Biochimica et Biophysics Acta, 795 (1984) 493-498

Elsevier 493

BBA 51767

CERULENIN EFFECT ON PHOSPHOLIPID METABOALISM IN MYCOBA CTERIUM SMEGMA TIS ATCC 607

SANDEEP MAHAJAN and GOPAL K. KHULLER *

Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh - 16OOI2 (India)

(Received November 2nd, 1983)

(Revised manuscript received May 23rd, 1984)

Key words: Cerulenin; Phospholipid metabolism; Pulse chase; Cardiolipin; Phosphatidylinositol mannoside; Phosphatidy

lethanolamine; (M. smegmatis)

Phospholipid metabolism in the presence of a subinhibitory concentration of cerulenin was studied in Mycobucte&m smegmds ATCC 607 by pulse labelling and subsequent chasing of radioactivity in phos- pholipids using [32P]orthophosphate. Cerulenin inhibited biosynthesis of total phospholipids to a significant level which is reflected equally in ah the phospholipid components (phosphatidylethanohunine, phosphati- dylinositol mannosides and cardiolipin) within the time of exposure. On chase, alteration in degradation of ah phospholipid components was observed on ceruienin treatment, in comparison with control cells. Differences seen in the metabolism of phospholipids in cerulenin-treated and control cells are discussed.

Introduction

Mycobacterial lipids have been the subject of intensive studies because of their involvement in pathogenicity and their immunostimulative prop- erties [l]. In general, phospholipids have been suggested to play an important role in maintaining the structure and functions of membranes [l-3]. The major phospholipids of mycobacteria are PI mannosides, PE and cardiolipin [4]. To investigate the biochemical importance of lipids in bacterial membranes, several methods have been employed, viz., use of fatty acid auxotrophs [S-7], altered growth conditions (e.g., growth temperature [1,8,9], ethanol supplementation [lo]) and use of 3-de- cynoyl-N-acetylcystamine, which inhibits synthesis of unsaturated fatty acids [ll]. In recent years, cerulenin (2S,3R)-2,3-epoxy-4-oxo-7,1O-dodeca-

* To whom correspondence should be addressed. Abbreviations: PPO, 2,kIiphenyloxazole; POPOP, l&bis-(2- (S-phenyloxazylyl))benzene; PI, phosphatidylinositol; PE,

phosphatidylethanolamine.

OOOS-2760/84/$03.00 0 1984 Elsevier Science Publishers B.V.

dienoylamide, an antibiotic produced by the fungus Cephalosorium cerulens, has been proved to be another potentially useful and more convenient modulator of lipid composition in microorganisms [12]. It is known to inhibit /%ketoacyl-ACP syn- thetase l(condensing enzyme) of fatty acid syn- thetase complex in a wide variety of orgnisms, including bacteria, fungi and yeasts [13]. Its use has now been advocated for studying the biogene- sis of membrane and organelles [12,14]. The effects of cerulenin on metabolism and function of fatty acids in bacterial membranes have been reported [15-171. However, no information has been hitherto available on the effect of cerulenin on phospholipid metabolism due to inhibition of fatty acid synthesis in the bacteria, hence this study was carried out in Mycobacterium smegmatis.

Materials and Methods

Materials. Carrier-free [ 32P]orthophosphate was obtained from Bhabha Atomic Research Centre, Bombay, India. Antibiotic cerulenin was purchased

494

from Makor Chemicals, Jerusalem, Israel. Bacterial strain and cultivation. Mycobacterium

smegmqtis ATCC 607 obtained from the National Collection of Type Cultures, London was main- tained on Lowenstein Jensen Medium [18] and grown in surface culture in modified Youman’s medium (0.5% potassium dihydrogen phosphate/ 0.05% magnesium sulphate/ 0.5% asparagine/ sodium titrate/4% glycerol (pH 7.2-7.4)) at 37 o C. Cells were harvested by filtration in mid-log phase after 4 days of inoculation [lo].

