Role of Cyclic Adenosine Monophosphate in Phospholipid Synthesis in Mycobacterium

smegmatis ATCC 607 Harpinder Kaur and G.K. Khuller*

Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India

ABSTRACT: The present study was undertaken to examine the influence of intracellular levels of cyclic AMP on phospholipid synthesis in Mycobacterium smegmatis ATCC 607. The cyclic AMP levels were modulated by growing cells in the presence of activator (forskolin) and inhibitor (atropine) of adenylate cy- clase, the synthesizing enzyme of cyclic AMP. Forskolin-grown cells exhibited a 1.4-fold increase in the level of cAMP while a similar decrease (1.8-fold) was seen with atropine-grown cells. These altered levels of cAMP in turn affected the total content, composition and synthesis of phospholipids. Total phospholipid content increased and decreased in cells grown in the presence of forskolin and atropine, respectively. These observations were further supported by alterations in [14C]acetate incorporation as well as in activities of glycerol kinase and glycerol-3-phosphate acyltransferase, the key enzymes of phospholipid synthesis. Pro- tein phosphorylation mechanism seems to be involved in phos- pholipid metabolism as the activities of protein kinase increased and decreased in cells grown in forskolin or atropine cells. Our results demonstrate a correlation between phospholipid synthe- sis and intracellular levels of cAMP in M. smegmatis. Lipids 30, 345-349 (1995).

Cyclic AMP participates in cellular function both in prokary- otic and eukaryotic organisms (1,2). Cyclic AMP in different mycobacterial species is present in large amounts in cells and culture fluid as reported earlier (3). Much of the evidence of cAMP involvement in regulation Of various processes is based on the methods involving manipulation of its intracel- lular levels (4). In Mycobacterium smegmatis, this nucleotide has been shown to enhance the incorporation of [14C]acetate into lipids when the cells were incubated with dibutryl-cAMP (5) or forskolin (6) while in M. phlei, no relationship was ob- served between the changes in phospholipid composition and cAMP (7). However, no systematic study has been carried out to demonstrate whether a correlation exists between intracel- iular levels of cAMP and lipid biosynthesis in mycobacteria. Therefore, the present study has been carried out with

*To whom correspondence should be addres~d.

Abbreviations: CL, cardiolipin; KRB, Kreb's Ringer buffer; PE, phos- phatidylethanolamine; PIM, phosphatidylinositol mannosides.

forskolin and atropine, activator/inhibitor of adenylate cy- clase to find out the relationship between cAMP and lipid biosynthesis in M. smegmatis ATCC 607. Further, prelimi- nary experiments were also carried out to determine the role of cyclic AMP-dependent protein phosphorylation in relation to phospholipid metabolism.

MATERIALS AND METHODS

[ 1-14C]Glycerol-3-phosphate (specific activity 4.75 mCi/mmol -~, [3H]ATP (specific activity 13.5 Ci/mmol), [3H]adenosine 3',5'-cyclic monophosphate (specific activity 2000 mCi/mmol), [y-32p]ATP (specific activity 3000 Ci/mmol), and [l-14C]acetate (specific activity 56.2 mCi/mmol) were purchased from Bhabha Atomic Research Centre (Bombay, India). [3H]Glycerol (specific activity 2.5 Ci/mmol) was ob- tained from Amersham (Buckinghamshire, United Kingdom). Atropine, forskolin, cyclic AMP-dependent protein kinase in- hibitor (catalogue number P-5015) and acetyl CoA were ob- tained from Sigma (St. Louis, MO). Other chemicals used were of analytical grade.

Organism and growth conditions. Mycobacterium smeg- matis ATCC 607 (M. 607) obtained from NTCC (London, United Kingdom) was grown in modified Youman's medium as a static culture at 37~ (8). Cells were harvested in their mid log-phase and processed for different studies.

Determination of optimum concentration of atropine and forskolin. Mid log-phase cells were incubated with different concentrations of atropine (2 to 10 mM) and forskolin (1-7 pM) in Krebs Ringer buffer (KRB) (pH 7.2) for 3 h at 37~ The cells were collected by centrifugation at 4~ and a ho- mogenate for adenylate cyclase assay was prepared by soni- eating the cells at 4~ in 40 mM Tris-HC! buffer (pH 7.5). The homogenate was centrifuged at 5,000 x g for 20 min to remove cell debris, and the supernatant was used for enzyme assay. The activity of adenylate cyclase was assayed by the method of Saiomon et al. (9).

