Modification of Lipid Composition in a Dimorphic Fungus, Candida Albicans During the Yeast Cell to Hypha Transformation

Jpn. J. Med. Mycol. Vol. 23, 159-165, 1982 ISSN 0583-0516

Modification of Lipid Composition in a Dimorphic Fungus, Candida albicans during the Yeast Cell to Hypha Transformation

Koh Yano*, Tomiyasu Yamada, Yoshiko Banno, Takashi Sekiya and Yoshinori Nozawa Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500, Japan

[Received for Publication: February 1, 1982] Alterations in phospholipid and fatty acid composition of Candida albicans were examined during the yeast cell to hypha transformation. The major phospholipids separated by thin layer chromatography were phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol/phosphatidylserine. Large increments of relative amounts of phosphatidylcholine and phosphatidylethanolamine with a profound decrement of phosphatidylinositol/phosphatidylserine were found to occur within 3 to 5 h after the induction of the conversion. There was a great increase in linoleic (18;2) acid in all phospholipids, especially in phosphatidylcholine, with a compensatory decrease in the monounsaturated fatty acids. These alterations were considered to be confluent in the direction of increasing the degree of unsaturation, as ascertained by measuring the unsaturation indices.These alterations in cellular lipid composition were observed to coincide with the rapid elongation of the germ tubes. These results suggest that modification of membrane lipid composition might be asso- ciated with the transformation from yeast cell to hypha in C. albicans.

A number of studies on the analysis of fungal available in which modification of cellular lipid lipids have been made with special reference to ac- composition during transformation of dimorphic climation to environmental conditions1-4), in at- fungi was examined together with the morphologi- tempts to shed light upon a relationship between cal changes. In the present study, we studied phos- cellular lipid composition and morphogenesis5, 6). pholipid and fatty acid compositions of a dimor- In 1971, Gordon et al. 5)stated that in Mucor gene- phic fungus, Candida albicans, during the yeast vensis, a dimorphic fungus, its morphology is not cell to hypha conversion as related to the morpho- directly related to lipid composition. On the con- logical change observed with scanning electron trary, Manocha6) recently reported with another microscope, and found that cellular lipid composi- dimorphic fungus Paracoccidioides brasiliensis tion of C. albicans demonstrated profound fluc- that cellular lipid composition of the organism tuations during the morphological transition. may play a role in the dimorphic behaviour, show- MATERIALS AND METHODS ing differential distribution of the major phospholipids and fatty acids between the yeast and Organism. C. albicans strain 3125, which was mycelial forms of this fungus. These analyses were supplied by Dr. S. Watanabe, Shiga University of performed on lipids extracted separately from the Medical Science, was used throughout this investi- two forms of these fungi when they reached the gation. The organism was maintained on Sabou- same stage of growth and were in morphologically raud glucose (2%) agar slants by transfer every 2 stable conditions. To our knowledge, no report is weeks at 25C. Culture media. For growth of yeast-form cells, *To whom inquires should be addressed. Sabouraud broth with a slight modification 160 真菌誌第23巻第2号昭和57年

