An overview of lipids of Candida albicans

Prog. Lipid Res. Vol. 29, pp. 65-85, 1990 0163-7827/90/50.00 + 0.50 Printed in Great Britain. All rights reserved © 1991 Pergamon Press pie

A N O V E R V I E W O F L I P I D S O F C A N D I D A A L B I C A N S

PRASHANT MISHRA a n d RAJENDRA PRASAD*

School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, lndia

C O N T E N T S

I. INTRODUCTION 65 II. LIPID PROFILE 65

A. Sterols 65 B. Fatty acids 63 C. Glycolipids ~8 D. Phospholipids ~] E. Sphingolipids 69

III. METHODS FOR ANALYSIS OF CANDIDA LIPIDS 159 IV. A NOTE ON TAXONOMIC SIGNIFICANCE gO V. CELLULAR DISTRIBUTION ~

A. Cell wall B. Plasma membrane U

VI. ENVIRONMENTAL STRESSES AND LIPID COMPOSITION 75 A. Age of the culture 75 B. Oxygen tension 76 C. Carbon sources 76 D. Growth temperature 77 E. Media additives 78

I. Choline and ethanolamine 78 2. Hydroxylamine 79 3. Ascorbate and hydroquinone 79 4. Aqueous garlic extract (AGE) 80 5. Cerulenin 81 6. Alcohol 81

VII. LIPIDS IN STRUCTURE AND FUNCTION 81 VIII. CONCLUSIONS 83

ACKNOWLEDGEMENTS 83 REFERENCES 83

I. I N T R O D U C T I O N

The spurt in Candida research started with its prevailing existence in immunologically compromised patients. Lipids of Candida albicans, being the prime target of antimycotic drug action, have attracted considerable interest. 22'24'31'32'91 Accordingly, several mutants of C. albicans defective in lipid metabolism have been exploited to understand the mechanism of drug action. ~-36 However, the fact that C. albicans can switch from one morphological form to another and can evade antifungal drugs warrants further attention. In addition, due to the recent realization that lipids of C. albicans may be involved in morphogenesis 7'~3's5 and adherence to host, 26-28 the study of lipids of this yeast and various environmental factors that can influence it has gained enormous importance.

In this review, we shall discuss the lipid profile of C. albicans and the array of environmental factors which can influence it. Briefly, the role of individual lipid(s) in membrane structure and functions has also been discussed.

II. L IP ID P R O F I L E

A. Sterols

In contrast to animal cells, yeast and fungi contain ergosterol as their major sterol. 65 Ergosterol differs from cholesterol in having two additional double bonds (A 7, A 22) and

*To whom all correspondence should be addressed.

JPLR 29/2--A 65

66 P. Mishra and R. Prasad

Ergosterol [holesferol

FIG. I. Structure of ergosterol and cholesterol.

a beta-oriented methyl group at C24 (Fig. 1). Ergosterol comprises 49-94% of all sterols present in C. albieans. 47m'~ Sterols are known to basically modulate the fluidity of membranes but in yeast at least four different levels of its functions are known. Based on the amount of ergosterol required by sterol auxotroph(s) of Saceharomyces cerevisiae, these functions have been designated as sparking, critical domain, domain and bulk. 82 It was observed that sterol auxotrophs of S. cerevisiae have restricted growth in presence of cholestanol unless supplemented with a minute amount of ergosterol. This phenomenon has-been coined as a "sparking" function of sterols and is probably involved in nonmembrane related functions. Supplementation of lanosterol, where a low level of ergosterol (10 times that necessary for sparking of cholestanol) is required for growth, has been designated as "critical domain" level of sterol and is unable to regulate overall membrane fluidity. This function is distinguishable from "domain" or "bulk", as plasma membrane isolated from auxotrophs grown on domain or bulk levels underwent no lipid thermotropic transition while those obtained from plasma membrane of cells grown on critical domain level showed thermotropic transitions: 2 Due to lack of sterol auxotrophs, however, such studies are lacking in C. albicans. The sterol composition of a number of polyene/azole resistant C. albicans mutants has been s t u d i e d 3.-36'47'71 but the role of ergosterol in the structure and function of C. albicans membrane could not be established since, in those studies, whole cell lipid composition was analyzed. A detailed lipid composition of plasma membrane isolated from antifungal sensitive and resistant strains is expected to provide definitive information about the mechanism of action of antifungals.

Sterol composition changes significantly during germination of C. albicans. For example, the free sterol content continued to increase over the initial stages of germ tube formation but decreased at later stages. 93 On the other hand, the sterol-ester fraction decreased throughout germination until it attained the concentration found in growing blastospores. A detailed analysis of C. albicans lipids revealed that the lipids of yeast form contained a larger proportion of free sterols than the myeelial lipids. 29 The total sterol content present in mycelial lipid was much higher than in the yeast form and was mainly present in the bound form as steryl glycosides, esterified steryl glycosides and steryl esters. The major steryl glycoside in the mycelial form was characterized as cholesterol mannoside. In a more' recent study, ergosterol and lanosterol contents were found to be higher whereas zymosterol content was lower in the mycelial form. 85 Furthermore, squalene content was found to be about ten times higher in the mycelial than in the yeast form. 85

Shepherd and his coworkers have studied the effect of different sterol supplementations on germ tube formation) 5 Of the different sterols tested, only ergosterol was found to prevent germ tube formation of C. albicans by completely inhibiting the membrane-bound chitin synthase, an enzyme known to be involved in germination. ~5 Cannon and Kerridge,'3 while examining various sterols of different morphological forms of C. albicans, detected no significant difference between yeast and mycelial forms; however, they could observe an interesting correlation between sterols and morphogenesis with the use of sterol biosynthesis inhibitors. Ketoconazole, when present at minimum inhibitory concentrations under mycelia forming conditions, yielded stunted mycelia. 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 ketoeonazole was added in yeast-forming conditions, a low content of ergosterol and an increased amount of 14-methylated sterol was observed while stunted mycelia had a low ratio of 4,14-dimethylsterols to 4-methyl sterols. However, terbinafine, which has a similar effect to ketoconazole on morphology,

Lipids of Candida albicans 67

resulted in inhibition of ergosterol biosynthesis and increase in squalene but no 4-methyl or 4,4-dimethylsterols were detected. It would thus appear that the amount of ergosterol in the membrane, rather than the precursors of its synthesis, is probably crucial for phenotypic divergence, ~

