Emerging role of lipids of Candida albicans, a pathogenic dimorphic yeast

Biochimica et Biophysica Acta, 1 ! 27 (1992) 1- 14 ~_'~ 1992 Elsevier Science Publishers B.V. All rights reserved 111}05-2700/92/$05J}0

B B A L I P 539O5

Review

Emerging role of lipids of Candida aibicans, a pathogenic dimorphic yeast Prashant Mishra a j. B o l a r d b and R a j e n d r a Prasad

~'

" School of Lift" Sciences, Jawaharhd Nehru Unirervity, New Delhi (hulia) and I, Lahoratoire tit" Physique et Chimie Bitmzoleculaire, CNRS Unit'ersitJ Pierre et Marie Curie, Paris (France)

(Received 12 M a r c h 1992)

Key words: Lipid; P a t h o g e n ; {('am/ida)

Contents !.

Introduction ..............................................................

I

II.

Lipid metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

!!1.

R o l e of sterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Membrane transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. M o r p h o g e n e s i s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Enzymatic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3 5 6

Role of phospholipids ....................................................... A. M e m b r a n e t r a n s p o r t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 7

IV.

B. Morphogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8 • .................

V.

R o l e of fatty acids

VI.

Role o f lipids in anti-('amh,ht drugs action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Polyenc antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Azole antifungals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. C o m b i n a t i o n o f p o l y e n e antibiotics and azole derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . D. O t h e r anti-Camli, la d r u g s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

S

.......................................................... 9 II) II 12 12

Vii. Summary ................................................................

12

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

12

!. Introduction Dimorphic Candida albicans is an opportunistic pathogen, which causes candidosis when host defenses are impaired [1,2]. With the increasing use of immunosuppressive drugs and the spread of AIDS, its infection has become even more prevalent. The fact that a very

Correspondence to: R. Prasad, School of Life Sciences, Jawaharlal Nehru University, New Delhi-l10067, India.

limited number of effective and safe systemic antiCandida drugs are known and that lipids provide potential targets for many antifungals, have generated immense interest in studying diversc facets of its lipids. U n l i k e other yeasts, especially Saccbaromyces cererisiae, very little is understood about the composition and turnover of different lipids of C. aibicans [3-6]. Only recently such studies were undertaken and, as a result, the list of functions which arc dependent on lipids of Candida, is continuously increasing (Table !). Keeping pace with research in this field, a few review

articles encompassing different aspects of lipids of C. albicans have appeared [4-7]. The present review, however, mainly focuses upon the emerging role of lipids of C. albicans. Some functions, like cellular permeability, enzymatic activity, morhogenesis, cell cycle, adherence and virulence of C albicans, are already shown to be affected by fluctuations in lipid composition and therefore, dealt in with some detail. The metabolism of iipids of C. albicans has been the subject of a recent review [6], therefore, a detailed account

of it is not included in this review. For details regarding lipid metabolism of C. albicans the reader is referred to Refs. 4-7. The discussion on the role of individual lipid classes in the known functions is preceded by a brief account of its composition.

11. Lipid metabolism

Phospholipid metabolism and its regulation has been studied in great detail in S, cerecisiae, however, such

TABLE !

Li~t of known lipid.mediutt'd fim¢iions in Candida albicans Changes in lipid composition achieved by

Allcration in lipid composition

Affected function

Reference

I, Growth conditions Suppl¢mentation of growth media with alkanes of different chain length

gross lipid changes

amino acid transport polyene sensitivity radiosensitivity

[49,114] [84]

Growth up to prolonged stationaw phase

alteration in phospholipid composition

cellcycle

[f~O]

Ergosterol supplementation

ergosterol level

inhibition of germ tube formation and chitin synthesis

[44]

block of PS-decarho~lation and PS-accumulation

amino acid transport

[54]

Cerulcnir

inhibition of fatty acid synthesis

block of germ tube formation and chitin synthesis

t! i ]

Uydtoquinon¢

increased level of e. ~osterol

amino acid transport polyene sensitivity

[25,114]

~.,corbic acid

decreased level,, ergosterol

amino acid transport polyene sensitivity

[25. ! 14]

Aqueous garlic ¢,xtract

decreased lc~cl of PS and block of fatty acid synthesis

growth and viability

[83]

Polyen¢ antibiotic

decreased level of ergosterol

inhibition of membrane enzymes inhibition of amino acid transport and K ÷ release

[89] t! 14]

Azol{

ste.~l composition

inhibition of amino acid transport

[19]

C7 E4 C4 Di0

defective in demethylase and accumulates 14 methyl sterols

membrane permeability glycerol uptake, growth and respiration assimilation of metabolites chitin synthase antibiotic sensitivity

[22,24]

erg.41

accumulate various sterol intermediates due to defect in ergosterol biosynthesis, as shown in Table ill

[38] [ 14]

Cer~Jlenin-resistan~ mutants

no change in [:~H]acetate incorporation into lipids

adherence to BECs

[80]

Fatty acid auxotroph A'44

,1'~ desaturase defective mutants results in altered fatty aoji composition

growth and polyene sensitivity

[ ! 3]

2, Specific drugs or inhibitors Hydro~lamine hydrochloride

3. Mutants defective in lipid biosynthesis erg.2, e~-20 erg-37, erg.40

Modified from Ref. 4 with permission.

TABLE I1

Lipid composition of C albicans Lipids

Range

Reference

Total lipid (ttg/mg of d,-y wt.) Total phospholipid Total sterols Triaeylglyceride Free fatty acids

105-175 39- 51 49- 55 9- 11 1

[29] [29] [29] [29] [29]

Polar lipids (% composition) Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol Phosphatidylserine Phosphatidic acid Cardiolipin Sphingolipids

25- 56 11- 33 5- 20 9- 24 9 8 4-10

Sterols (% composition) Ergosterol Zymosterol 24,28-Dehydro-ergosterol 3.,~.Hydroxy-24.methylcholesterol 4,4-Direct hylzymosterol