Pulse-labelling of phospholipids. Cells harvested by filtration were washed with physiological saline and resuspended under sterile conditions in phos- phate-free Youman’s medium (KH,PO, was re- placed by 0.24% Tris). The cells were incubated at 37 ‘C for 60 min to minimise the endogenous phosphate pool. Further, the cells were treated with cerulenin (1.6 pg/ml final concentration) for 10 h. Carrier-free [ 32P]orthophosphate (2.0 mCi/7.0 g wet cells per 100 ml) was then added and incubation continued for 150 min. At the time intervals indicated, aliquots of culture were re- moved and added to equal volumes of 15% trichlo- roacetic acid. The cells were harvested by filtration and lipids were extracted. Radioactivity was counted in a Packard Tricarb Liquid Scintillation Counter using a toluene-based scintillation fluid containing 0.4% PPO and 0.05% POPOP. The individual phospholipids were separated by thin- layer chromatography and the radioactivity in each phospholipid component was determined.

Chase of radioactivity in prelabelled phospholi-

pids. Pulse-labelling of phospholipids in control cells and cells treated with cerulenin for 10 h was done as described above for 120 min. The labelled cells were then harvested by filtration, washed with high phosphate medium (0.4 M) and resus- pended in normal Youman’s medium containing 0.04 M phosphate. Cerulenin-treated cells were resuspended in normal Youman’s medium con- taining 1.6 pg/ml of cerulenin. Incubation was continued for 24 h and, at indicated time intervals, aliquots of culture were removed, added to equal volumes of 15% trichloroacetic acid, filtered and the lipids were extracted. Radioactivity in ex- tracted lipids was counted as described above.

Extraction, purification and quantitation of lipids. Extractions, purification and quantitation of phos-

pholipids were done according to the method re- ported earlier [I$].

Results and Discussion

The minimum inhibitory concentration of cerulenin for Mycobacterium smegmatis ATCC 607 was determined to be 1.8 pgg/ml. Since this con- centration would stop metabolic processes in the cells, a subinhibitory concentration of 1.6 pg/ml was used in this study. To determine the ap- propriate time of treatment that should be given to cells, cerulenin and [ 32P]orthophosphate were ad- ded together in the medium and cells were in- cubated for 12 h. Specific radioactivity in total phospholipids showed (Fig. I) a low rate of incor- poration of [32P]orthophosphate with time on cerulenin treatment as compared to the control. After 10 h of exposure, more than 50% inhibition was noted in the incorporation of label into the

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Fig. 1. Incorporation of [32P]orthophosphat: into total phos- pholipids of control and cerulenin-treated M. smegmntis ATCC 607. Cells in log phase were harvested by filtration, washed and suspended in phosphate-free Youman’s medium. Cells were kept in a shaking bath at 37 o C for 60 min. Cerulenin (1.6 pg/ml) and [32P]orthophosphate (2.0 mCi/7.0 g wet cells per 100 ml) were added together to cell suspensions and incubated for 12 h. Aliquots were removed at time intervals indicated, added to equal volumes of 15% trichloroacetic acid, fiitered, and lipids were extracted. Radioactivity in lipids extracted was

counted using toluenebased scintillation fluid. +----- *, con-

trol cells; A -A, cerulenin-treated cells.

phospholipids. Hence, in subsequent experiments to examine the effect of cerulenin on the metabo- lism of phospholipids, mycobacterial cells were pre-exposed for 10 h to cerulenin.

After a 10 h exposure of cells to cerulenin, pulse-labelling of phospholipids with [ 32P]ortho- phosphate was followed for 150 min (Fig. 2). The net synthesis of phospholipids was observed to be significantly lower in cerulenin-treated cells when compared to the rate seen in control cells. Among the individual phospholipids of control cells (Fig. 3a), the highest specific activity was observed in PE, followed by PI mannosides and cardiolipin as reported previously [19]. Similar patterns of net synthesis were observed in cerulenin-treated cells (Fig. 3b) but with decreased rates of synthesis. The extents of inhibition noted after 150 min of pulse for cardiolipin, PE and PI mannosides were 60, 58 and 54%, respectively. It is clear from these studies that phospholipid synthesis may have been af- fected because of the inhibitory action of cerulenin

