Calmodulin mediated activation of acetyl-coa carboxylase during aflatoxin production by Aspergillus parasiticus

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1 Letters in Applied Microbiology 2000, 30, Calmodulin mediated activation of acetyl-coa carboxylase during aflatoxin production by Aspergillus parasiticus J. Praveen Rao* and C. Subramanyam Department of Biochemistry, Osmania University, Hyderabad, AP, India 35/99: received 2 November 1999, revised 22 November 1999 and accepted 7 December 1999 J. P R A V E E N R A O A N D C. S U B R A M A N Y A M The relevance of Ca 2 -calmodulin-mediated processes in channelling acetate for a atoxin formation was investigated by studying the in uence of tri uoperazine (an anticalmodulin agent) on [ 14 C]-acetate incorporation and activity of acetyl-coa carboxylase in Aspergillus parasiticus NRRL Culturing the organism in presence of 014 mmol l 1 tri uoperazine resulted in 55% decrease of [ 14 C]- acetate incorporation into a atoxin B 1, along with an 80% decrease in acetyl-coa carboxylase activity at periods corresponding to maximal a atoxin production. Concomitant decrement (35%) in the activity of glucose-6-phosphate dehydrogenase indicated decreased availability of reduction potential (NADPH) required for a atoxin biosynthesis. The ability of calmodulin to activate and tri uoperazine to inhibit acetyl-coa carboxylase activity in a dose-dependent manner was also noted under in vitro conditions. The combined results suggest calmodulin-mediated activation of acetyl-coa carboxylase as an important event for a atoxin production. INTRODUCTION A atoxins are well-recognized toxic fungal secondary metabolites that have deleterious effects on human and animal health due to their carcinogenic, mutagenic, immunogenic and teratogenic properties and their entry into food chains (Smith et al. 1995; Steyn 1995). Despite the large number of anti-a atoxigenic compounds studied, the mechanism of action of only a few have been investigated (Maggon et al. 1977; Zaika and Buchanan 1987; Jayashree and Subramanyam 1999). Further research into anti-a atoxigenic properties of different compounds would be bene cial not only for comprehending the regulatory phenomena involved in mycotoxigenesis, but also may contribute towards developing newer strategies for controlling a atoxin contamination of foods and feeds. Earlier studies from our laboratory have revealed the requirement of Ca 2 for a atoxin production (Praveen Rao and Subramanyam 1999) and inhibition of a atoxin production by tri uoperazine suggesting the importance of calmodulin-dependent protein phosphorylation/dephosphorylation for the onset of a atoxin biosynthesis (Praveen Correspondence to: C. Subramanyam, Department of Biochemistry, University College of Science, Osmania University, Hyderabad , AP, India ( csubramanyam@hotmail.com). *Present address: Division of Agriculture and Agricultural Life sciences, Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-Ku, Tokyo , Japan. Rao et al. 1998). In order to unravel the anti-a atoxigenic property of tri uoperazine and to understand the relevance of Ca 2 -calmodulin-mediated regulation of a atoxin biosynthesis, studies were conducted towards (i) monitoring the incorporation of [ 14 C]-acetate into a atoxins, (ii) assaying the activity of acetyl-coa carboxylase, the enzyme involved in channelling the precursor acetate towards a atoxin synthesis, and (iii) assaying the activity of other enzymes indicative of primary metabolism at different growth periods corresponding to trophophase (2-d growth period; corresponding to exponential growth) and idiophase (3-d growth period; corresponding to maximal a atoxin production). The present communication describes results that implicate acetyl-coa carboxylase as the target enzyme in uenced by tri uoperazine, indicating the regulatory importance of Ca 2 and calmodulin in a atoxin production. MATERIALS AND METHODS Organism and growth conditions Toxigenic strain of Aspergillus parasiticus (NRRL-2999) was obtained from Department of Agriculture, Northern Regional Research Centre, Peoria, Illinois, USA and maintained on Potato Dextrose Agar (PDA; 2% dextrose and 1% agar) slants. Spores ( ml 1 ) were cultured in 100-ml conical asks containing 20 ml basal medium = 2000 The Society for Applied Microbiology