Extraction and estimation of intracellular cAMP. Cells (0.3-0.5 g wet weight) were suspended in hot 0.1N HCI, and the suspension was kept in a boiling water bath for 10 min. Each sample was chilled, centrifuged at 12,000 x g for 10 min

Copyright �9 1995 by AOCS Press 345 Lipids, Vol. 30, no. 4 (1995)

346 H. KAUR AND G.K. KHULLER

at 4~ and pH of the supernatant was adjusted to 7.0. The su- pernatant was again centrifuged at 12,000 • g for 10 min to remove precipitated material (10). Intracellular cAMP was then measured by the method of Takeda et al. ( I I).

Quantitation o f lipids. Lipids were extracted by the method of Folch et al. (12). Phospholipids were quantitated by the method of Marinetti (13). Total phospholipids were separated from total lipids by thin-layer chromatography in acetone while individual phospholipids were separated by one-dimensional thin-layer chromatography on silica gel H (14) impregnated with 0.18% ammonium sulfate in chloro- form/methanol/7N ammonia (65:25:4, by vol) as developing solvent. The respective fractions were scraped off and trans- ferred into vials containing a toluene-based scintillation fluid for radioactivity determination.

Uptake studies. Cells grown in the presence of optimum concentration of atropine (6 mM) or forskolin (5 pM) were harvested and suspended in KRB (pH 7.2). [J4C]Acetate (25 pCi/100 mL) pulse was given for different time intervals ranging from 15 to 90 min. After appropriate incubation, cells were filtered and lipids were extracted.

Fatty acid synthesis. Fatty acid synthesis by intact cells was studied according to the method of Buttke and Ingrain (15). [14C]Acetate was added to the control, forskolin, and atropine grown cultures, at a concentration of 10 laCi/g cells/15 mL KRB buffer and incubated at 37~ for 60 min. The cells were filtered, washed with normal saline and lipids were extracted by the Folch procedure (12). Transesterification of lipids was done using 2% H2SO 4 in methanol by the method of Silbert et al. (16). The methyl esters were extracted with pentane and concentrated under vacuum prior to analysis. Radioactivity was counted using a toluene-based scintillation fluid.

Enzyme assays. The homogenate for adenylate cyclase, phosphodiesterase and glycerol-3-phosphate acyltransferase was prepared by sonicating cells at 4~ in 40 mM Tris-HCl buffer (pH 7.5). For glycerol kinase, the cells were homoge- nized in 10 mM Tris-HCl buffer (pH 8.0) with 250 mM su- crose. The homogenate for protein kinase activity assay was prepared in 50 mM-Tris maleate buffer (pH 7.0). The ho- mogenates were centrifuged at 5,000 • g for 20 min to re- move cell debris, and the resulting supernatants were used for enzyme assays.

The activities of adenylate cyclase and phosphodiesterase were measured according to the methods of Salomon et al. (9) and Aboud and Burger (I 7), respectively. For the activity of acyltransferase, the assay system used was of Okuyama et al. (18), and the filter paper disc method of Goldfine (19), as modified by van den Bosch and Vagelos (20), was employed. The activity of glycerol kinase was determined by the method of Kasinathan et al. (21). The activity of protein kinase was measured as described by Banerjee and Sarkar (22).

RESULTS

The role of cAMP in phospholipid synthesis of M. smegmatis was examined by modulating its intracellular cAMP levels

with the help of forskolin and atropine. These compounds alter the activity of adenylate cyclase and thus increase or de- crease the cAMP levels in the ceil.

Initially, the optimum concentrations of both forskolin and atropine were determined by measuring the activity of adeny- late cyclase in the presence of different concentrations of ei- ther atropine (0-10 mM) or forskolin (0-7 luM) in the assay mixture. The activity of adenylate cyclase increased with in- creasing concentration of forskolin while the reverse was ob- served in case of atropine (Table I). Maximum increase (56%) and decrease (48%) in adenylate cyclase activity was observed at 5 pM of forskolin or 6 mM of atropine, respec- tively. Therefore, these concentrations were used for further studies.