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Fig. 2. Change in specific activity of total phospholipids of control and cerulenin-treated M. smegmatis ATCC 607. Cells in

log phase were harvested by filtration, washed and suspended

in phosphate free Youman’s medium. Cell suspensions were

kept in a shaking bath at 37 OC for 60 min. Cerulenin was

added to cells (1.6 pg/ml final concentration) and incubated

for 10 h. At the end of incubation period, [32P]orthophosphate

was added (2.0 mCi/7.0 g wet cells per 100 ml) and incubation was continued for 150 mm. At time intervals indicated, aliquots were removed and added to equal volumes of 15% trichloro- acetic acid, filtered, and lipids were extracted. Radioactivity in lipids extracted was counted using toluene-based scintillation fluid. a- 0, control cells; A -A, cerulenin-treated

C&S.

c (b)

5 zo-

; 15-

u

IO-

5-

0 15 30 60 90 120 150

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Fig. 3. Change in specific activity of individual phospholipid

components of M. smegmatis ATCC 607. Thin-layer chro-

matography of lipids extracted from aliquots taken at different

time intervals of pulse experiment (as detailed in Fig. 2) was

performed using Silica gel H (impregnated with 0.18%

(NH,),SO,) and solvent system containing

chloroform/methanol/7 N ammonia (65 :25:4, v/v) to sep-

arate the phospholipid components. Spots were identified and

scraped directly into vials for counting radioactivity. (a) control

cells; (b) cerulenin-treated cells; l - 8, PE; A -A, PI

marmoside; n ---- W, cardiolipin.

on fatty acid biosynthesis. Further, it has been shown that phospholipid synthesis is regulated at the phosphatidic acid level in mycobacteria [20] probably through acyltransferases. Decreased rates of synthesis were also seen when the growth tem- perature of M. smegmatis was shifted from 37 to 27 ‘C [19] and on ethanol supplementation [21].

Chase of radioactivity in M. smegmatis was done in labelled cells for 24 h after removing the extracellular [ 32P]orthophosphate (Fig. 4). In con- trol cells, continuous incorporation of the label into phospholipids was observed for up to 6 h, followed by 66% loss of radioactivity up to 24 h. In contrast, there was continuous incorporation of

496

01 1 I I

0 4 6 8 12 16 20 24

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Fig. 4. Chase of radioactivity in total phospholipids of control and cerulenin treated M. smegmatis ATCC 607. Log-phase cells harvested by filtration were washed and suspended in phos- hate-free Youman’s medium. Cells were kept in a shaking bath at 37 ’ C for 60 mm. Cerulenin (1.6 ag/ml final concentration) was then added to cultures and incubated for 10 h. At the end of the incubation period [ 32P]orthophosphate was added (2.0 mCi,G’.O g wet cells per 100 ml) and cells were pulsed for 120 mm. At the end of this period cells were harvested by filtration, washed with high phosphate medium (0.4 M) and resuspended in normal Youman’s medium containing 0.04 M phoshate. Cerulenin-treated cells were resuspended in normal Youman’s medium containing cerulenin (1.6 pg/ml final concentration). Chase of radioactivity was done for 24 h and, at time intervals indicated, aliquots of culture were removed, added to equal volumes of 15% trichloroacetic acid, filtered and lipids were extracted. Radioactivity in lipids extracted was counted. 0 - 0, control cells; A---- A, cerulenin-treated cells.

32P into phospholipids in cerulenin-treated cells up to 16 h, followed by 14% loss of radioactivity during the subsequent period. Synthesis of phos- pholipids observed even after the removal of [32P]orthophosphate from the medium occurs due to the presence of a large pool of 32P-labelled precursors formed inside the cell, as suggested previously [19,22]. The present study shows that this pool gets exhausted more rapidly in control cells (6 h), while it continued to exist up to 16 h because of slow utilization of 32P-labelled pre- cursors in cerulenin-treated cells. This contention is further supported by the decreased rate of incor- poration of the label into phospholipids on cerulenin treatment (Fig. 2). Decreased degrada- tion (low turnover) seen in the temperature-shift studies [19] and in the presence of ethanol [21] in M. smegmatis has also been attributed to the slow-

ing down of metabo]ic processes in the ccl]. Fig. 5a

shows loss of radioactivity in individual phosghos-

Hipids of control ce]]s. After the initial incorpora-

tion of label into phosphoiipid components, loss of

radioactivity was seen to be maximal in PE (NE%),

followed by PI mannosides (46%) and cardiolipin (35%). Phosphatidylethanolamine and PI manno- sides were noted to be the most dynamic compo- nents of M. smegmatis [19], while in M. phiei they

are stable components [22]. In contrast, cerulenin-treated cultures (Fig. 5b)

showed continuous incorporation of label in PE and cardiolipin up to 16 h, followed by 12 amend