B. Fatty Acids

Similar to higher eukaryotes, the fatty acid composition of C. a.lbicans shows a preponderance of C16 and C18 fatty acids. The structure of major fatty acids found in C. albicans is depicted in Fig. 2. The branched chain fatty acids, cyclopropane and cyclopropene acids, which are common components of bacterial lipids, are absent in C. albicans as well as in other yeasts. Polyunsaturated fatty acids, which are not found in S. cerevisiae, have been detected in C. albicans. The absolute requirement of fatty acids for C. albicans has been shown by isolation of an auxotrophic mutant requiring unsaturated fatty acid for its growth. .7

The fatty acid composition can vary markedly depending on the morphological form of C. albicans. Both polar and nonpolar lipid fractions from the mycelial form contain higher levels of polyunsaturated fatty acids (18:2 and 18:3) but lower levels of oleic acid (18: 1) than corresponding fractions from the yeast form. Sadamori observed that the content of palmitate (I 6: 0), palmitoleate (I 6: 1), stearate (18 : 0), oleate (18: 1) and linoleate (18:2) were higher in the mycelial form of C. albicans, g5 This led him to suggest an immaturity in lipid composition of the mycelial form compared to the yeast form as metabolites of nascent palmitic acid, viz. 16: 1, 18: 0, 18: 1, 18: 2 were higher in the mycelial form.

Involvement of fatty acids in the adhesion and virulence process of C. albicans has recently been suggested by using cerulenin resistant mutants. 3g These mutants have been shown to exhibit decreased virulence and adherence capacities. 3~ Biochemical characteriz- ation of these mutants suggested that the resistance in at least two mutants was partly due to the result of mutation in one of the genes encoding fatty acid synthase which differed in the two strains. ~6 Other aspects of fatty acids such as temperature-dependent changes and variations associated with plasma membrane isolated from yeast and mycelial form are discussed in sections VB and VID.

HO x ¢0 HO x ~0 HO\ #0 HO\ ~0 HO HO\ ~0 HO\ ~0 \ ~0 c

~H2 ~H2 ~H2 ~H2 CH 2 ~H2 ~H2 ~H 2 ~H 2 ~H ~ H2 CH CH CH CH 2 CH 2 CH CH 2 I1 I1 11 CH CH CH

I I ~H2 ~H2 ~H2 ~H2 CH 2 ' ~H2 qH2 qH2 ~H2 CH 2 CHI, CH, I ~H2 ~H2 ~H2 ~ H2 ~H 2 CH, CH, CH3 ~H 2 CH21 CH21 ~H2 ~ H2 ~ H2

CH 2 CH 2 CH 2 CH 2 CH 2 CH I I I I t

CH 3 CH 3 CH 2 CH 2 CH 2 CH, CH2 ~H2 ~H2 ~H2 CH 3 CH3 CH 3 CH3

Myrfstlc acid Palmihc acid Palm~tolemc Stearic acid Ole~c acid Linoleic acid Linolenlc acid (1~:0) (16:0) acid (16:1~9c~s) (18:0) (18:1~9 cis) (18:2 ~9'12c~s,c is) fl8:3~9'12'15 cm,c~ c~)

FtG. 2. Structure of major fatty acids of C. albicans.


19 OH CH~

1 I 5 7 / ~ 11 13 15 17

H 0 , . , " ~ U \ 0 " " T ~ ~"~ 6 8~ ~ 10 12 14 16 18

o ~ N H HO nu 1" I~, , 3' 5' 7' 9' 11' 13' 15' 7'

~" 0 ~ ~ 8 , OH

Cerebros,de

FXG. 3. Structure of cerebroside isolated from C. albicans. Reproduced by permission of Elsevier Science Publishers BV (Biomedical Division) Amsterdam. Chem. Phys. Lipids 43, 1-12 (1987).

C. Glycolipids

Glycolipids represent an important part of total lipids of C. albicans. 29"52 Structural studies on cerebrosides isolated from the yeast form of C. albicans 3125 revealed that glucose is attached to ceramide in a E-configuration and the major fatty acid of ceramide is 2-hydroxystearic acid (Fig. 3). The long chain base was identified as 9-methyl C18 sphinga-2,8-diene. 5~ Other major glycolipids present in C. albicans are steryl glycosides (both free and esterified forms) while digalactosyl diacylglycerol and monogalactosyl diacylglycerol constitute a minor fraction of lipids, z9 There appears to be some specificity with regard to morphological forms. For example, ceramide monohexosides and steryl glycosides are found exclusively in yeast and mycelial forms, respectively. 29

Matsubara and his coworkers extracted cerebrosides from both yeast and mycelial forms and used a combination of physical methods, e.g. proton magnetic resonance, gas chromatography-mass spectrometry, to ascertain the differences in cerebrosides isolated from the two morphological forms. 52 Their analyses revealed that the cerebrosides of the two morphological forms are not different and that glycolipid structure is not modified during dimorphism. However, Ghannoum and coworkers, 29 in an earlier study, did observe differences in glycolipid composition. They had shown that the yeast form contains ceramide hexosides while mycelial glycolipid consists of mainly steryl glycosides. The importance of individual glycolipids isolated from total lipids of C. albicans on adherence of yeast cells to buccal epithelial cells has been evaluated recently. 28 Among various glycolipids, ceramide monohexoside, ceramide dihexoside and steryl glycosides were found to significantly reduce the adherence while other glycolipids had no significant effect on adherence (Table 1).

D. Phospholipids

Phospholipids, which resemble a typical eukaryotic mixture of phospholipids, consist of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). Similar to other eukaryotic systems and yeasts, phospholipids of C. albicans are essential components of cell membrane. PC, which ranges from 25 to 56%, accounts for a major portion of the total phospholipids. 7'21'29'93 The percentage of

TABLE 1. Effect of Individual Glycolipids on the Adherence of C. albicans to Buccal Epithelial Cells

Mean adherence + SE

Glycolipids Without With (pg/ml assay mixture) lipid lipid

Percentage reduction

Ceramide monohexosides (6) 529 + 29 276 + 24 Ceramide dihexosides (7) 529 + 29 268 + 21 Steryl glycosides (11) 529 + 29 243 + 20 Monogalactosyl diacylglycerols (48) 529 + 29 579 + 25 Digalactosyl diacylglycerols (29) 529 + 29 510 + 28 Sulfoquinovosyl diacylglycerol (18) 529 + 29 524 + 24

(48, p < 0.001) (49, p < 0.001) (54, p < 0.001)

NS NS NS

Data are expressed in mean number of yeast cells adhering to 100 BEC. NS---Not significant. Reproduced by permission of Grosse Verlag GMBH, Berlin. Mykosen 30, 371-378 (1987).


PE varies between 12 and 35%, and the percentage of PS between 7 and 24% and that of PI ranges between 6 and 16%. The structures of major phospholipids of C. albicans are shown in Fig. 4. The fluctuations in phospholipid composition during various stages of growth and differentiation and the role of phospholipids in membrane structure and function have been dealt with in Sections VIA and VII.