After determining the optimum concentration, cells were grown in the presence of these concentrations, and the growth was monitored. No significant change in the growth pattern of M. smegmatis was observed with optimum concentration of forskolin (5 IJM) and atropine (6 mM). To examine the ef- fect of cAMP on phospholipid synthesis, levels of intracellu- lar cAMP were determined in cells grown in the presence of these compounds. Increased levels of cyclic AMP (40%) was exhibited in forskolin grown cells while a decrease (44%) was observed in atropine-grown cells (Table 2). These altered lev- els of cyclic AMP were probably due to the effect of these compounds on adenylate cyclase as the activity of this en- zyme increased (60%) and decreased (45%) respectively in cells grown with forskolin or atropine (Table 2). However, the activity of phosphodiesterase remained largely unaltered by either treatment.

The effect of altered levels of cAMP on phospholipids of M. smegmatis was examined (Table 3). It was found that total content of phospholipids increased in forskolin-grown cells while a decrease was observed in atropine-grown cells. On examining the individual phospholipid components, signifi- cant increases in phosphatidylinositol mannosides (PIMs) and cardiolipin (CL) were observed in cells grown with forskolin while all the phospholipid components (PIMs, phos-

TABLE 1 Effect of Forskolin and Atropine on Adenylate Cyclase Activity of Mycobacterium smegmatis ATCC 607 a

Adenylate cyclase (nmol/mg protein/min)

mM Atropine laM Forskolin

2 16.21 • 0.55 c 1

4 14.07 • 0.67 d 2

6 10.87 • 0.12 d 3

8 11.64 • 0.39 d 4

10 13.27 • 0.52 c 5

6

7

Control 20.73 • 1.20

23.00 + 0.41 b

25.27 + 0.40 d

27.61 + 0.92 `4

31.65 + 0.68 d

33.14 + 1.19 d

30.03 + 0.77 d

27.98 + 0.82 ~

aValues are mean + SD of three (atropine) or four batches. bp < 0.05.

cp < 0.01. dp< 0.001.

(forskolin) different

Lipids, Vol. 30, no. 4 (1995)

CYCLIC AMP AND MYCOI3ACTERIAL PHOSPHOLIPID SYNTHESIS 347

TABLE 2 Activities of cAMP Metabolizing Enzymes and Intracellular Levels of cAMP of Mycobacterium smegmatis ATCC 607 Grown in the Presence of Atropine or Forskolin a

Adenylate cyclase Phosphodiesterase Intracellular cAMP levels

Cells (nmol/mg protein/min) (nmol/mg protein/min) (nmol/g dry wt)

Control 20.87 • 2.32 1.54 • 0.10 18.67 • 1.31 Atropine 11.17 • 1.25 c 1.79 • 0.05 b 10.54 • 1.21 c Forskolin 32.74 • 3.06 c 1.30 • 0.22 b 25.94 • 1.98 c

aValues are mean • SD of three different batches grown for 4 d in the presence of 6 mM atropine or 5 pM forskolin. bNot significant. cp < 0.01.

TABLE 3 Phospholipid Composition of Mycobacterium smegmatis ATCC 607 Grown in the Presence of Atropine or Forskolln a

Total phospholipids PE

Cells (mg/g dry wt) PIM (mg/g dry wt) CL

Control 25.42 _+ 1.13 9.21 • 0.78 4.64 • 0.42 11.33 • 0.68 Atropine 19.25 + 1.01 c 6,63 + 0.68 c 3.89 • 0.24 b 9.21 • 0.68 c Forskolin 30.79 • 0.99 c 11.48 • 0.79 b 5.20 • 0.548 14.16 • 1.02 b

aValues are mean + SD of five different batches grown for 4 d in the presence of atropine (6 mM; 5 batches) or forskolin (5 pM; 3 batches). bp < 0.05. cp < 0.01. aNot significant; PIM, phosphatidylinositof mannosides; PE, phosphatidylethanolamine; CL, cardi- olipin.

phatidylethanolamine and CL) decreased in atropine-grown cells. These changes in the phospholipids were further con- firmed by examining the rate of incorporation of [14C]acetate into phospholipids and total lipids (Fig. 1). A net stimulation in the rate was observed in forskolin-grown cells as compared to control cells while the rate was decreased in the atropine- grown cells.