14.6 17 42 12 10

Fatty acids(%composition) 14:0 14:1 16:0 16:1 18:0 18:1 18:2 18:3

4 21131011114-

[7,29,48,491

[301

[ 13,14,30,68,74] 6 27 17 14 41 36 7

studies in C. albicans have been rather limited. Nevertheless, most of the enzymes involved in the synthesis of major phospholipids and the existence of the CDPpathway in C. albicans have been identified [8-10]. The evidence accumulated so far suggests that the synthesis and degradation of phospholipids are similar to the most commonly studied yeast, S. cerevisiae. Since sterols are the major target of many antifungals, the sterol biosynthesis has received much attention. It seems that biosynthetic pathways of sterols are common between S. cerevisiae and C. albicans up to the point of the biosynthesis of episterol. Episterol is then converted to ergosterol through different pathways in both yeasts. Some of the enzymes of sterol biosynthesis, which are a potential target of antifungal drugs, have been studied in great detail. The genes coding for these target enzymes have also been cloned and sequenced. In contrast, the fatty acid metabolism of this pathogenic yeast is relatively less well understood [11]. Iii. Role of sterols

Ergosterol is the major sterol (49-94% of total sterol) present in C. albicans, while zymosterol 24,28dehydro ergosterol 34~-hydroxy-24-methylcholesterol

and methylated steroi 4,4-dimcthylzymosterol only constitute a minor fraction [12-15] (Tabie ll). Sterols are known to modulate the fluidity of membranes [16]. In yeasts, however, at least four different functions have been ascribed to sterol. Based on the amount of ergosterol required by sterol auxotrophs of S. cerevisiae, these functions have been designated as 'sparking', 'critical domain', 'domain' and 'bulk' [17]. Sterol auxotrophs of S. cerevisiae had restricted growth in dae presence of cholestanol, unless supplemented with a minute amount of ergosterol (1-10 ng/ml). This phenomenon has been coined as a 'sparking' function of sterols and is likely to be involved in non-membranerelated functions. The sterol auxotroph RD5-R was able to grow in media containing 5/xg/ml 9f lanosterol plus 100 ng/ml of ergosterol [17]. The low level of ergosterol (100 ng/ml) required is insufficient to regulate overall membrane fluidity, however, as suggested by Rodriguez et al., it is needed in restricted areas for 'critical domain' function. This function is distinguishable from 'domain' or 'bulk', as plasma membrane isolated from auxotrophs grown on domain or bulk levels underwent no lipid thermotropic transitions, while those obtained from plasma membrane of cells grown on critical domain level showed thermotropic transitions [17]. Due to insufficient information no such conclusions can be drawn in the case of C. aibicans. However, alterations in sterol composition ha~c been shown to affect certain functions of C. albicans, which are discussed below. Alteration in sterol composition can be obtained either by direct action of azole derivatives on C. albicans or by selection of mutants in the presence of polyene antibiotics. The exact mechanism of action of these drugs will be discussed in section VI, devoted to the role of lipids in anti-Candida drug action. Briefly, the antifungal activity of azole derivatives is due to their interference with ergosterol biosynthesis [18]. Polyenes, on the other hand, are known to specifically bind with ergosterol of the plasma membrane of fungal cells, resulting in ,~Itered permeability and subsequent death of the organism. III-A. Membrane transport

Recently triazole ICI 153066, an azole derivative, has been used to illustrate the importance of ergosterol and its metabolic intermediates in membrane transport systems of C. albicans [19]. Similar to other azoles, it is a potent inhibitor of the 14 a-demethylase system and, as a result, its presence (1 /zg/ml) in growth media leads to the accumulation of 14-methylated sterols. While 6 h treatment with this antifungal led to the accumulation of lanosterol, longer exposure el up to 24 h predominantly resulted in accumulation ot' 24methylenedehydrolanosterol. In the conditions leading to the accumulation of lanosterol, uptake of L-glutamic

acid, L-lysine, L-leucine and L-serine was shown to be reduced, as compared to those cells which had predominantly ergosterol. Kinetically, the reduction in uptake was attributed to reduced l/m~,xvalues because the apparent K m values for these amino acids remained unaffected by fluctuations in ergosterol levels [19]. That the accumulation of lanosterol has considerable effect on membrane properties had already been demonstrated in an earlier study by Van den Bossche [20]. The relea~ of entrapped glucose from ergosterol-containing unilamellar vesicles composed of phosphatidyicholine (PC), phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) (5:3: l) was reduced by more than 50% but when lanosterol was replaced by ergosterol, the release ot' glucose was similar to that observed with vesicles composed of phospholipids alone [21], Most likely the bulky C-14 methyl group of lanosterol protrudes from the steroi c~-face in the lipid-lipid contact region, which diminishes van der Waals contacts and allows greater flexibility to fatty acyi chains [16], The secondary effect of azoles on fatty acid composition needs special mention. The observed shift to more unsaturation in the phospholipids of C albicans grown lbr 6 h may reflect an attempt by the yeast cell to dial membrane fluidity in response to the azoleinduced alteration of ergosterol synthesis. C. aibicans, if grown for a longer period of time (16 h) with 14 a-demethylase inhibitor, is unable to maintain the synthesis of unsaturated fatty acids, which may be due to the inhibition of A'~ desaturase [16]. As a result of azole antifungal-induced ergosterol depletion, accumulation of methylated sterols and subsequent secondary changes in fatty acids to a more unsaturated form and perturbations in membrane structure and function arc likely. The effect of imidazole and triazole derivatives discussed above in membrane functions strengthens this point. The availability of polyene resistant t,,g mutants of C albicans, which are detective in ergosterol biosyn. thesis (Table !!!) also offers an opportunity to analyse the importance of other intermediates of its biosyn. thesis in the structure and function of membranes. Therefore, in order to explore the role of sterol and its derivatives in membrane transport, erg mutants of C. albicans have been employed. These mutants accumulate various intermediates of sterol biosynthesis due to the defect in their biosynthetic enzymes (Table I!!) [22]. Since accumulated sterol intermediates are able to sustain the growth of e,g mutants, they do not have any auxotrophic requirement tor sterols. However, adaptive changes in phospholipid and fatty acyl composition are also associate,.t with these mutants [23]. Consequently erg mutants exhibit higher plasma membrane order parameters than their ergosterol-producing parent strain [~]. Using these mutants, Pesti et al. [~] demonstrated that the depletion of ergosterol re-

TABLE Ill Enzymath' defi'cts hi ergosterol-deficient mutants of C ,dbicans

Mutant strain

Accumulated intermediate

Enzymatic defect

eta-2 e~-!6 :'