E. Sphingo/ipids

Sphingolipids are commonly found as membrane constituents, of animals, 94 higher plants ~ and fungi I~ and are rarely found in bacteria. 81 These lipids are known to be associated with cell-cell interactions and growth and differentiation of animal cells. The isolation of a S. cerevisiae mutant requiring a long chain base for growth and synthesis of phosphosphingolipids has categorically suggested a vital role of these lipids in yeasts, u3 Sphingolipids have also been reported as a major lipid component of the plasma membrane of C. albicans which is absent from the mycelial form. 5~ Shepherd and his coworkers have observed that sphingolipid content increases with growth of C. albicans and its content is highest when cells are starved. 93

III. M E T H O D S FOR A N A L Y S I S OF C A N D I D A LIPIDS

The extraction of lipids from intact yeast cells has posed methodological problems mainly because of the presence of a tough cell wall. The amount of lipid extracted varies depending on the type of method used. The variable values reported for lipid contents could also be due to, in part, incomplete extraction. It is known that only one-third of lipids can be extracted even after prolonged soaking of the yeast cells in chloroform/ methanol (2: 1, v/v) or by continuous extraction with a variety of hot solvents. 5 The remaining lipids of the cell can be subjected to further extraction by freeze drying of the cell, mechanical disruption or by acidic extraction. 33"49 The complete extraction of lipids from yeast cells can be ensured by saponifying the residual material after extraction and

Name Structure

0 II

Phosphohdylchohn¢ (P C) 0 CH 2- O - C - R 1

R z - C - O - C H 0 I ÷ / C H 3 CHz-O - g- o - CH2- C . f . ~ C . 3

I_ 0 CH~ 0

Phosphat,dylethanolQm,ne (PE)~ ~H2-O - ~ - R 1

R 2 - C - O - C H I 0 CH2-O - i ~ - 0 - CH2- CH2- ~H 3

o -

Phosphahdyhnosltol (PI) 0

C H 2 - O - ~-R1 OH OH

,o, C H 2 - O - iP_--

0

Phosphahdylser tne (PS) 0

0 CH2-O - ~-R1 II I

R2"- C - O - C H O 0 II Ii

CH2-O -- p - O - C H 2 - C H - C --(3- - I

O NH 3 + 0 +l R COrdlohpln (CL) 0 011 ~ H 2 - O - C - I

0 C H 2 - O - C-R1 R 2 - C - O - C H it I J

R2-C - 0 -CIH i01 O11 CH 2 - O - P - O - CH 2 - CH - CH2-O-I~I - O - CH2

I _ 6 - O OH O

FIG. 4. Structure of major phospholipids of C. albicans.


analyzing the presence of long chain fatty acids by gas liquid chromatography. 83 So far, the lipid extraction procedures used for C. albicans have not taken into account the complete extractiofl of lipids and inactivation of phospholipases during extraction. In general, the extraction procedure involves mixing C. albicans cells of the yeast or mycelial form in chloroform/methanol (2: 1, v/v) and essentially following Folch's extraction procedure. However, recently cells disintegrated with the use of glass beads and a cell disintegrator is also used for total lipid extraction. Methods for the analysis of various lipids and typical chromatograms of Candida lipid are given in Figs 5-9.

IV. A N O T E ON T A X O N O M I C S I G N I F I C A N C E

Lipids have been widely employed for the taxonomic classification of microorganisms; 39a however, little attention has been paid by mycological taxonomists for using lipids as criteria for classification of various fungi. This is mainly because of the lack of information on fungal lipids and well defined morphological distinction among fungi. Phospholipids of fungi predominantly consist of PC, PE, PS and PI and range from 3 to 7% of the dry weight of yeasts. Phospholipid composition is relatively constant and hence does not have taxonomic value. However, based on total lipid content, yeasts have been classified into three main groups, i.e. containing low lipids (< 5%), medium lipids (5-15%) and high lipids (> 15%)? o Because of large variation in total lipid content amongst species of one genus) °m this classification has not been accepted. Fatty acids, on the other hand, have been the choice of taxonomists due to their variation in different classes of microorganisms. Fatty acid profiles of different yeasts are relatively simple which mainly consist of monounsaturated fatty acids and may be accompanied by other fatty acids in relatively small quantities. It is interesting to note that some yeasts like Saccharomyces, contain only monounsaturated fatty acids and lack polyunsaturated fatty acids while Candida species are rich in linoleic acid. The long chain fatty acid composition of 22 species of Candida and 10 species of genus Pichia was determined to establish a relationship between these species.

• :,C-,,,., ~-.1". " ' " , ~ 1 ~ , ~

Sq + SE

TG

FA

L M

E

PG

FIG. 5. Typical chromatograms of sterols extracted from C. albicans grown on YNB medium supplemented with 2.5% glucose as a carbon source. The adsorbent was Silica Gel G and solvent system was petroleum ether (40-60%)/diethyl ether (3:1, v/v). The sterols were visualized by charring. TG, triacylglycerols; E, ergosterol; M, methyl sterol; L, lanosterol; SE, steryl esters; SQ,

squalene; FA, fatty acid; PG, phosphatidylglycerol.


Squalane

Calciferol

ol Erqosterol

Obt usllollol

Lanosterol

24- Methyldihydro lanoste¢o!

FIG, 6. Typical gas-liquid chromatogram of trimethylsilyl (TMS) derivatives of total sterols from C. albicans grown in YNB medium supplemented with 2.5% glucose on 3% SP-2100, column

temperature 230°C.

Based on the presence of linoleic and linolenic acid and the mean percentage of oleic acid, these species are classified into five different groups. ~°8 Sterols which account for 1-10% of the total yeast lipid consist mainly of ergosterol. In addition, yeasts contain a number of sterols (at least 14) which are of little significance for taxonomic purposes. Yeasts secrete lipids in their growth medium and the type of lipid excreted differs with change of conditions. At least four classes of extracellular lipids produced by yeasts have been characterized. However, its presence has not been exploited for taxonomic use. Lipids present in the cell walls of various fungi have also been found to vary but are taxonomically insignificant.


o

o

.'?.

FIG. 7. Typical gas-liquid chromatogram of fatty acid methylesters of total lipids isolated from C. albicans grown on YNB medium supplemented with 2.5% glucose as a carbon source on 10%

DEGS, column temperature 180°C.

