as select sequential AS and AR clinical isolates of C. albicans, were performed (Singh et al. 2011, 2012, 2013). These studies not only provided a comprehensive evaluation of lipids as the determinants of drug resistance but also showed that though each AR isolate possessed a characteristic lipid composition, there were consistent differences in several lipid classes. Development of azole tolerance also impelled the remodeling of molecular species of lipids. The fact that lipidomic response of match pair isolates was associated with simultaneous overproduction of efflux pump membrane proteins suggested for a possible common regulatory mechanism between the two phenomena. Such a common link has already been observed in case of S. cerevisiae and C. glabrata, where genes encoding efflux pumps, such as ScPdr5 or CgCdr1 and CgCdr2 play an important role in regulating lipid levels (Shahi and MoyeRowley 2009). For example, in Pdr5 and Yor1, membrane efflux proteins of S. cerevisiae help in maintaining the asymmetrical distribution of PC and PE, which in turn alters the trafficking of other membrane proteins (Johnson et al. 2010). Loss of Rsb1 (integral membrane protein), Pdr5 and Yor1 also result in altered phytosphingosine (PHS) susceptibility and affects localization and trafficking of other membrane transporters, like Tat2 (tryptophan permease), in S. cerevisiae (Johnson et al. 2010). In another example, nulls of YBT1 (vacuolar ABC transporter), are

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found to be defective in vacuolar transport of NBD-PC (7Nitro-2,1,3-benzoxadiazol-PC, a PC analog) (Gulshan and Moye-Rowley 2011). Evidence present also demonstrates a critical role of PS decarboxylases (Psd1/Psd2) and PI transfer protein (Pdr17) in maintaining vacuolar phospholipid homeostasis and regulation of MDR (Gulshan et al. 2008, 2010). There are not enough instances to confirm coregulation of lipids and MDR in Candida, although Psd1 has been found to be upregulated in some AR clinical isolates (Singh et al. 2012). Crosstalk between lipids, mitochondria, cell wall (CW) integrity and virulence In C. albicans, PL biosynthetic pathways have been implicated in the maintenance of CW integrity and virulence (Chen et al. 2010). In a 2010 study, Chen et al. have discussed the role of PS and PE in the virulence of C. albicans. In that study, the possibility of these aminophosphoglycerides levels being linked to dysfunctioning of mitochondria, CW integrity and filamentous growth of C. albicans is presented. It however, remained unclear as to how these events are co-regulated in C. albicans. A recent study involving detailed lipidomics of select sequential isolates of C. albicans demonstrated a close link between lipid homeostasis, mitochondrial membrane dysfunction and CW integrity (Singh et al. 2012). The study illustrated as to how gradual corrective changes in Candida lipidome coincided with the development of fluconazole tolerance. Of particular significance were the changes in PM micro-domain-specific lipids such MIPC and ergosterol, and in a mitochondrial-specific GPL, PG (Singh et al. 2012). PG is the precursor of cardiolipin (CL), which has multiple roles such as in promoting cell growth, anaerobic metabolism and mitochondrial biogenesis (Schlame et al. 2000). In S. cerevisiae, PGS1 (PG phosphate synthase) gene is linked to CW biogenesis (Zhong et al. 2005, 2007). Further evidence shows that in S. cerevisiae and C. glabrata, mitochondrial mutants with defect in PG and CL biosynthesis are hypersusceptible to a CW targeting drug, caspofungin (Shingu-Vazquez and Traven 2011). A decrease in mitochondrial membrane action potential could be correlated to the PG levels and CW integrity in several genetically matched clinical AR isolates of C. albicans. The study mentioned above provided an instance of a cross talk between mitochondrial lipid homeostasis, CW integrity and azole tolerance (Singh et al. 2012) (Fig. 3). In S. cerevisiae, the Pdr pathway is the link between the mitochondrial membrane structure and lipids (Shingu-Vazquez and Traven 2011), but not much is understood in C. albicans in this regard, which presents us with an area that is worth pursuing.

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Fig. 3 A cartoon depicting possible events associated with fluconazole resistance. The resistance to fluconazole in C. albicans results from an interplay amongst overexpressed efflux pump proteins and tightly regulated lipid metabolic networks. It appears that fluconazole entry into the cell over a period of time leads to certain transcriptional changes that includes an overexpression of MDR genes including CDR1, CDR2 and MDR1 (shown by different colors), that may impact specific changes in lipid metabolism, suggesting that the two events might be co-regulated. The changes in lipid homeostasis provide an apt membrane environment for proper localization of membrane efflux pump proteins. Further, mitochondrial lipid imbalances appear to affect its function affecting the cell wall (CW) integrity and may confer resistance phenotype to Candida cells. Thick broken purple line shows that the link is not established, however, these events might be co-regulated. N nucleus, PM plasma membrane

Conclusions Recently, high throughput MS-based methods have allowed deeper insights into the complexity of lipids of pathogenic yeast C. albicans and its implications in antifungal resistance. Comparative lipidomics of several clinical azole-resistant isolates of C. albicans has led to the characterization of previously undetected lipid changes and molecular species which separate them, and thus, have emphasized on the power of advance high throughput analytical tools. It is expected that more intense lipidomics of yeast cells of interest would reveal newer lipids, their regulatory circuitry and physiological roles. Acknowledgments The work from authors (RP) laboratory discussed has been supported in part by grants from the Department of Biotechnology (BT/PR11158/BRB/10/640/2008, BT/PR13641/Med/ 29/175/2010, BT/PR14879/BRB10/885/2010, BT/01/CEIB/10/III/12).

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