Zymosterol, Fccosterol Zymostcrol, Epistcrol, Ergosta-7-en-3-~.ol Ergosta- 7,22-dien-3-/J-ol Zymosterol, Episterol, Ergosta-7-en-3-~-ol Zymosterol, Episterol, Ergosta-7-en-3-B-ol Zymosterol, Fecosterol l.ailO.Mcnll Zymostcrol, Episterol, Ergt~sta-7.cn-3/3-ol Ergosta. 722-dic n-3o/J-ol

A'~aT-isomcrasc 5,6-dchydrogcnasc

erg-20

crg-37 ,e~:~-4|) eta-41 t,

22,23-dehydrogenase 22,23-dehydrogenase ,anALisomerase 5,~-dchydrogcnase

('ompik.d from Ref 23, with permission. " isolated from 33 ERG'. h Isolated from 35 E R G ' ,

suited in reduced assimilation of glycerol, methylglucoside, m.-lactic acid, L-sorbose, L-arabinose, ribitol and decreased permeability of glycerol [22-24]. Prasad et al. (unpublished observations) have recently observed that the transport activities of several amino acids in erg2 and erg 16 mutants are reduced, as compared to their wild type. The level of plasma membrane ATPase and proton flux associated with the uptake of amino acids, however, did not differ significantly between erg mutants and their wild type. Therelbre, the observed reduction in uptake of amino acids was not due to a difference in energy status of the mutant and wild type strains but, most likely, was related to ergosterol depletion. That the fluctuation of ergosterol level affects amino acid transport was also evident when C atbicans was grown in hydroquinoneor ascorbate-supplemented media [25]. The hydroquinone.grown cells had higher levels of ergosterol, while ascorbate-grown cells had lower levels, as compared to normal cells. Although the mechanism by which ascorbate or hydroquinone alters the level of sterols is not clear, it is known that alterations in sterol composition were specific and had a selective effect on the amino acid uptake of C. albicans [25]. While the uptake of L-lysine, glycine, L-proline, L-glutamic acid, L-methionine and L-serine increased in ascorbate-grown cells (with lower level of ergosterol), the uptake of these amino acids was reduced in cells grown in hydroquinone (with higher level of ergosterol). The transport activities of some of the amino acids namely, L-phenylalanine and L-leucine, however, remained unaffected by ergosterol fluctuations [25]. It must be emphasized that the observed effect on amino acid transport in ascorbate-grown cells and erg mutants cannot be compared. While ascorbate-grown cells had low levels of

ergosterol (16% less), erg mutants (erg2 and et~g16) do not have any ergosterol, in addition, due to the accumulation of various ergosterol biosynthetic intermediates, the erg mutants exhibited a higher plasma membrane order parameter, suggc:;ting that the mutants had a more rigid membrane [22-25]. The mechanism by which sterois can selectively influence the acti,.~ity of certain transporters is not known. Most likely the surrounding lipids affect the conformation of a certain transporter which, in turn, could alter accessibility to their binding sites. Alternatively, the effect of an altered lipid microenvironment around various transporters may be selective [26]. The existence of ergosterol-rich and poor domains in the yeast plasma membrane could also contribute to such selectivity [3,27]. lli-B.

Motphogem'sis

As mentioned earlier, C all, icons is a dimorphic yeast which can exist either as ellipsoidal buds (yeast lbrm) or elongated hypha (mycelial form), depending upon a host of factors [2,28]. The ability of C. albicans to exist in two forms has received much attention, as both forms are implicated to its pathogenicity [28]. The exact role of the morphological forms in pathogenesis, however, could not be demonstrated, since both forms are found in the infected tissues. Nevertheless, an understanding of the molecular basis of dimorphism is essential for the development of effective and saf,." antifungals. The germination-related changes in steroi composition have revealed that free steroi content progressivcly increased during germination but reduced at later stages [29]. in contrast, the sterol-ester fraction decreased during germination till it attained the concentration found in growing blastospores [29]. Ghannoum et al. [30] have shown that the total sterol content present in mycelial iipids was much higher than in the yeast form. The sterol was mainly present in the bound form as a steryl glycoside, esterified steryl glycoside and steryl ester. On the other hand, free sterols were the main constituents of lipids of yeast form [30]. In a later study [31], ergosterol and lanosterol contents were found to be higher in the mycelial form whereas the content of zymosterol was higher in the yeast form. Squalene was about ten times higher in the mycelial than in the yeast form [31]. Since squalene and lanosterols arc the precursors of ergosterol, the increase of their content in myceliai form is regarded as immaturity of sterols in mycelial cells. The polyenc resista,at mutants of C. aibicans, KD 4700 and KD 4900, which were isolated by ultravioletmutagenesis, are defective in hyphai growth [32]. The analysis of their stert~ls has revealed that, although the total sterol content of the mutant strains was similar,

their composition distinctly differed from their parent strain. Both the mutants have shown that the amount of ergosterol was reduced, with concomitant ihcrcasc in proportions of 14-methy~sterols (e.g., lanosterol; 24methylenelanosterol; 4, 14-dimethylzymosteroi and obtusifoliol). This suggested a probable relationship between the accumulation of 14-methyl sterois and morphogenesis and was further substantiated when clotrimazole was used [32]. C!otrimazole, an imidazole antifungal which leads to the accumulation of 14-methyl sterols, also caused l epression of hypha! growth in normal cells and thus was able to mimic the mutant cells with regard to sterol composition and morphogenesis. Therefore, there appears to be an intrinsic relationship between sterol composition and morphogenesis, however, the exact mechanism is yet to be resolved. Cannon and Kerridge [33] have observed an interesting correlation between sterols and morphogcnesis by using stcrol biosynthesis inhibitors [33]. Ketoconazolc, whcn present at nfinimum inhibitory concentrations under mycclia-forming conditions, yielded stunted mycclia. Under similar conditions, however, the drug had no effect on a mutant which was unable to form mycelia but continued to grow in yeast form. When ketoconazole was added in ycast-forming c{)nditions, a low content of ergosterol and an increased amount of 14-methylated sterol were observed, while stunted mycelia had a Iov~er ratio of 4, 14-dimethylsterols to 4-methyl sterols. However, tcrbinafine, which has an effect similar to ketoconazole on morphology, resulted in inhibition of ergosterol biosynthesis and increase in squalene but no 4-methyl or 4, 14 dimethyisterols wcrc dctected. Thus, it is apparent that the ~.inlount of ergosterol in the membrane rather than the precursor of its synthesis, is probably crucial for phcnotypic divergence [33]. in most cases, the steroi composition has been determined in whole cells, which does not provide the idea of the nature of sterols in plasma membranes. Since the plasma membrane provides the site of interaction of many antifungais, synthesis of ceil-wall enzymes and a barrier for external medium, it would be worthwhile to study sterol composition of the plasma membrane. Nevertheless, the observed difference in sterol composition in different morphological forms may suggest their role in morphogenesis but represents a poorly understood area. In addition to the yeast mycelial transition, C. all#cans also exhibits phenotypic switching of its colony morphology [34-37]. A spontaneous high-frequency white to opaque transition of its morphology is well documented [35-37], which probably provides one of the mechanisms by which this pathogen can evade host defenses [38]. In the white phenotype, cells form colonies which are white and smooth while in the opaque phenotype, colonies are larger, flatter and