V. CELLULAR DISTRIBUTION

A. Cell Wall

The presence of lipids in cell wall of C. albicans has been reported by many workers.~0.~4.2s.43.gz The cell wall lipid content ranges from 0.5 to 10% of dry wall material, depending on the strain and the morphological form (Table 2). Both freely extractable (0.5-1.1%) and bound forms of lipids (0.3-0.6%) have been found. 43 Poulain suggested the presence of lipids bound to peptidoglycan matrix of cell wall. 7z The purified cell walls of the mycelial form of C. albicans has about twice as much lipid as either the juvenile or yeast form (Table 2). Sterol esters, triacylglyceroi, sterols, fatty acids and phospholipids are present in cell walls of all the three forms of C. albicans. Phosphatidylglycerol and sphingolipids, on the other hand, are only present in juvenile and mature forms. Sterols and steryl esters comprise 40-60% oflipids of cell wall of C. albicans. Despite these reports, there have been doubts if lipids isolated from cell wall of C. albicans are indeed its components or present as a contamination of plasma membrane during isolation. These doubts seem to have recently been resolved by Ghannoum and his coworkers. 2s By isolating pure cell wall fraction from C. albicans KCCC 13878, they have shown that cell wall has distinct fatty acids as its components. The exact role of cell wall lipids is unknown. However, they are suggested to confer rigidity or protection against drying. H2

Various classes of lipids of cell wall were found to inhibit the adherence of Candida species to buccal epithelial cells (BEC), suggesting that surface lipids from yeast cells may be involved in their mutual adherence. ~s Individual phospholipids, sterols and steryl esters isolated from C. albicans significantly blocked its adherence to BEC whereas triacylglycerol and free fatty acid were ineffective. 2s It was observed that the ability of phospholipids in blocking adherence is not related to their acyl moieties since neither palmitic nor oleic acid

PA D

Lipids of Candida albicans

" I I I P ~ - • ; -e

CL

PE

D _

PG

CMH

NL

)

73

PS

PI

PC

Ii

I

I

I !

~ r I ~

I

FIG. 8. Typical chromatogram of polar lipids extracted from C. albicans. The adsorbent was Silica Gel G and solvent systems were chloroform/methanol/7 ~t ammonium hydroxide (65:30:4 (v/v/v)) and chloroform/methanol/acetic acid/water (170:25:25:4 (v/v/v/v)). The lipids were visualized by charring. S, start; PI, phosphatidylinisitol; PS, phosphatidylserine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; CMH, ceramide

monohexosides; CL, cardiolipin; FA, fatty acid; NL, neutral lipids.

showed any effect on adherence. In addition, ceramide mono- and dihexosides isolated from the yeast form of C. albicans also inhibited the adherence. Subsequent experiments have shown that total lipid extract of cell wall and from whole epithelial cells (presumably

TABLE 2. Lipid Composition of Cell Wall of C. albicans

Total lipid"

Readily Strata extractable Bound Ref.

C. albicans 0.5-1.1 0.3-0.6 43 C. albicans 582 b 2.1 - - 10 C. albicans 582 ¢ 1.8 - - 10 C. albicans 582 a 4.5 - - 10 C. albicans c 0.6-10.6 - - 14 C. albicans r 4.5-5.5 - - 14 C. albicans ATCC 2.0 - - 92

Fatty acids 8 % Wt C. albicans 14:0 27.8 28 C. albicans 16:0 29.9 28 C. albicans 16: 1 I 1.0 28 C. albicans 16: 2 tr 28 C. albicans 16:3 5.7 28 C. albicans 18:0 12.1 28 C. albicans 18:1 I 1.4 28

a % Dry weight. b Juvenile. cYeast-like mature. d Filamentous mature. c Vcast blastospore. fMycelium. 8Reproduced by permission of Grosse Verlag GMBH, Berlin. Mykosen 30, 371-378 (1987).


i

MG

P k

FIG. 9. Typical chromatogram of apolar lipids of C. albicans. The adsorbent was Silica Gel G; the solvent system was bexane/diethyl ether/acetic acid (75: 5:1 (v/v/v)). The lipids were visualized by charring. TG, triaeylglycerols; FA, fatty acids; DG, diaeylglycerols; S, sterols; MG, monoacyl-

glycerol; PL, phospholipids.

containing the same mixture of lipids) blocked adherence. Thus, surface lipid of fungus as well as of host appears to be involved in their mutual adherence. Strains of yeast isolated from patients with invasive Candida infection demonstrate better adhesion than those from carriers. In some instances, their virulence to animals can be correlated to their adherence.

Adherence plays a decisive role in setting up an infection. Thus, any experimental evidence displaying involvement of lipids in adherence of C. albicans necessitates a careful and critical analysis of lipids present in microorganism and host during the process of infection.

B. Plasma Membrane

Plasma membrane acts as a selective permeability barrier for many hydrophilic substances and is of special importance since it also acts as a primary target of polyene antibiotic action. In addition, it is also the site of enzymatic actions involved in cell wall synthesis and thus likely to be involved in morphogenesis of this organism. There are few studies where lipid composition of pure plasma membrane has been analyzed because of the difficulty in isolation and purification of plasma membrane.

The available data on chemical composition of C. albicans plasma membrane reveal that yeast and mycelial forms contain 43 and 31% of lipid, respectively. Phospholipids account for 7 and 10% of dry weight of membrane from the yeast and mycelial forms, respectively. Plasma membrane isolated from the mycelial form mainly consists of triacylglycerol and free fatty acids and lesser quantities of both free and esterified sterols. The major


TABLE 3. Lipid Composition of Plasma Membrane of C. albicans

Yeast form Mycelial form Lipids (%) (%)

Total lipid 43 + 3 31 _+ 3

Neutral lipids Sterol ester 40 _ 4 28 __+ 1 Triacylglycerol 24 + 3 36 _+ 2 Free fatty acid 17 + 3 27 + 2 Free sterols 19 + 2 9 + 1

Phospholipids Phosphatidylethanolamine 70 + 4 50 + 4 Phosphatidylserine 11 _+ 2 - - Phosphatidylcholine 4 _ 1 50 _+ 4 Sphingolipids 15 -t- 4 - -

Sterols (ester form) Zymosterol 32 + 5 36 +__ 3 Ergosterol I0 + 2 10 + 1 24, 28-dehydroergosterol 10 + 2 12 + 1 3-fl hydroxy-24 methyl

cholesta-5,7 diene 7 + 2 6 + 0 3-fl hydroxy 24 methyl

cholesta-7 ene ND 6 + 0 Fecosterol 4 _ 1 ND Episterol 8 + 3 19 + 2

Fatty acids 16:0 19+4 24+0.5 16:1 8+1 16-t-I 16:2 2 + I tr 17:1 tr 2 + 0 18:0 5+ 1 7 + 0 18:1 26+3 26__+0 18:2 30+3 18+ I 18:3 8+3 6+1

Compiled and reproduced by permission of Society for General Microbiology, Great Britain, J. Gen. Microbiol. 86, 115-132 (1975).

phospho l i p id in p l a sma m e m b r a n e o f the yeast form was found to be PE fol lowed by PC. These two phospho l ip ids were in app rox ima te ly equal amoun t s in p lasma membranes isola ted f rom the mycel ial form. 5~ Asymmet r i ca l d i s t r ibu t ion o f phospho l ip ids between the two halves o f the m o n o l a y e r is an es tabl ished p h e n o m e n o n o f bacter ia l and animal cell membranes . Except in a few instances, such in format ion is not avai lable for any yeast, including C. albicans. In view of the impor t ance o f i ipids in adherence and different iat ion, it would be wor thwhi le to invest igate the asymmetr ica l d is t r ibut ion o f l ipids in C. albicans.