(medium A) consisting of 4% glucose, 1 % poly- roform:acetone:methanol:acetic acid:water peptone and 1 % yeast extract was used. For induc- (3:4:1:1:0.5, v/v) in the second direction). After tion of hyphae, a chemically defined medium charring the developed plate with 50 % H2SO4, the (medium B) prepared as described by Tani et al. 7) areas corresponding to individual phospholipids modified by the substitution of L-methionine for were scraped off the plate and their phosphorus casamino acids was employed. This medium con- content was determined by the method of Rouser tained per 100 ml of distilled water: glucose 1. 65 g, et al. 12).For determination of phospholipid acyl L-methionine 100 mg, KH2PO4170 mg, MgSO4 chain composition, the developed plate was 7H2O 13 mg, KC 143 mg, FeC 13. 6H2O 0. 25 mg, sprayed with 8-anilino-l-naphthalenesulfonic acid MnSO4 4H2O 0, 25 mg, Na2HPO4 450 mg and magnesium salt (ANS-Mg), and spots of individual biotin 0, 6, ug. phospholipids were scraped and methylated with Preparation of cells. Stock cultures on Sabou- 1 ml of a 10 % boron trifluoride methanol complex raud glucose agar slants were diluted with medium in sealed ampoules at 100C for 10 min, Quantita- A to 2, 5-3. 0 x 104 per ml. One ml of the suspen- tive analysis of fatty acids was carried out by gas- sion was inoculated into 200 ml each of medium A liquid chromatography (Shimadzu GC-6A type gas in a 500 ml Erlenmeyer flask, and then incubated chromatograph) using a column of 10% diethy- at 37C with gentle shaking in an enclosed hori- leneglycol succinate (DEGS) al 175C. Weight per- zontal shaker for 10 h or to the early stationary centage was calculated by a Shimadzu chromato- phase. pac-EIA integration system. To transform to hyphae, 1. 2-1. 5 x 107 yeast Scanning electron microscope. Washed cells cells in the early stationary phase were transferred were immediately fixed for 1 h with a cold (4C) into 200 ml medium B, and then incubated at solution of 4% glutaraldehyde-2 % osmium tetro- 37C with gentle shaking. Aliquots were taken at xide (1:1) mixture, each solution being prepared selected intervals for morphological observation in 0.1M phosphate buffer (pH 7, 2). The fixed and lipid analysis. Cells were harvested by 5 min samples were subsequently washed in distilled centrifugation at 1, 500 x g and washed free of water and dehydrated in a graded acetone series. media with distilled water. The dehydrated cells immersed in absolute ace- Lipid extraction and analysis. Total lipids tone was quickly transferred to the specimen were extracted from the washed cells according to holder of a Hitachi HCP-1 critical point drying the method of Bligh and Dyer8) with chloro- device, dried using liquid CO2 and examined in a form/methanol (2:1, v/v). In order to obtain a JSM-U3 scanning electron microscope. maximum recovery of extraction, the suspension of washed cells in chloroform/methanol (2:1, v/v) RESULTS was incubated at 37C for 4 h under nitrogen be- Morphological changes during the Y-H, con- fore the chloroform layer was collected. Phospho- version. Morphological transition from the yeast lipid phosphorus content was determined by using cell to hypha was observed with the scanning elec- the method of Bartletts) with the modification of tron microscope and its process is displayed in Fig. decomposition in 70% perchloric acid as described 1, Fig. lA is a picture of typical yeast form cells in by Marinettila). Phospholipid composition was the early stationary phase. The yeast to hypha con- examined by two dimensional thin layer chro- version (Y-H conversion) was observed soon-after matography on 20x20 cm plates made of Silica yeast cells were transferred into medium B. First, a Gel H using the following solvent system; chloro- small projection emerged at one site of each form:methanol:7 N ammonium hydroxide solu- spherical yeast cell and then within 1 to 2 h a short tion (90:54:11, v/v) in the first direction and chlo- germ tube developed as an extention of the small roform :methanol :acetic acid:water (90:40:12:2, projection (Fig. 1B). Yeast cells were no longer ob- v/v) in the second direction. For better separation served at this time, indicating that they were of phosphatidylinositol from phosphatidylserine, almost synchronously transformed to hyphal cells. 2. 5% magnesium acetate impregnated Silica Gel The morphological transition was completed H plate (20 x 20 cm) was developed with chloro- within 4 to 5 h after the transfer, and the cells be- form:methanol:l3 N ammonium hydroxide solu- came the typical hyphae in appearance (Fig. 1C). tion (65:35:5. 5, v/v) in the first direction and chlo- With further incubation, the filamentous cells ap- Jpn. J. Med. Mycol. Vol. 23 (No. 2), 1982 161