11% loss of radioactivity during the next g h examined, respectively. Radioactivity of PI man-

01 I I 1 I I I f

0 4 6 6 12 16 20 24

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Fig. 5. Chase of radioactivity in individual phospholipid com- ponents of M. smegmatis ATCC 607. Thin-layer chromatogra- phy of lipids extracted from aliquots taken at different time intervals of chase experiment (aa detailed in Fig. 4) was per- formed as described in fig. 3. (a), control cells; (b), cernlenin- treated cells; O- 0, PR, A -A, PI mannoside; O-U, cardiohpin.

TABLE I

EFFECTS OF CERULENIN ON PHOSPHOLIPID COMPOSITION

Cells in log phase were given treatment with cerulenin (1.6 pg/mI final concentration) and harvested after 24 and 48 h of further

incubation. Lipids were extracted and quantitated. Values are means + S.D. of four different batches and are expressed as mg/g dry

wt of cells. The results were considered to be significant with P < 0.05. The levels of significance are denoted by a P i 0.05;

b P .c 0.01; ’ P i 0.001. d Non-significant values.

Duration of Total Total Phosphohpid composition

incubation

with cerulenin

lipids phospholipids PI mannosides PE cardiolipin

24 h

Control

Treated

93.03 f 3.53 20.01+ 1.73 7.70 + 0.31 3.62+0.16 8.44kO.19

95.83 + 2.82 d 20.90 + 0.69 d 8.40i0.20d 3.51*0.11 d 8.98 f 0.13 d

48 h

Control

Treated

104.70 + 2.12 24.59 + 1.20 11.12+0.78 3.44 + 0.29 10.03 + 0.61

110.94k3.53 a 30.96 + 2.07 b 13.44 i 0.55 b 4.70+0.17 c 13.61 k 0.47 ’

nosides became almost stable after 8 h of removal and many membrane-located enzyme activities of label from the medium. The stability observed have been observed to be associated with these in PI mannosides, which is otherwise a dynamic macromolecules [3]. Thus alterations observed in component in control cells, might be necessary in phospholipid composition on cerulenin treatment maintaining the structural integrity of the mem- will provide a novel method for studying the func- branes in cerulenin-treated cells. Low turnover tions ascribed to phospholipids in the membranes. observed in phospholipid components on cerulenin In brief, this study demonstrates that cerulenin treatment is probably due to the changed physio- exerts an inhibitory effect on the biosynthesis and logical state of the cells. Phospholipid metabolism degradation of phospholipids and alters the phos- has previously been shown to be sensitive to the pholipid composition of M. smegmatis. However, physiological state of the cells in Bacillus subtilis further work at the enzymatic level is warranted to [23] and in Af. smegmatis grown in the presence of provide knowledge about the mechanism of ethanol [21]. cerulenin action on phospholipid metabolism.

Since chase studies revealed alteration in turnover of total and individual phospholipids, compositional analysis of phospholipids was done to determine whether or not the effect of cerulenin was reflected in .the phospholipid content of the cell. Table I shows the phospholipid composition of M. smegmatis treated with cerulenin. There was accumulation of phospholipids after 48 h of ex- posure to cerulenin. Phospholipid content in- creased by 25% in cerulenin-treated cells in com- parison to control cells. This accumulation was reflected significantly in all the phospholipid com- ponents (PE, PI mannosides and cardiolipin) and it can be attributed to the low catabolism of these components on cerulenin treatment as seen by chase studies (Fig. 4).

Acknowledgement

S.M. thanks the Council of Scientific and In- dustrial Research for awarding a Senior Research Fellowship.

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