Ergos te ro l is the main free sterol o f p lasma m e m b r a n e ob ta ined f rom both yeast and mycel ial forms while the relat ive amoun t s o f o ther precursors differed in these two forms. Differences in fat ty acid compos i t i on o f p lasma membrane ob ta ined f rom both yeast and mycel ial forms has also been observed (Table 3).

vI . ENVIRONMENTAL STRESSES AND LIPID COMPOSITION

There are several factors which influence lipid compos i t ion o f organisms. Yeast is no except ion, ra ther it is more amenab le to l ipid f luctuations. 75'79 C. albicans l ipids are also affected by a host o f o ther factors. Some o f the factors which affect the l ipid compos i t ion o f C. albicans cells and its m e m b r a n e are discussed in the fol lowing sections.

A. Age of the Culture

Lipid compos i t i on o f C. albicans is significantly influenced by cul ture age. S u n d a r a m and his coworkers observed an increase in the total l ipid and phospho l ip id content o f


C. albicans with growth whereas the levels of sterol, triacylglycerol and free fatty acids remained unchanged. 93 Some minor variation in individual phospholipid content was also recorded by these workers. Combs et al. also did not observe any significant difference in fatty acid profile when lipid composition of cultures of different age groups were compared except for an increase in 18:iso component with age. 17 However, recent studies have revealed that 12 hr cultures of C. albicans consisted mainly of polar lipids, viz. phospholipids and glycolipids, while older cultures (96 hr) of both yeast and mycelial forms showed substantial accumulation of apolar lipids. 29 Besides culture age, the specific growth rate of C. albicans is also known to affect the lipid content. For example, the total lipids and phospholipids of three-day-old cultures of fast growing strains of C. albicans were comparable to seven-day-old cultures of slow growing strains. 23

Using a polyene antibiotic, filipin, as an in situ cytochemical probe for membrane sterols, Takeo inferred that the structural organization of plasma membrane of stationary phase cells of C. albicans differs from that of exponential phase cells. 96 The plasma membrane of exponential phase revealed numerous filipin induced deformations while most of the areas of plasma membrane of stationary phase resisted such deformations. 96 Plasma membrane structures of S. cerevisiae and C. albicans were studied during growth by freeze-fracturing and it was observed that undifferentiated regions of the plasma membrane of C. albicans were severely deformed by filipin whereas plasma membranes of small buds were mildly deformed, suggesting high and low levels of ergosterol, respectively. The bottom part of the invagination and the plasma membrane of the neck between the mother and daughter cells usually lacked such deformation. 97

B. Oxygen Tension

Oxygen tension has a crucial effect on the composition of yeast lipids, especially of sterols and fatty acids. Incubation of C. albicans cells in nonaereated conditions for 120 hr resulted in a decrease in unsaturated fatty acids (UFA), particularly 18:1 and 18:2, while the content of saturated fatty acids (SFA), e.g. 16:0, 18:0, increased under these conditions. In addition to the culture age, these changes have been partially attributed to low oxygen tension, j7 Similar to S. cerevisiae, the synthesis of UFA was also suppressed in C. albicans under anoxic conditions. While S. cerevisiae cells under strict anaerobic conditions become auxotrophic for sterois and UFA, 2'3 such a requirement has not been shown for C. albicans. 75

Rose and coworkers have recently observed that C. albicans accumulates an appreciable amount of long chain alcohols when grown aerobically or anaerobically.~4 However, their synthesis was favoured under anaerobic conditions. It was suggested that saturated long chain alcohol arises by reduction of CoA ester of long chain carboxylic acids that otherwise should be channelled to other neutral lipids. This switch was also associated with the cessation of growth. ~a By selection of growth media and operation conditions, the yield of fatty alcohols (C14-C18) has been increased, j~5 Although efforts to increase the yield of fatty alcohol by supplementing fatty acids in the medium were unsuccessful, ~a5 the potential of C. albicans for conversion of fatty acids into alcohol remains to be tested in detail.

C. Carbon Sources

The ability of yeast to convert different carbon sources into lipid varies with species. 8°'~2 Unlike S. cerevisiae, species of Candida and Torulopsis are able to utilize alkanes of C~0 to Cj8 chain length, 86'99 but most of the hydrocarbon-utilizing yeasts are unable to metabolize shorter chain alkanes (C5 to C9). A primary oxygenase involving cytochrome P-450 is induced at an early stage which hydroxylates n-alkanes for their further utilization. 95'~L6 The growth of Candida on various hydrocarbons resulted in altered lipid composition, reflecting varying metabolic capacities of different strains. 55'77

When C. albicans cells were grown on alkanes of varying chain lengths (Ct0 to Cjs), growth response was variable. There was no growth in the presence of decane (C~0) and

Lipids of Candida albicans

TABLE 4. Lipid Composition of Glucose and n-Alkane Grown C. albicans Cells"

77

n-Alkancs

Component Glucose Ca3 Ct4 Ci5 Ci6 Ct7 Cts

Total lipid b 100 160 200 250 380 450 470 Ergosterol b 15.0 22.0 20.0 17.0 18.0 15.5 15.0 Total glycerides b I0.0 9.0 12.0 11.5 9.0 13.5 13.0 Total phospholipid c 15.0 36.0 35.0 22.0 14.0 8.0 12.0

(12.5) (25.0) (20.3) (20.0) (12.5) (7.8) (9.0) PC ¢ 6.0 20.0 20.0 9.5 5.5 4:5 5.9

(5.2) (12.8) (8.7) (11.6) (5.9) (3.7) (4.1) PE c 3.8 10.0 8.7 5.8 4.6 2.6 3.7

(2.5) (4.2) (3.7) (4.2) (2.8) (1.9) (2.4) PS + PI¢ 1.8 5.0 5.0 4.7 2.8 UN a 1.6

(3.1) (6.7) (6.2) (3.8) (3.8) (1.8) (2.1) Cardiolipin ¢ 0.8 UN UN UN UN UN UN

(1.0)