Fig. 1. Scanning electron microscopic observations of Candida albicans. (A) Typical yeast form cells in the early stationary phase. Pictures of C. albicans taken at 1. 5 h (B), 4. 5 h (C), and 12 h (D) after the early stationary phase yeast cells were transferred into medium B illustrate the representative stages of the morphological transition from the yeast cell to hypha. Each bar indicates 10, um. peared to conglomerate with each other as shown thentic standards. Major phospholipid compo- in Fig. ID. nents of yeast cells were phosphatidylcholine (PC, Alteration in phospholipid content during the 28%), phosphatidylethanolamine (PE, 15%), and Y-H conversion. During the process of conver- a fraction (PI/PS, 33%) of phosphatidylinositol sion, as germ tubes developed and elongated, the (PI) plus phosphatidylserine (PS). Attempts were culture dry weight increased progressively (Fig. 2). made to separate PI from PS and it was found that In contrast, the phospholipid content decreased in the former comprised approximately 70% of the a reciprocal manner. This may be in part due to total of these two phospholipids. Phosphatidic acid an increment in weight of cell walls primarily com- and cardiolipin were minor constituents. posed of glycoproteins such as glucomannan-pro Fig. 3 illustrates changes in the percentage com- n teinn and glucan-protein 13). position of major phospholipids during the Y- H Alterations in phospholipid polar head group conversion. The levels of both PC and PE in- composition. Individual phospholipids separated creased by approximately 10% within 3 to 5 h on thin layer chromatographic plates were identi- after the transfer while that of PI/PS declined fied by comparing their Rf values with those of au- from 33% to 7% in a reciprocal fashion. There- 162 真菌誌第23巻第2号昭和57年

after, relative amounts of PC and PE decreased gradually while that of PI/PS increased greatly and at 12 h after the transfer the distribution of these major phospholipids returned to their initial levels before the shift to medium B. Fatty acid composition of total lipids. The major fatty acids detected by gas-liquid chromatography in total lipids were palmitic (16:0), oleic (18:1) and linoleic (18:2) acids. The minor constituents include palmitoleic (16:1), heptadecenoic (17:1), stearic (18:0) and linolenic (18:3) acids. Marked changes were observed in the proportional distribution of fatty acids during the Y-H conversion, as shown in Table 1. It is of considerable interest to note that, within a short time of period (1.5h) after incubation in medium B, there was already a striking increase in linoleic acid (18:2) and a compensating decrease in oleic acid (18:1). Such replacement of monoene by diene led to an Fig. 2. Changes in dry weight of cells (/) and phos- increased unsaturation index. pholipid content (0) during the yeast cell to hypha Fatty acyl composition of major individual conversion. Dry weight of cells was measured after phospholipid classes. The extracted lipids were freezing-drying. Each point represents the mean separated by thin layer chromatography and three from two different experiments. major phospholipid fractions (PC, PE and PI/PS) were examined for their fatty acid composition. The fatty acid profile appeared to be characteristic of each phospholipid. While both PC and PE contained primarily oleic (18:1), palmitic (16:0) and linoleic (18:2) acids, the PI/PS fraction had a greatly higher proportion (42%) of palmitic acid. In contrast, linolenic acid (18:3) was a minor component. Thus, the PI/PS fraction was least unsaturated as indicated in U. I, and, in comparison, PC was highest in the degree of unsaturation. Al- terations in fatty acid composition of each phospholipid were followed during the YiH conversion (Table 2). PC exhibited the most pronounced changes. At 3 to 5 h after the transfer, linoleic acid showed an approximately 20% increase and the level of linolenic acid also increased from 1 % to 13%. These increases were offset by a compensatory decrease in the monounsaturated fatty acids in which the oleic acid contributed about a 20% decrease. These results led us to consider that de- saturation of 18:1 to 18:2 might have been brought about during the early period of the conversion. The similar trends of acyl group changes Fig. 3. Alterations in phospholipid polar head group observed in PC were also reflected in PE and composition during the yeast cell to hypha conver- PI/PS. However, the degree of fluctuations was sion. Symbols: (a PC; (A) PE; (Q) PI/PS. Each much less pronounced both in PE and PI/PS. In point represents the mean from three different ex- order to determine which phospholipid plays a periments. dominant role in modifying the degree of unsatu- Jpn. J. Med. Mycol. Vol. 23 (No. 2), 1982 163

Table 1. Alteration in fatty acid composition of total lipids of C. albicans during the yeast cell to hypha transformation

a Given as average of percentage of total fatty acids from three different experiments. b U. I. (unsaturation index) was calculated from [(percentage of each unsaturated fatty acid) x (number of double bond)].