ous reasons have been ascribed for such an increase in chitin synthase activity in mutants. Since the membranes of et;g mutants are more rigid [23], it might also result in increased accessibility of an activation factor or trypsin to the zymogenic form of chitin synthase, thereby enhancing the number of active enzymes and its specific activity. The possibility that altered lipid composition of erg mutants could lead to changes in the conformation of the enzyme favourably to allow greater activity, should also be considered. These findings were consistent with earlier observations of Chiew and his co-workers, as they observed that, among various sterols supplemented, viz. ergosterol, lumisterol, stigmasterol and cholesterol, only ergosterol supplementation prevents germ-tube formation in C. albicans [44]. Inhibition of germ-tube formation was associated with complete inhibition of membrane-bound chitin synthase activity. On the other hand, sterol solvents viz. methanol and ethanol, stimulated chitin synthase activity and inhibited germ-tube formation. Since chitin synthase is known to be involved in germination, presumably ergosterol could regulate morphogenesis by modulating its activity [44]. In addition to the role of sterols in various functions as discussed above, it has been shown to block adherence of C albicans to epithelial cells [45]. Sterols, steryi esters, as well as steryl glycosides, were effective in reducing the adherence of C albicans to buccal

opaque or grey. These cells also differ in their requirements for pH-regulated dimorphism as well as cell-wall morphology. Thus analysis of their lipid composition is expected to provide information regarding the role of lipids in phenotypic switching (Table IV). Recently, sterol composition of white and opaque forms has been analyzed [39]. Higher proportions of free sterols were found in white cells when compared with opaque cells. Steryl glycosides and steryl esters, however, were higher in opaque cells. Gas liquid chromatographic analysis showed that the sterol components of white and opaque cells also differed (Table IV).

Ill.C, Enzymatic actit'ity Chitin synthase is an enzyme involved in the biosynthesis of chitin, an important structural component of the coil wall of C. atbicans. This enzyme is Ioca'.~d in the plasma membrane [40] as well as in cytoplasmic particle chitosomes which contain the zymogenic form of it [41]. The enzyme is embedded in a lipidic environment in both locations [42], therefore, modulation in its activity due to an altered lipid environment is very likely. Using erg mutants of C albicans, Pesti and his co-workers have demonstrated the role of ergosterol vis ~ vis chitin synthase and morphogenesis. The total and specific activity of chitin synthase was higher in ergosterol-deficient mutants of C albicans [43]. VariTABLE IV

Cmnin,rison o,f liphls ]hint whiu' and Ol~tque /,hemnypes o[ C a/bicans l~ipid class

Mid.exponential cultures

Stationary cultures

white "

opaque "

white "

opaque ~'

Apolar lirdds Ste~l esters Alkyl esters Triac~Igly~rols Fatty acids 1,2 and 1,3 diglyceridcs Slerols i- and 2-monoglycerides

II),0 ± 0,7 12,(1± 0,9 10,5 ± 0,8 10,8 ± 0.6 7, I ± 0,2 12,2 ± 1,0 9,0 ± 0,5

14,8 ± I, I 10.3 ± 0,7 7.2 ± 0,3 4.5 ± 0.2 I, ! ± 0,(17 6,3 ± 0,4 7.8 ± 0.5

6,8 ± 0.5 9.8 ± 0,h Tr 6.4 ± 0,3 8,4 ± 0.5 10,5 ± 11.9 6.5 ± 0.2

15.3 ± 1.3 2. I :E11.05 3.8 2: 0. I 1.8 :E 0.02 6.7 ± 0.4 7.5 ± 0.6 9,2 ± 0.7

Polar lipids Monosalactt~wldiacylglycerol Stcryl glycosides Ceramide monohexoside Phosphatidylethanolamine Phosphatidylglyc~rol Phosphatidylcholine Digalactos,vldiacYlslyce mi Pho~phatidylim~sitol Phosphatidyl,~rine Phosphatidic acid Cardiolipin

Tr 1.4 ± 0,02 !,6 ± 0.(11 8,6 ± 0,8 12,8 +_ 1.4 4,0 ± 0,09 ND Tr Tr Tr Tr

Tr 15,5 ± 1.3 Tr 12. I ± 0.9 Tr 13,5 ± !. I ND Tr Tr Tr 6.9 ± 0.3

6.0 5:11,119 6,0 ± 0.3 4.2 ± 0. I 9.6 ± 0.6 Tr I 6.5 ± 1.5 2.3 ± 11.1}8 2.9 + 0.06 1.4 ± 0.01 Tr 2.7 ± 0.02

4.8 ± 0. I 15.6 ± I. I 6.4 ± 0.5 7.4 + 0.5 5.6 ± 0. I I 1.8 + 1.0 ND Tr Tr 2.0 + 0.06 Tr

o Values are expressed as % (w/w) of total lipids, each value is the mean ± S.D. of three determinations. Tr = Traces. N D = Not detected, Data taken from Re£ 39 with permission.

epithelial cells, suggesting a putative role of sterols in adherence of this fungus [45,46]. Ergosterol has also been shown to enhance the recovery of mutagenized cells of C. albicans [47]. The enhancement of survival rate was dependent on the stage of cell growth, as it was only observed when non-budding cells were treated with chemicals or ultraviolet radiation [47]. IV. Role of phospholipids