"Figures in parentheses represent values obtained from crude plasma membrane fraction derived from cells grown under different growth conditions. bMilligrams content per gram protein. ePercentage of total lipid. dUN, Undetected. Reproduced by permission of Academic Press Inc., U.S.A. Adv. Lipid Res. 21, 187-242 (1985).

dodecane (C12) while the g rowth rate was slow in smal ler chain- length a lkanes (Ct3 and C~4) as c o m p a r e d to cells g rown in longer chain- length a lkanes (CI6, CIT, Cts). as However , as c o m p a r e d to g lucose-grown cells, the g rowth rate o f all a lkane-grown cells was slow, with a lower yield o f cells. The slow growth was associa ted with a g radua l increase in the to ta l l ipid content . Mish ina and coworkers have also observed an increase in to ta l l ipid con ten t when C. tropicalis and C. lipolytica cells were grown on different a lkanes . 54 There was no significant change in to ta l e rgos tero l in C. albicans grown on C~7 and Cls alkanes; however , it was 2 0 - 4 0 % more in cells g rown on a lkanes o f lower chain lengths. To ta l phospho l ip ids were a b o u t two-fo ld higher in C~3-, C~4- and C~5-grown cells, while the conten ts were low in Ct6-, C~7- and CIs-grown cells (Table 4). The fat ty acid compos i t ion o f an a lkane -g rown cell and its m e m b r a n e fract ions depends upon the type o f a lkane used as the ca rbon SOUrCe. 66'78"87 These differences in to ta l l ipid as well as in its compos i t i on were

also reflected in c rude p la sma m e m b r a n e f ract ions i sola ted f rom a lkane-g rown C. albicans (Table 4). Therefore , l ipid changes observed in a lkane grown cells were main ly associa ted with the m e m b r a n e fract ion. 8~'s7

D. Growth Temperature

Based on the op t ima l g rowth tempera tures , yeasts are g rouped as psychrophi l ic , mesophi l ic and the rmo to l e r an t strains. 6'n°'ltl On the basis o f the above classification, C. albicans falls into the mesophi l ic group. However , as c o m p a r e d to S. cerevisiae and some

TABLE 5. Effect of Temperature on Fatty Acid Composition of Candida albicans

Percent fatty acid of total Growth

Species temperature 14:0 15:0 16:0 16:1 17:1 18:0 18:1 18:2 18:3

C. albicans IFO 1385 37°C 3.0 tr 36.5 15.4 5.8 13.8 23.5 2.0 tr Serotype A 25°C 0.8 tr 22.1 9.4 - - 5.5 28.3 28.5 5.4

C. stellatoidea IFO 1583 37°C 2.2 2.0 38 .4 13.2 3.3 10.5 28.5 2.0 - - Serotype B 25°C 0.7 0.6 24.0 5.7 - - 8.0 20.7 29.2 10.2

C. albicans IFO 0692 37°C I.I 0.5 28.8 12.1 - - 4.7 30.1 19.3 2.0 Serotype B 25°C 1.8 1.3 23.0 8.6 2.2 7.6 21.8 21.2 8.5

Reproduced by permission of The Faculty Press, Cambridge, Great Britain. Microbios 51, 37-42 (1987).


TABLE 6. Effect of Choline and Ethanolamine Supplementation on Phospholipid Composition of S. cerevisiae and C. albicans

i

Yeast strain

S. cerevisiae C. albicans Percentage of phospholipid

Addition PC PE PI+PS CL+U PC PE PI+PS CL+U

None 41.2 18.5 25.4 2.9 38.9 25.8 19.2 4.0 +Choline 55.0 16.7 20.7 1.5 41.0 20.5 17.7 4.6 +Ethanolamine 39.2 31.3 22.7 4.7 37.2 25.6 17.2 3.4

Reproduced by permission of Academic Press Inc., U.S.A. Adv. Lipid Res. 21, 187-242 (1985).

other species of Candida, relatively little is known about the effect of temperature on growth and lipid composition of C. albicans. Kobayashi and his coworkers 46 have recently observed that lowering the growth temperature of different strains of C. albicans from 37 to 25°C resulted in considerable increase in the content of the polyunsaturated fatty acids 18:2 and 18:3 (Table 5). The effect of temperature on other lipids is not known although, in view of temperature-dependent dimorphism of C. albicans, a detailed analysis of the lipid composition with respect to growth temperature would be worthwhile.

E. Media Additives

The supplementation of growth media with different additives has been a common method to manipulate the lipid composition of Escherichia coli 18-~° and S. cere- visia, ss'58'59'75'76 However, such methods have not been fully exploited in C. albicans. Very few studies have indicated the influence of growth additives on the lipids of this yeast as described in this section.

1. Choline and Ethanolamine

Transport of extracellular choline and its incorporation in PC is known to be an obligatory process ~z4~.42 and thus it has been postulated that eukaryotic cells are largely dependent on the extracellular supply of choline to synthesize PC. ~'73:°9 Contrary to S. cerevisiae, the inability of C. albicans cells to become enriched with PC or PE upon supplementation of choline or ethanolamine, respectively (Table 6), constitutes a major difference between these two yeasts' genera? °2'~°3 This difference was ascribed to either differences in uptake or biosynthetic pathway of choline/ethanolamine. No significant difference in choline uptake and its phosphorylation by choline kinase (E.C. 2.7.1.32) was observed in these two yeasts) °2 However, none of the ~4C choline incorporated into total phospholipids could be recovered in C. albicans, while most of it could be quantitated in PC spot of S. eerevisiae lipids. It was suggested that CTP:chol inephosphate cytidyl transferase (E.C. 2.7.7.15) may be the regulatory step in PC biosynthesis of C. albicans. In addition, the CDP choline pathway of C. albicans may not be fully active as compared to S. cerevisiae.

TABLE 7. Lipid Composition in Normal, Hydroquinone and Ascorbic Acid Grown C. albicans Cells

Lipid composition (mg/g Protein)

Cells Total l ipid Phospholipid Ergosterol

Normal 100 _+ 7.05 26 _+ 2.65 15.0 -/- 0,32 Hy Cells a 98 _+ 4.80 26 _+ 3.08 19.0 _+ 0.58 As cells b 100_+3.26 27+2.18 12.5+0.36

aCells grown in hydroquinone supplemented media. bCells grown in ascorbic acid supplemented media. Reproduced by permission of Elsevier Science Publishers BV (Bio- medical division), Amsterdam. Biochim. Biophys. Acta 555, 42-55 (1979).


\.

F~

(

~ , . , "%.., FxG. 10. Scanning electron microscopy ofC. albwans KCCC 14172 grown m absence (a) or presence (b) of aqueous garhc extract. Bar, 2 #m. Reproduced by permission of Society for General

Microbiology, Great Britain. J. Gen. Mtcrobiology 134, 2917 2924 (1988).