Table 2. Alterations in fatty acid composition of major phospholipids of C. albicans during the yeast cell to hypha transformation

a Given as average of percentage of total fatty acids from four different experiments. b U. I. was calculated as described in Table 1.

ration during the Y-*H transformation, the un- of 114 at 4. 5 h after the transfer and then saturation index was examined. PC's U. I, in- decreased. PI/PS fraction, which is much lower in creased from 94 to a maximum value of 140 at 3 h U. I, as compared with PC and PE, did not show a after the transfer and it gradually decreased there- large increase of U. I. as observed with PC. These after. As for PE, the index reached the maximum results suggest that PC is a principal participant in 164 真菌誌第23巻第2号昭和57年 regulating the degree of unsaturation which might result from aging of hyphal cells. changes during the Y-H transformation of C. The present study demonstrates an association albicans. between morphology and lipid (phospholipid and fatty acid) composition of C, albicans. However, a DISCUSSION causal relationship between the two phenomena Sentheshanmuganathan et a1. 14)suggested that remains as yet unclear. Also, since phospholipid biosynthesis of phospholipid is a form determinant unsaturation was reported to occur at the active in a dimorphic fungus, Trignopsis variabitis. phase of growth of C. lipolytica 17),the possibility Manocha et at. 15)argued against this suggestion by exists that the changes in lipid composition ob- showing that the yeast form of another dimorphic served here merely reflect a common phenomenon fungus Paracoccidioides brasiliensis contains as which occurs during a transition from a non-grow- low an amount of phospholipids as the mycelial ing to growing state. In this context, a working form. A rapid decline in phospholipid content in hypothesis of Bartnicki-Garcia18) on dimorphism yeast to hypha conversion in C. albicans observed from his extensive studies on a dimorphic fungus, in the present study, therefore, suggests that it Mucor rouxii is relevant. He proposed that fila- seems not to be a principal determinant in mor- mentous development occurs when protoplasm is phogenesis. Rather, a more important finding preferentially deposited in the polarized direction obtained here is the large increment in the relative whilst yeast-like development results from the ex- percentages of both PC and PE with a concurrent pansion of protoplasm uniformly in all directions, increment in the degree of unsaturation in fatty although the mechanism which determines the acyl groups. These changes occured during the polarization remained open to question. This pro- early period (3-5 h) after the induction of the mor- posal does not compromise the interpretation of phological transition. Furthermore, it is note- our data since, as described above, the increased worthy that elevation of unsaturation index took membrane fluidity due to enhanced unsaturation place when the cells underwent the rapid morpho- of fatty acyl chains of phospholipids during the logical change by elongation of germ tubes. Taken Y-iH conversion of C. albicans might involve in an into account evidence's) that the unsaturation expansion of protoplasm to a polarized direction index is somehow implicated in the physical state which is governed by unknown factor(s). of membrane lipids and that a greater index In addition, an investigation using a mutant of implies that the membrane lipid bilayer becomes strain 3125 which is incapable of undergoing mor- more fluid, it is not unreasonable to assume that C. phological transition is required to obtain a clear albicans renders its membranes more deformable picture of nutritional effects per se of the medium by increasing membrane fluidity. Thus, cells are on C. albicans. Nevertheless, we would like to able to adapt efficiently to ongoing morphological believe that the present study opens a way to fur- changes. Consistent with our results are the find- ther understanding of the morphogenesis in C. ings described by Manocha6) using Paracocci- albicans. dioides brasiliensis. The lipids of the mycelial form of this fungus were in general shown to be more ACKNOWLEDGEMENTS unsaturated than the corresponding yeast form. The strain of Candida albicans was kindly sup- Since the dimorphic conversion of P. brasiliensis is plied by Dr. S. Watanabe, Shiga University of known to be thermally induced, it is possible that Medical Science. We wish to thank Dr. K. Iwata, temperature per se has a great influence on the Meiji College of Pharmacy, for his critical reading degree of unsaturation of membrane phospho- of the manuscript. lipids; lowering temperature leads to increase in REFERENCES unsaturation as indicated with other cell types. However, in the present study, we grew the yeast 1) McMurrough, I. and Rose, A.H.: Effectsof tem- form and induced the Y-H conversion of C. albi- perature variation on the fatty acid compositionofa cans at the same temperature (37C) and effects of psychrophilic Candida species. J. Bacteriol., 114: temperature on lipid composition, therefore, 451-452, 1973. could be excluded. The recovery to the initial 2) Mumma, R. O., Fergus, CL. and Sekura, RD.: The lipids of thermophilic and mesophilic fungi. profile of lipid composition at 12h after transfer Lipids, 5: 140-103, 1969. Jpn. J. Med. Mycol. Vol. 23 (No. 2), 1982 165