Phospholipids of C. albicans, as well as of other yeasts, represent a typical mixture common to other eukaryotic systems. PC, PE, phosphatidylserine (PS) and phosphatidylinositol (PI) are the major phospholipids of C. albicans [7,29,48,49]. The amount of PC in intact cells ranges from 25 to 56% of the total phospholipids, which accounts for a major portion. The percentage of PE varies between 11 and 33% and that of PS between 9 and 24%, while that of PI ranges between 5 to 20% {Table !1). The significant variations observed in phospholipid contcn: and composition could partly be due to strain variations and methods employed for lipid extraction and estimation [4]. It must be emphasized that pure preparations of plasma membranes have not been employed to study the phospholipid composition, therefore, the exact proportion of individual phospholipids may vary from what has been reported so far. S. cerevisiae represents an extremely useful system for studying the regulation of eukaryotic metabolism

because of the opportunity to combine biochemical and genetic approaches. In contrast, for C. aibicans, which is a diploid, the techniques of classical genetics are not easily tractable. Some recent preliminary s t u d ies have indicated that the major steps of phospholipid biosynthesis in C. albicans may be similar to S. cerevisiae, however, differences at the level of regulation have not been ruled out [8-10,50]. Although, several mutants of S. cerevisiae, defective in phospholipid biosynthesis, are known [51-53], however, no well characterized mutant of C. albicans is available. As a result, it has hampered studies of phospholipid metabolism as well as of its functional roles in C. aiOicans. In the following sections we will enumerate some of the established roles of phospholipids. IV-A. Membrane transport

in the absence of mutants of C. albicans defective in phospholipid metabolism, it is difficult to dial the phospholipid composition in a predictable fashion. However, Trivedi et al. have been able to specifically enrich the plasma membrane fraction of C. albicans with PS by using hydroxylamine, a specific inhibitor of PSdecarboxylase [54]. PS is decarboxylated almost as rapidly as it is formed in yeast cells [54]. As a result of PS-decarboxylase blockage, the steady state accumulation of PS was increased by 4-fold in hydroxylaminegrown C. albicans cells. The increase in PS level was associated with a simultaneous decrease in PE content

TABLE V PhospholipM coniposition (,4) and amino acM mmsport (B) of C. alhicans grown in presence of hydroxyhmlim'. A. Phospholipid

Phosphatidylserine Phosphatidylinositol Phosphatidylcholine Phosphatidylethanolaminc Cardiolipin + uncharactcrised

% of total phospholipid without hydroxylamine 6.6 14.! 39,3 22.5

with hydroxylamine 22.4 12.8 24.8

18.0

23.0

17.0

B. Amino acids

# mol accumulation"/mg protein

L-Phenylalanine L-Methionine i.-Leucine L-Serine Glycine L-Glutamicacid L-Proline L-Lysine

without hydroxylamine 0.531 0.875 11.717 !.225 11.854 0.438 0.366 0.812

a 20 min accumulation. Data taken from Ref. 54 with permission.

with hydroxyamine O. 178 0. i 67

O.334 I.I17 O.124 {).275 {I.358 0.770

Pert mt reduction in Iransport activity 66.5 81).9 53.4 9O.4 85.5 37.2 2.2 5.2

(Table VA). PS, being a negatively charged phospho~ lipid at neutral pH, once accumulated, is expected to affect vital functions. Indeed, when transport of different amino acids was investigated in PS-enriched cells, it was observed that accumulation of most of the amino acids was reduced however, there were few amino acids where uptake remained insensitive to PS fluctuations (Table VB). It is possible that activities of transport proteins related to different amino acids vary in their sensitivity towards fluctuations in phospholipid levels which may account for different responses. IV.R Morphogenesis Phospholipids may turn out to be of special interest in the dimorphism of C. albicans since they are important membrane constituents, whose composition may influence morphology and, consequently, affect interaction with anti-Candida drugs. There are several reports which describe total lipid composition of yeast and mycelial forms of C. aibicans. All these reports, unfortunately, show great variations in their observations, which could partly be due to different growth media and a variety of methods employed to induce germ-tube formation. C. albicans cells in chemically defined medium (Lee's medium) are induced to form a germ tube if grown at 37°C [55]. Yeast growth is, instead, favoured if exposed to 30°C. Based on the incorporation [t4C]acetate into phospholipid fractions of cells grown either at 30°C or at 37°C, Ballman and Chaffin observed that both phospholipid content and its composition remained more or less constant, irrespective of its morphological forms [48], in another study, changes in phospholipid composition have been determined during growth, starvation and germination of C. albicans. An increase in total phospholipid content per cell was observed dining starvation, During germination a decline in phospholipid content in the early hours was followed by an increase in its content at later stages (after 4 h) till it attains a level similar to growing blastospores. Although changes in phospholipid content were drastic, only minor changes in composition were observed. Fluctuations in phospholipid composition was also observed by Ghannoum and co-workers in yeast and mycelial cultures grown for 12 h and 96 h. The changes in phospholipid composition were more pronounced in 96 h cultures than in 12 h cultures [3(}]. Recently, PI has been implicated in various functions in yeasts and other higher eukaryotic systems [56-60], Its involvement in mammalian cells as a secondary messenger is also well documented, where hormones and growth factors activate GTP binding proteins (G proteins) which, in turn, stimulates specific phospholipase C, leading to the cleavage of phosphatidylinositol 4,5-bisphosphate into a diacyiglycerol and