2. Hydroxy lamine

Hydroxylamine hydrochloride blocks PS-decarboxylase (E.C. 4. !. 1.65) and results in the accumulation of PS in E. coli 62 and S. cerevisiae. 57"~°5 Prasad and his coworkers have successfully employed this inhibitor to enrich the plasma membrane of C. albicans with PS. When hydroxylamine was added in growing cultures of C. albicans, they accumulated 1.5- to 3.5-fold more PS than the normal cells. The accumulated PS in plasma membrane of C. albicans provided an opportunity to study the effect of such an enrichment of PS on the structure and function of this yeast. ~°5

3. Ascorbate and Hydroquinone

Yeast cells are known to exhibit altered sterol contents when grown in media supplemented with various compounds, e.g. ascorbic acid, hydroquinone, methylene blue and potassium persulphate. 44 Using a similar approach, it was observed that C. albicans


TABLE 8. Lipid Composition of C. albicans Grown in Absence of Aqueous Garlic Extract

Presence or

Plus AGE Lipids Without AGE (0.4 mg/ml)

Apolar compounds Steryl esters 18.0 + 0.7 7.0 + 0.2 Alkyl esters 2.0 + 0.1 2.0 + 0.05 Triacylglycerols 5.5 + 0.2 3.0 + 0.1 Fatty acids 2.0 + 0 1.5 + 0.05 Diglycerides 3.0 + 0.1 8.0 + 0.3 Sterols 5.0 + 0.1 8.5 + 0.6

Polar compounds Esterified steryl glycosides ND 8.0 5:0.7 Monogalaetosyldiacylglycerols ND 7.0 + 0.5 Steryl glycosides 1.5 + 0 ND Ceramide monohexosidses 3.0 + 0.1 ND Phosphatidylethanolamine 11.5 5:0.8 13.0 5:1.1 Phosphatidylglycerol Tr 1.5 _ 0. I Phosphatidylcholine 21.5 5: i. 1 9.0 5:0.8 Phosphatidylserine 13.0 5:0.9 19.5 +__ 1.0 Phosphatidylinositol 8.0 + 0.3 7.0 + 0.4 Phosphatidic acid 6.5 + 0.4 5.0 + 0.3

Fatty acids 14:0 3.0__+0.2 2.5-t-0.1 14:1 2.7 ___ 0 1.5 + 0.05 16:0 11.6__+ 1.0 26.2+2.3 16:1 Tr Tr 16:2 11.0+1.8 8.8+1.2 18:0 15.0__+ 1.5 14.8+__0.9 18:1 27.2+ 1.1 37.0+3.4 18:2 13.6+__0.8 9.2+0.3 18:3 15.9 + 2.1 Tr

Reproduced by permission of Society for General Microbiology, Great Britain. J. Gen. Microbiol. 134, 2917-2924 (1988).

cells grown in the presence of hydroquinone resulted in a 26% increase in total ergosterol levels while it was reduced by 16-17% in cells grown in the presence of ascorbic acid (Table 7). Such changes in ergosterol level were found to be specific since supplementation of ascorbate or hydroquinone did not alter any other lipids of C. albicans. 89

4. Aqueous Garlic Extract (AGE)

A G E inhibits the growth of a large number of fungi including yeast. 4'8 In addition, a protective effect of garlic against pathogenic yeasts, especially C. albicans, has also been found. 74 Such a protective effect of A G E has been attributed to its ability to block lipid synthesis.I The effect of A G E on the morphology of C. albicans has revealed that cells grown in the absence of A G E were generally smooth walled, spherical to elongated in shape (Fig. 10a), while cells grown in the presence of 0.4 mg/ml A G E appeared deformed and distorted, resulting in cell collapse and cytoplasmic debris (Fig. 10b). Besides morphological changes, C. albicans grown on A G E preferentially accumulate PS with a lower proport ion of PC ~5 (Table 8). Since the synthesis of PC depends upon the decarboxylation of PS to PE, which is subsequently methylated to PC, it was suggested that A G E probably interferes with the enzymes catalyzing the PC synthesis. In addition, A G E is also known to interfere with the fatty acid metabolism especially by inhibiting the biosynthesis of UFA (Table 8). The mechanism by which A G E affects fatty acid metabolism is not clear. Since oxygen consumption is reduced by AGE, therefore, it is possible that the lack of molecular oxygen could affect the degree of unsaturation. In addition, the antagonism of anticandidal activity by thiol compounds suggest that garlic extract could also exert its effect by inactivating essential thiol groups. 25 The finding that the AGE treatment of C. albicans resulted in the accumulation of a glycolipid (steryl glycoside) is interesting because this molecule may have a role in regulating the fluidity


of membrane. 25 Recent observations that AGE inhibits adherence of C. albicans to BEC 26 and that iipids are important in adherence process 27,z8 are worth exploring.

5. Cerulenin

Cerulenin, an antifungal agent, is a potent inhibitor of fatty acid synthase systems of various microorganisms. 63 This antibiotic specifically blocks fl-ketoacyl thioester synthase 63 by binding to the functional cysteine-SH in the active centre .of the condensing enzyme domain. 64 Although it was reported that cerulenin can block the synthesis of both fatty acids and sterols, 6~'~°7 the latter conclusion, however, could not be confirmed, z403° Cerulenin prevented germ tube emergence of C. albicans and this effect could be reversed by the addition of palmitate. 37 Thus, fatty acid biosynthesis appears to be necessary for C. albicans germination. Chitin biosynthesis, which normally accompanies germ tube emergence, was also inhibited by cerulenin. Therefore, it was inferred that cerulenin perturbs the normal lipid environment and thus interferes with the lipid-enzyme interaction necessary for the optimal enzyme activity of chitin biosynthesis. 37 Recently, cerulenin resistant mutants of C. albicans with varying adherence capacity have been isolated. 3s Thus, fatty acids may not only regulate morphogenesis but also appear to have a role in setting up an infection.

6. Alcohol

The effect of alcohol on growth and morphology of various fungi is fairly well established and is known to regulate the morphogenesis of Mucor rouxii, Rhizopus arrhizus, Zygorhynchus rnoelleri, Neurospora crassa and C. albicans. 5°'99' ~00 The dimorphic transition of C. tropicalis has been found to be associated with the accumulation of extracellular glutathione. The addition of myoinositol along with alcohol reduced the production of glutathione and prevented morphological changes. ~17 The cellular content of PI was also extremely low in ethanol grown culture (log phase) of C. tropicalis. Accordingly, in C. tropicalis grown in the presence of ethanol, a high turnover of PI (as judged by pulse chase experiments with 32p) has been observed. 9s Such a high turnover of PI was not detected in filamentous cells or in yeast-like cells if cells were grown with ethanol plus myoinositolJ °6 The involvement of turnover of PI in cell growth and division of S. cerevisiae is emerging 39 but such information is completely lacking for C. albicans. In view of the dimorphic nature of C. albicans and the well established role of PI in signal transduction, 9 this aspect deserves further attention.