The levels of phospholipids in cells change only when the activity of either synthesizing or degradative enzymes are al- tered. The activities of glycerol kinase and glycerol-3-phos- phate acyltransferase, the key enzymes of phospholipid syn- thesis, increased and decreased in forskolin and atropine- grown cells, respectively (Table 4). Further, the activity of glycerol-3-phosphate acyltransferase depends upon the syn- thesis of fatty acids. Therefore, in vivo fatty acid synthesis was monitored, and it was found to increase (34%) in forskolin-grown cells and decrease (46%) in atropine-grown cells (Table 5).

In order to study the mechanism of action of cAMP, the phosphorylating capacity of M. smegmatis was examined. A crude mycobacterial cell extract phosphorylated histone, in- dicating the presence of protein kinase activity (Table 6). On addition of different amounts of cAMP to the assay mixture, the activity of protein kinase increased with increasing con- centration of cyclic AMP. There was maximum activation at 2 pM, indicating the presence of cAMP-dependent protein ki- nase in M. smegmatis (Fig. 2). The protein kinase activity was completely inhibited in the presence of specific cAMP-depen- dent protein kinase inhibitor (20-60 ~tg/I-tL), further indicat-

ing its presence in M. smegmatis. The phosphorylating activ- ity of forskolin- and atropine-grown cells was increased and decreased, respectively, compared to controls (Table 7), which may be due to the increased and decreased intracellu- lar levels of cAMP in these cells (Table 2).

D I S C U S S I O N

In prokaryotes, the role of cyclic AMP in the synthesis of in- ducible enzymes is well established but its effect on the regu- lation of phospholipid metabolism is not known. In this study, the influence of cyclic AMP on phospholipid synthesis was determined by modulating the intracellular levels of cAMP in M. smegmatis. Cells grown with forskolin and atropine ex- hibited increased and decreased levels of cAMP, respectively (Table 2). The raised levels of cAMP in forskolin-grown cells was due to the activation of adenylate cyclase by forskolin (Table 2) which is known to bind the catalytic subunit of the enzyme (23). Atropine decreased the activity of adenylate cy- clase (Table 2), but its mechanism is unknown. Forskolin has been shown to increase the cAMP levels in human platelets (24) while atropine decreased its levels in Neurospora crassa (4).

Forskolin, which increased the cAMP levels, further en- hanced the total content and synthesis of phospholipids while the opposite was seen with atropine, which lowered the lev- els of cAMP (Table 3). These results show a direct correla- tion between intracellular cAMP and phospholipid synthesis in M. smegmatis similar to the observations in M. gypseum

Lipids, Vol. 30, no. 4 (1995)

348 l't. KAUR AND G.K. KHUI.I ER

200

150

~ lOO

X

E ~ 50

120

Forskolin Control

-41- Atropine **

/

15 30 60 90 Time (rain)

(A)

--o- ForskoUn Control

41- Atropine I00-

80-

~ 60 ~ X

20 **

(B) 0 , I I I

15 30 60 90 Time (min)

FIG. 1. Incorporation of [14Clacetate into total lipids (A) and phospho- lipids (B) extracted from control cells (A), forskolin (@), a t ropine (11) grown cells. After suspending the cells in Krebs Ringer buffer, [14C]ac- etate (25 pCi/lO0 mL buffer) was added, and aliquots were taken at dif- ferent time intervals ranging from 0-90 rain. Lipids were extracted, and the radioactivity incorporated into lipids was counted as described in the Materials and Methods section. The values are mean • SD of three different batches. Asterisk denotes values that are significantly different from control. *P< 0.05, ** P < 0.01, ***P< 0.001 based on Student's t- test.

TABLE 4 Activities of Enzymes of Phospholipid Biosynthesis of Mycobacterium smegmatis ATCC 607 Grown in the Presence of Atropine or Forskolin a

Enzyme Control Atropine Forskolin

Glycerol kinase 319.30 + 12.99 129.28 :i: 4.40 c 521.50 • 17.66 c (nmol/mg protein/h) Glycerol-3-phosphate 26.01 • 3.06 11.58 • 3.00 b 56.41 _+ 5.27 c (nmol/mg protein/h)

'Walues are mean • SD of three different batches grown for 4 d in the pres- ence of 6raM atropine or 5 I.tm forskolin. bp < 0.01.

cp < 0.001.