inositol 1,4,5-triphosphate. These two components, inositol 1,4,5-triphosphate and diacylglycerol, act as secondary messengers [59]. The addition of glucose to cultures of S. ceret'isiae was found to increase PI turnover [61]. C. tropicalis grows in filamentous form when cultivated in defined media containing ethanol. The addition of myo-inositol, along with ethanol, has been shown to prevent the morphological transition [62]. Subsequently, it has been observed that cells which were grown with ethanol exhibited an increased rate of PI turnover [63]. Such enhanced metabolism of PI was not observed in fully developed filamentous cells or in yeast-like cells grown without ethanol, thus implying the role of PI in morphogenesis. In view of the observation that steady state levels of PI were low in evagihating cells of C. albicans under pH-regulated regimen (Prasad, R., unpublished observation), it would be worthwhile to follow the turnover of individual phos. pholipids during phenotypic divergence. IV-C Cell cycle Prolonged exposure of C. albicans cells to stationary-phase yields synchronized Gcarrested unbudded singlets. This is due to zinc depletion in growth media [64,65] and is probably the only known method by which C. albicans cells could be synchronized at the G l phase of cell cycle [5]. Using synchronized cells, the status of phospholipids during the progression of C. aibicans from G I to S phase was analyzed [66]. Phospholipid composition of exponentially growing and Gcarrested cells of C. albicans did not reveal any significant difference in the phospholipid head group ratio of individual phospholipids. When Gcarrested prolonged stationary phase (PSP) cells of C. albicans were allowed to grow in a fresh medium, the cells could initiate new buds and grow synchronously tbr at least one generation, after a lag of about 2-5 h. The contents of individual phospholipids were found to increase at the time of bud emergence, which was also reflected in an increased phospholipid to protein ratio at that time [b6]. There was no increase in phospholipid levels prior to bud emergence. However, proteins continued to be synthesized even before one could detect either morphological change or DNA synthesis. Therefore, the drop in phospholipid levels observed prior to bud emergence was due to the synthesis of proteins which were synthesized at the beginning of new growth. A stepwise increase in phospholipid content, coinciding with the bud emergence of C. albicans, was observed [66]. Phospholipid catabolizing enzymes (phospholipases) are of special interest since they appear to play an important role in the pathogenesis of C. albicans [6772]. The activity of phospholipase A is not only associated with the membrane but is released into the sur-

rounding medium as well [67,68,70]. It is likely that lysophospholipids formed due to phospholipase action may act as a membrane disruptive agent and help to invade host tissues in the process of virulence of C. albicans. However, this aspect is purely speculative. V. Role of fatty acids

Palmitic (16:0), stearic (18:0), oleic (18:1) and linoleic (18"2) acids are predominant fatty acids of Candida lipids. In general, yeasts lack polyunsaturated fatty acids but Candida species are unique in containing linoleic (18: 2) and linolenic (18:3) acid (Table II) [13,14,30,73,74]. The isolation of fatty acid auxotroph [13] suggests that fatty acids are essential for C. albicans. Their role, however, is poorly defined. Cerulenin inhibits fatty acid biosynthesis in Candida by specifically blocking functional the cysteine-SH group of/~-ketoacyl thioesterase synthase [75-77]. The resistance to cerulenin could develop either due to a mutation in the gene encoding the acyl synthase subunit or the drug may become impermeable due to surface alterations of resistant mutants. Notwithstanding the fact that the exact mechanism of action of cerulenin is far from clear, mutants of C. albicans resistant to it have been isolated and have yielded useful information about the role of fatty acids in adherence and virulence [78-81]. The mutant strains differ from wild type in several phenotypic traits, including alterations in phospholipid and fatty acid composition and exhibit decreased adherence and virulence. The data imply that changes in adherence and virulence are due to altered fatty acid synthesis [81]. Cerulenin is also known to block chitin l-;osynthesis by perturbing the normal lipid environment [I 1]. An Arrhenius plot has shown a break in the rate of incorporation of mannose from GDP mannose by purified plasma membranes of C. albicans, which again indicated that the enzyme is sensitive to lipid environment [82]. It is thus possible that the fatty acyl composition, by modulating enzymatic activity, may have a role in cell wall biosynthesis [11,82,83]. The composition of the fatty acids varies with the morphological form of C. albicans. Both polar and non-polar fractions from the mycelial form contain higher levels of polyunsaturated fatty acids (18:2 and 18:3) and lower levels of oleic acid (18: 1) than corresponding fractions from the yeast form [30]. The higher contents of palmitate (16:0), palmitoleate (16:1), stearate (18:0), oleate (18: 1) and linoleate (18:2) in mycelial form of C. albicans were observed by Sadamori [31]. The analysis of fatty acyl composition in pH-regulated dimorphism of C. albicans has also yielded useful information [Prasad, R., unpublished observation]. The ratio of SFA to UFA showed morphology-dependent

changes. The ratio was increased around the time of evagination in both populations destined to form bud and mycelium. However, after evagination the ratio remained constant in mycelia-forming populations, while it dropped in bud-forming populations. Recently, the fatty acyl composition of white and opaque cells has been analy,..ed [39]. Comparison of white and opaque phenotypes shows that white cells had higher proportions of palmitoleic (16: 1) and stearic (18:0) but lower proportions of linoleic (18:2) acid than opaque cells. The unsaturated to saturated fatty acid ratio of the total lipid was higher in opaque cells. Fatty acid composition of individual phospholipids, triacylglycerols and non-esterified fatty acids also differed with the phenotype. Unsaturated fatty acids in opaque cells resemble more those in the mycelial culture. The fluidity of membrane is dependent on fatty acyl composition, hence variation in fatty acids between the two phenotypes may be reflected in the altered structure and function of the membrane. This aspect, however, remains to be resolved. Amino acid transport was found to be selectively affected following changes in the lipid composition of C. albicans grown on alkanes of different chain lengths [49]. On the basis of the results obtained with various metabolic inhibitors, there did not seem to be much difference in the mode of energy coupling for the uptake of amino acids. Based on altered lipid composition the amino acid uptake could be grouped into two categories. The uptake of one group of amino acids namely, L-proline, L-lysine, L-serine and L-methionine was reduced in alkane grown cells, while the uptake of other group of amino acids, namely, i,-glutamic acid, i,-phenylalanine, l,-leucine and glycine remained unaffected [49]. AIkane-grown C. albicans cells, when exposed to various doses of y-radiation, do not exhibit the same extent of reduction in uptake of amino acids as observed in normal glucose-grown cells [84]. Similar resistance towards y-radiation was also seen in oxygen uptake. Cell survival was also significantly greater in irradiated, alkane-grown cells as compared to the irradiated normal glucose-grown cells [84]. A few earlier reports using E. coli had shown that iipids affect the extent of damage to cellular DNA by ionizing radiation, which, in turn, affects cell survival [85,86]. It was concluded that the resistance acquired by alkane-grown C. albicans cells towards radiation damage was due to altered lipid composition and was not related to altered growth rate [84]. However, the lipid changes observed, though associated with the plasma membrane [49], were not very specific for any particular class of lipids, and therefore, the lipid molecules responsible for offering protection or resistance towards radiation could not be ascertained [84].