VII. L I P I D S IN S T R U C T U R E A N D F U N C T I O N

While the role of lipids in membrane structure is well established for other microorganisms, ts-2°'53 relatively little is known about this aspect in yeast. 75'76 There are even fewer reports on the structural role of lipids of C. albicans. One of the approaches to obtain information about the involvement of lipids in the structure of membrane comprises selective degradation of lipids followed by studying its effect on the structure. Using this approach, Prasad and his coworkers have shown that phospholipase A selectively cleaves PE (40%) and PS plus PI (50%) while most of PC remained inaccessible to the enzyme in spheroplasts isolated from exponentially growing C. albicans. The structural alterations subsequent to phospholipid cleavage were assessed by a fluorescent probe, ANS. ~°4 As compared to untreated spheroplasts, the relative fluorescence intensity of ANS was reduced in phospholipase A treated spheroplasts. This decrease was found to be associated with an increase in apparent dissociation constant and reduced number of binding sites.~°4 Another approach was used by Pesti and his coworkers where ergosterol-less mutants of C. albicans were employed to study the structural role of ergosterol and its precursors. ~ Although these mutants did not exhibit any ultrastructural alterations, as was evident from freeze-etch electron microscopy, using an ESR probe (5-doxylstearic acid) the

JPLR29/2--B


T ~ 9. Effect of Lipid Composition on the Membrane Function of C. albicans

Changes in lipid composition Alteration in lipid Affected achieved by composition function Ref.

1. Growth conditions Supplementation Gross lipid

of growth media changes with alkanes of different chain length

Growth up to Alteration in prolonged phospholipid stationary phase composition

Alcohol PI

Temperature Fatty acid composition Ergosterol Ergosterol

supplementation level

2. Specific drugs or inhibitors Hydroxylamine

hydrochloride

Cerulenin

Block PS- decarboxylation and results in PS accumulation

Inhibits fatty acid synthesis

Hydroquinone Increased level of ergosterol

Ascorbic acid Decreased level of ergosterol

Aqueous garlic Lowers PS and blocks extract fatty acid synthesis

Polyene Sterol levels antibiotics

3. Mutants defective in lipid biosynthesis erg 16 Defective in 5, 6

dehydrogenase and accumulates Zymosterol, Episterol, Ergosta-7-en-3 ~-ol, Ergosta-7,22 diene-3-ol

erg 2 Defective in AS-A 7 isomerase and accumulates Zymosterol and Fecosterol

erg 20 Defective in erg 37 22, 23 dehydrogenase

and accumulates Zymosterol, Episterol, Ergosta-7 en-3-pol

Blocks germ tube 37 formation and chitin synthesis

Amino acid transport, 89, 90 polyene sensitivity

Amino acid transport, 89, 90 polyene sensitivity

Growth and viability 25

Amino acid transport, 90 K + release

Miconazole 67 sensitivity

Membrane 69, 70 permeability, glycerol uptake

Growth and respiration Assimilation of glycerol, methyl- o-glucoside, DL-lactic acid, L-sorbose, L-arabinose and ribitol

Antibiotic 71 sensitivity

Adherence 38 to BECs

Growth and 47 polyene sensitivity

Amino acid transport, 88,89 polyene sensitivity, radiosensitivity 45

Cell cycle 21

Blocks the morphogenic 50 transition

Morphogenesis 46 Inhibits germ tube 15

formation and chitin synthesis

Solute transport 93

erg 40 Defective in As-A~ isomerase and accumulates Zymosterol and Fecosterol

erg 41 Defective in 5, 6 dehydrogenase and accumulates Zymosterol, Episterol, Ergosta-7 en-3 p-ol, Ergosta-7,22-diene-3-/~ ol

C7 Defective in FA demethylase and C4 accumulates DI0 14 methyl sterols Ccrulenin No change in 3H

resistant acetate incorporation mutants into lipids

Fatty acid A 9 desaturase defective auxotroph A'44 mutant results in altered

fatty acyl composition


differences in membrane order parameter between wild type and sterol mutan t strains o f C. albicans were evident. 6a The mutants exhibited higher plasma membrane order parameter than their ergosterol producing parental strain (33 erg ÷) suggesting that the parental strain had a more fluid membrane. Studies are not yet sufficient to envisage a structural role o f individual membrane lipids.

The study o f membrane lipids o f C. albicans in relation to their functions is a relatively new field. 6° As a result, the role o f lipids in the function o f yeast membrane has not yet been unequivocally established. Table 9 lists some o f the membrane functions o f C. albicans, e.g. nutrient t ransport , growth and differentiation, polyene sensitivity and adherence, etc. where the role o f lipids is emerging. 6°

Vlll . CONCLUSIONS

The foregoing discussion reveals that C. albicans, similar to other yeasts, is also amenable to lipid manipulations. These fluctuations in lipid composi t ion o f C. albicans can be induced either by manipulat ion o f environmental condit ions or isolating mutants o f lipid biosynthesis or t reatment o f C. albicans with phospholipases. However, these methods have not been fully exploited to predict a detailed relationship between lipids and the structure and function o f the C. albicans membrane. Nevertheless, what emerges f rom the work already published is that the membrane lipids o f this pathogenic yeast are inextricably associated with its structure and function. Most o f the informat ion is based on lipids obtained f rom the whole cell o f various strains o f C. albicans. The observed variation in results is probably due to species variation and /or due to environmental factors which vary in batch cultures. A prerequisite to avoid variation is to ensure complete extraction o f lipids and to use cells grown in cont inuous culture conditions. Since plasma membrane provides the site o f interaction for many ant imycotic drugs, synthesis o f cell wall enzymes and barrier for external medium, it is essential to reexamine the available data on intact cells by using pure plasma membrane preparat ions for lipid analysis. Such studies are likely to provide precise role(s) o f lipids in various functions.

Acknowledgements--The authors would like to thank Dr M. A. Ghannoum, Kuwait University, for providing photographs of his observations on lipid analysis and Dr S. Kaur for critically reading the manuscript. Data from our laboratory included in this review was partially financed by The University Grant Commission (F.3-273/90(SR-II) and The Department of Science and Technology (SP/SO/D-38/89), New Delhi.

(Receit,ed 1 7 August 1990)

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An overview of lipids of Candida albicans

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