TABLE 5 Fatty Acid Synthesis by Mycobacterium smegmatls ATCC 607 Grown in the Presence of Atropine or Forskolin a

Incorporation of [ 14C]acetate into total fatty acids

Cells (cpm/g dry wt)

Control 8654 • 983 Forskolin 11630 + 1041 b (+34%) Atropine 4630 • 484 c (-46%)

aValues are mean • SD of three different batches grown for 4 d in the pres- ence of 6 mM atropine or 5 pM forskolin. Values in parentheses indicate I~pprCent increase or decrease.

< 0.05. cp < 0.01.

TABLE 6 Protein Kinase Activity in Mycobacterium smegmatis ATCC 607 a

Protein kinase Cells (pmol/mg protein/min)

Control 87.3 + 7.31 Control + inhibitor b No activity Forskolin 139.2 + 12.01 c Atropine 52.3 • 3.5 c

"Values are mean + SD of four different batches. bcAMP-dependent protein kinase inhibitor (conc. 20-60 lag/200 laD. cp < 0.001.

160

140

.~ " 120

~ 80

~ 40

2o

0 I I I I 1 2 3 4

Concentration of cAMP (gM)

FIG. 2. Effect of cAMP on protein kinase activity in cell extracts of My- cobacterium smegmatis. Protein kinase activity was measured by com- paring the transfer of [32p]phosphate from [T-g2p]ATP to histone in the presence of different concentration of cAMP. Assay was carried out in 50 mM Tris-maleate buffer (pH 7.0), containing 20 mM MgCI2, 0.25 mg histone, 150 IJM [7-32p]ATP, 10 mM DTT, 2.5 mg/mk bovine serum al- bumin, and an appropriate amount of enzyme preparation. Values are mean + SD of four different batches. Asterisk denotes values that are significantly different from control (without cAMP). *P < 0.05, **P < 0.01 based on Student's t-test.

(25). The earlier report (5) that dibutryl cAMP increased the incorporation of [14C]acetate into lipids of M. smegmatis fur- ther supports our observations with forskolin.

The increased and decreased activities of glycerol kinase and glycerol-3-phosphate acyltransferase in forskolin- and atro- pine-grown cells (Table 4) also correlates with the altered synthesis of phospholipids in these cells. We observed that

Lipids, Vol. 30, no. 4 (1995)

CYCI.IC AMP AND MYCOBACTERIAI. PHOSPHOLIPID SYNTHESIS 349

the activity of these enzymes depended on cAMP, which is consistent with earlier reports (24,26). Further, the activity of glycerol-3-phosphate acyltransferase depends upon the syn- thesis of fatty acids, which also was found to exhibit increases or decreases in cells grown in forskolin or atropine (Table 5). These results are in agreement with the earlier observation (3) that dibutryl cAMP increased the incorporation of [14Clac- etate into fatty acids by M. smegmatis. Thus, the increased or decreased synthesis of fatty acids in response to the altered levels of cAMP may ultimately be responsible for the altered synthesis of phospholipids.

In eukaryotes, action of cAMP is mainly mediated through cAMP-dependent protein kinase (27). As M. smegmatis is be- lieved to lack cAMP receptor protein (28), the possibility of the presence of protein kinase in this organism was examined. A crude extract of the mycobacteria was found to possess pro- tein kinase activity by its phosphorylation of histone (Table 6). This has also been demonstrated in other bacterial species (29). The addition of exogenous cAMP enhanced the protein kinase activity, indicating that at least some of the protein ki- nase present in M. smegmatis is cAMP-dependent. Similar findings were reported earlier for Escherichia coli (30), Strep- tococci (31 ), and Saccharomyces cerevisiae (32). Increased or decreased activity of protein kinase in cells grown in forskolin or atropine, respectively (Table 6), is probably due to the altered expression of the protein in mycobacteria, and this aspect warrants further study.

In summary, we can conclude from the present results that phospholipid biosynthesis in M. smegmatis is influenced by cyclic AMP and that its effects are probably mediated through protein kinases.

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[Received April 4, 1994, and in final revised form January 18, 1995; Revision accepted January 19, 1995]

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