10 VI. Role of membrane lipids in anti.Candida drugs action

The plasma membrane represents for drugs either the target of their action or a barrier to pass through before their internalization into the cell and the development of their subsequent activity. In these conditions the action of antifungal drugs may be perturbed by any modification of the membrane properties. We have seen that changes in lipid composition can be at the origin of such modifications. Actually, the influence of the lipid variation is well documented only for the action of drugs having the plasma membrane for targe' and, more precisely, for polyene antibiotics. Consequently we shall focus our discussion on them. VI.A. Polyene amibiotics Among polyene antibiotics, amphotericin B (AraB) and nystatin (Nys) are widely used (although presenting severe side effects). The consequences of their interaction with the plasma membrane of fungi, which leads to the development of their lethal action, are numerous (see, for instance Refs. 87-89). it is generally considered that their first effect is the formation of transmembrane pores, by binding to the molecules of ergosterol, and through these pores leak cellular components essential for the life of the cell. The exact nature of these pores is still in discussion [87]. They also interfere with membrane enzymes, such as proton A

Acetyt C O

t Squalene t Lanosterol

Azoles Fecosterol ~

er.g.g mutants

Ergosterol Phosphol i pi d s .~..._ . ~ ~ POIyenes

Altered ~nteracttons between plasma membrane components

l

Perturbed membrane functions Fig. I. Site of action of antifungal agents and the block of ergosterol biosynthesis by erg mutants.

ATPase, and disruption of the membrane structure may resulting from the antibiotic-induced lipid peroxidation (Fig. 1). Several studies were performed on model membranes to analyse the role of phospholipids' physical state (gel or liquid crystalline state), length and degree of unsaturation of the fatty acid chains and presence of ergosterol. Information (reviewed by Kerridge [87]) was, therefore, obtained at the molecular (amount of drug bound and changes in its configuration, nature of the pores formed) and at the functional level (characteristics of the induced permeability pathways). It should be noted that earlier studies were generally done on small, unilamellar vesicles, which have special propertie~ not, necessarily tound in cellular membranes, due to the small radius of curvature of the model. Only recently, a more realistic model of large, unilamellar vesicles has been used. it has been shown that pure phospholipid vesicles in liquid crystalline state, the state generally found in biological membranes, bind Arab poorly [90,91] and do not release K + in the presence of the drug [92], that in the presence of ergosterol in the membrane, complexes of stoichiometry I: 1 are formed with AraB, and that there is evidence for single channels [93]. it should be stressed that with cholesterol-containing membranes, mimicking mammalian cell membranes, the interaction appeared to be totally different, where neither the existence of a single channel nor any direct interaction between cholesterol and Arab could be proven. [93]. Until now no studies have been done on the polyene-induced inhibition of membrane enzymes or lipid peroxidation with model membranes. Cellular studies have analysed the correlation between the toxicity and the characteristics of the permeability induced by the drug, as observed in model membranes. While the role cf membrane lipids in relation to the formation of 'pores through binding of ergosterol has been rather well documented, much less is known about the inhibition of membrane enzymes [see 871. Studies on cells resistant to AmB action offer a good opportunity to discover the mechanism of action of the drug and have, therefore been well developed. That polyene-resistant strains of fungi in the laboratory could easily be developed, has certainly facilitated such studies. Hsu Chen and Feingold [94] described two types of polyene resistance in C. albicans: one type resisted polycn.e-induced leakage of intracellular contents and killing (type I) and the other resisted only polyene-induced killing (type ll). In both cases, however, resistance was accompanied by changes in sterol patterns. This duality at the level of resistance may be due to the e>'stence of the two mechanisms of toxicity already mentioned, i.e., permeability changes and lipid peroxidation. If the first mechanism is assumed to be

ll the dominant one, any changes in ergosterol composition would be expected to modify the response of the ceil. That was checked on the sterol auxotroph of S. cerecisiae [95], which offered a convenient way to control the ergosterol content of the cell. When the sterol auxotroph contained a high level of sterol, the cells were sensitive to the effects of nystatin, as monitored by both K + leakage from the cell and viability. When the sterol content was low, sensitivity to nystatin was markedly decreased. Accordingly, in several studies, the analysis of in vitro resistance of C. albicans mutants to polyene antibiotics revealed a lower ergosterol content than in the corresponding wild type, or even no ergosterol at all [7,14,23,96-102]. It was suggested, that the replacement of ergosterol by other sterol components could be associated with strong protein-lipid interaction in the membrane [103,104]. EPR studies of membrane fluidity indicated that a decreased level of ergosterol results in a lower-order parameter when 5-doxyl stearic acid is employed as a probe [105]. Spectroscopic studies indicated that AraB binds much less (Pra:~ad, R. and Bolard, J., unpublished data) to the erg mutant of C. albicans lacking ergosterol [2224]. Actually, all these results are difficult to interpret because the sterol composition is given for the total cell membranes. Data for plasma membranes, the actual target of the polyene antibiotics, are not available. Furthermore, the alterations in ergosterol content are accompanied by changes in sterol composition. In the absence of sound studies on the selectivity of the polyene antibiotics towards different sterois found in these membranes, no firm conclusion can be drawn. In contrast, AmB-resistant mutants have been isolated, which presented wild type sterol profiles [106,107]. That was also observed with mutants of Cryptococcus neoformans, which showed only minimal quantitative changes and no qualitative changes in the sterols [108]. As shown in C. gulliermondii [109], the correlation between the sensitivity to polyene antibiotics and ergosterol content may not be systematic. Even, inc,'eased levels of ergosterol were found in mutants selected after rautagenesis with N-methyI-N'nitro-N-nitrosoguanidine [110]. From al! these data it appeared, therefore, necessary to consider more carefully the consequences of the second mechanism of toxicity, that is lipid peroxidation. A laboratory-derived mutant of C. albicans (L) and a clinical strain of C. albicans (C), both lacking ergosterol, were isolated and shown to be less susceptible to AmB-induced cell membrane permeability to K + and lethality than was the wild type laboratory strain, which contained ergosterol [111]. Similar to the type 2 resistance observed by Hsu Chen and Feingold, the resistance of L and C strain to AmB-induced killing was much greater than the level of resistance to AraB-induced cell membrane permeability. Interestingly their level of catalase activ-