3) Raju, KS., Maheshwari, R. and Sastry, P. S.: Lipids platelets: changes in phosphatidylinositol, phospha- of some thermophilic fungi. Lipids, 11: 741-746, tidic acid and lysophospholipids. J. Clin. Invest.,66: 1976. 275-283, 1980. 4) Sumner, J. L., Morgan, E. D. and Evans, H. C.: The 12) Rouser, G., Siakotos, AN. and Fleischer, S.: Quan- effects of growth temperature on the fatty acid corn- titative analysis of phospholipids by thin layer chro- position of fungi in the order Mucorales Can. J. matography and phosphorus analysis of spots. Microbiol., 15: 515-520, 1969. Lipids, 1: 85-86, 1966. 5) Gordon, P. A, and Clark-Walker, G. D.: Fatty acid 13) Yamaguchi, H.: Effect of biotin insufficiency on and sterol composition of Mucor genevensis in rela- composition and ultrastructure of cell wall of tion to dimorphism and anaerobic growth. J. Bacte- Candida albicans in relation to its mycelial morpho- riol., 107: 114-120, 1971. genesis. J. Gen. Appl. Microbiol., 20: 217-228, 6) Manocha, M. S.: Lipid composition of Paracocca- 1974. dioides brasaliensis- comparison between the yeast 14) Sentheshanmuganathan, S. and Nickerson, W.J.: and mycelial forms. Sabouraudia, 18: 281-286, Composition of cells and cell walls of triangular and 1980. ellipsoidal forms of Trzgonopsis variabilis. J. Gen. 7) Tani, Y., Yamada, Y, and Kamihara, T.: Morpho- Microbiol., 27: 451-464, 1962. logical change in Candida tropicalas pK 233 caused 15) Manocha, MS., San Blas, G. and Centeno, S.: by ethanol and its prevention by myo-inositol. Bio- Lipid compositions of Paracoccidioades brasdiensis: chem. Biophys. Res. Commun., 91: 351-355, possible correlation with virulence of different 1979. strains. J. Gen. Microbiol., 117: 147-154, 1980. 8) Bligh, E. G, and Dyer, W. J.: A rapid method of total 16) Nozawa, Y.: Fluidity and functions of biological lipid extraction and purification. Can. J. Biochem. membranes. Seikagaku, 47: 52-58, 1975. Phys., 37: 911-917, 1959. 17) Kates, M. and Paradis, M.: Phospholipid desatura- 9) Bartlett, CR.: Phosphorus assay in column chro- tion in Candida lipolytica as a function of tempera- matography. J. Biol. Chem., 234: 466-468, 1959. ture and growth. Can. J. Biochem., 51: 184-197, 10) Marinetti, CV.: Chromatographic separation, 1973. identification, and analysis of phosphatides. J. Lipid 18) Bartnicki-Garcia, S.: Symposium on biochemical Res., 3:1-20, 1962. bases of morphogenesis of fungi. III. Mold-yeast di- 11) Broekman, M.J., Ward, J. W. and Marcus, A.J.: morphism of Mucor. Bacterial. Rev., 27: 293-304, Phospholipid metabolism in stimulated human 1963.