ity were 3.8-fold (L) and 2-fold (C) higher than that of the wild strain. It was, therefore, proposed that the resistance to oxidative-dependent cell damage corresponds to type 2 resistance. Following nitrous acid mutagenesis, two other AraB-resistant mutants were isolated. They presented wild type ergosterol and fatty acid profiles [106]. Only slight differences in AmB-induced K + leakage were observed between the mutants and wild type. However, the rautants were more resistant to the membrane disruptive properties of Triton X-100. The origin of the resistance should, therefore, be searched for in levels of catalase or subtle alterations of membrane structure. It should be stressed that if AmB-induced lipid peroxidation occurs, the lipid composition of C. albicans membrane may affect the activity of the drug, in particular the unsaturation of the fatty acid chains. Parameters other than ergosterol content or resistance of 'ke membrane to lipid peroxidation, may play a role in tile sensitivity of fungal cells to polyene antibiotics. Indeed, for instance, the onset of K + leakage induced by AmB in yeasts depends on the culture age: stationary-phase yeasts leak K + more slowly than exponential-phase yeasts (see Ref. 112 and references therein). However, it was shown that total ergosterol increases during growth but it was also the case for triacylglycerol, which would indicate that triacylglycerol in the cell wall plays a role in antibiotic sensitivity I113]. Accordingly, the importance of the cell wall was stressed because spheroplasts prepared from stationary-phase organisms have the same sensitivity as those prepared from exponentially growing organisms [ll2] Phospholipid composition has been shown to be also important. C. albicans cells, when grown on alkanes of different chain length, have significantly altered lipid composition. Concomitantly, different sensitivities of the cells to various polyenes were also observed I114]. However, increase in PS content does not affect the specific binding of AmB to membrane ergosterol (Prasad, R. and Bolard, J. unpublished results). Similar studies were done on S. cerevisiae cells enriched with PC and PE and demonstrated that phospholipidenriched cells acquired resistance to Nys, Arab and filipin [115]. In conclusion, it seems that the origin of the-resistance of C. albicans to polyene antibiotics cannot be limited to a decrease in ergosterol content. The mechanism may involve differences in catalase levels and subtle changes in the structural features of the membranes, which are still to be unraveled. Membrane lipid perturbations may be generated by the antifimgal drug itself and perturb its subsequent challenge. Indeed, ergosterol synthesis has been shown to bc reduced in C. albicans cells grown in the presence of Nys [25]. A similar effect was observed in the presence of sub-inhibitory doses of AraB. in addition, a

12 delay in the intracellular accumulation of fatty acids appearing during growth was also observed [116].

VI-B. Azole antifungals The antifungal activity of azoles, e.g., clotrimazole, miconazole, ketoconazole, itraconazole, etc., is due to their interaction with ergosterol biosynthesis. They are potent inhibitors of the P450-dependent 14-a-demethylase system. The difference in the extent of inhibition of fungal and mammalian P450"s accounts for the selective toxicity of azole for fungi. Although mammalian cholesterol synthesis is also blocked by azoles at stage 14, demethylation, the dose required to effect the same degree of inhibition is much higher than the inhibitory dose for fungi [18.87]. The inhibition in ergosterol synthesis and concomitant accumulation of 14-methyl. ated sterols in C. albicans has been shown with most of the azoles [18]. The accumulation of 14-a-mcthylergosta-8,24(28bdien-31/,6.a.dioi(3,6.diol) is of interest since the 3,6 diol is unable to support the growth of yeast cells. The lipid compositions of three clinically azole-resistant strains have been reported [7]. There are differences in the phospholipid and fatty acid compositions, but they follow no consistent pattern. The only consistent difference was in the ratio of total phospholipid to non-esterified sterol, which was approximately 2-fold lower in resistant strains.

VI-C. Combbmtion of polyene antibiotics and azole deri~'atit'es in the treatment of fungal infections, a combination of two different drugs is often used, for instance, azole derivatives and AraB. As azole derivatives inhibit sterol synthesi~ the sensitivity of the fungal cells to the risk of AraB to be diminished. Actually the combination was found either antagonistic or synergistic and its outcome was dependent on the duration of exposure of the cells to the drugs [I 17,118].

VI-D. Other anti-Candida drugs As for aculacein, an inhibitor of glucan synthesis, its activity on a resistant mutant of C aibicans and its susceptible strain was examined for cellular lipids. The higher saturation of the lipids in the resistant mutant may contribute to its resistance to the drug [119]. Despite the absence of experimental proof, one may anticipate that the activity of other antifungal drugs may be perturbed by lipid modifications of the plasma membrane of C. albicans. For instance we have seen that lipid alterations modify amino acid transport. A similar modification of peptide transport, still to be demonstrated, would perturb the activity of antifungai

drugs, e.g., polyoxins (nikkomycin and bacilysin) which enter the cell by using the principle of 'illicit transport'. Indeed C. albicans mutants resistant to the polyoxins have been isolated which are defective in amino acids and peptide transport [120]. A similar situation could be envisaged with fluocytosine resistance, which is transported by cytosine permease.

VII. Summary It is clear that C albicans lipids have gained tremendous importance in recent years. In addition to being a barrier for entrance of various metabolites, it also provides the site of action for the synthesis of enzyme(s) involved in cell wall morphogenesis and antifungal action. While alterations in lipid composition during a yeast to mycelia transition have been observed, in most of the studies, lipid fluctuations reported could have been due to various environmental factors involved in the induction of morphogenesis [4,5]. A clear understanding of lipid biosynthesis and metabolic blocks due to antifungal action is likely to shed further light on selective interactions of antifungals. Despite the multifacet role of lipids in various functions of this pathogenic yeast, their exact involvement is poorly understood. The situation is little better with regard to ergosterol and its metabolism. Ergosterol is, indeed, important for anti-candidal activity and appears to be involved in the morphogenesis of C. albicans. The fluctuation in phospholipid composition have led to altered properties of plasma membrane namely, membrane fluidity, transport activities and drug sensitivity, ~hieh suggest that-a critical level of individual phospholipid is important for proper functioning of the plasma membrane. What the exact role is of individual phospholipid is far from clear. Many unan~ swered questions relating to the role of PI and sphingomyelin in signal transduction, involvement of phospholipases in the maintenance of phospholipid composition, and role of lipid transfer proteins in assembly and asymmetry of lipids are some aspects which merit further work.

Acknowledgement The work from our laboratories, included in this review, was partially financed by lndo-French Centre for the Promotion of Advanced Research (NO. IFC/A-304-1/90/289).

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