2 278 J. PRAVEEN RAO AND C. SUBRAMANYAM (ph 60), supplemented with 5% glucose and trace metal stock (Adye and Mateles 1964). Varying amounts (0± 3 mmol l 1 ) of tri uoperazine (obtained from Sigma, St Louis, MO, USA) were included in the medium aseptically. The organism was grown at 28 C under stationary conditions for a period of 2 or 3 d corresponding to the trophophase and idiophase. At the end of the growth period mycelia were harvested by ltration and subjected to further analysis. [ 14 C]-acetate incorporation into aflatoxins The in uence of tri uoperazine on the incorporation of [ 14 C]-acetate was studied on 3-d-old preformed cultures coinciding with the stage of maximum a atoxin production, by modi cation of the method of Yao and Hsieh (1974) as described earlier (Praveen Rao and Subramanyam 1999). The mycelial pads (500 mg) were transferred to 50-ml conical asks containing 2 mci [ 14 C]-acetate (speci c activity 60 mci mmol 1 ; obtained from BRIT, Mumbai, India) in 10 ml basal medium and incubated at 28 C for a further period of 24 h in presence or in absence of varying concentrations of tri uoperazine. A atoxins extracted by adding equal volumes of chloroform to the culture medium were dried by rotary evaporation, dissolved in a benzene-acetonitrile mixture (98 : 2) and subjected to thin layer chromatography (TLC) on 075 mm thick silica gel G coated plates (particle size 75 mm). Fluorescent spots corresponding to appropriate reference standards of a atoxins, i.e. AFB 1, AFB 2, AFG 1 and AFG 2 (a gift from Dr B. Sashidhar Rao, Department of Biochemistry, Osmania University) on the TLC plate were scraped, dissolved in 3 ml of toluene cocktail (made of 03% 2,5-diphenyloxazole and 001% 1,4-bis [4-methyl-5 phenyl]-2-oxazolyl benzene in toluene) and counted in Beckman 1801 liquid scintillation counter (Fullerton, CA, USA). Preparation of cytoplasmic extracts Mycelia (2 and 3 days old) grown either in presence or in absence of tri uoperazine (014 mmol l 1 ) were homogenized at 4 C using acid-washed sand in buffer A (25 mmol l 1 Tris, ph 74; 1 mmol l 1 EDTA; 1 mmol l 1 NaF; and 1 mmol l 1 b-mercaptoethanol) for assay of pyruvate kinase, buffer B (02 mol l 1 phosphate buffer, ph 72; 1 mmol l 1 EDTA; 1 mmol l 1 NaF; and 1 mmol l 1 b- mercaptoethanol) for assay of glucose-6-phosphate dehydrogenase and phosphofructokinase and buffer C (01 mol l 1 phosphate buffer, ph 65; 1 mmol l 1 EDTA; 1 mmol l 1 NaF; and 1 mmol l 1 b-mercaptoethanol) for assay of acetyl-coa carboxylase. All the homogenates were clari ed by centrifugation at g for 30 min at 4 C. After determining the protein contents according to Bradford (1976), the supernatants were used for enzyme assays. Enzyme assays The activity of acetyl-coa carboxylase (EC ) was assayed essentially as described by Sumper (1981) employing the 14 CO 2 xation method by monitoring the formation of malonyl-coa. The in vitro in uence of tri uoperazine or calmodulin on the activity of the enzyme was determined by including these compounds at varied concentrations in the reaction mixtures of cytoplasmic extracts obtained from cultures grown in absence of tri uoperazine. Glucose-6- phosphate dehydrogenase (EC ) was assayed as described by De Moss (1955) by monitoring the increase in absorbance at 340 nm resulting from the reduction of NADP. Pyruvate kinase (EC ) was assayed as described by Bucher and P eiderer (1955) in a lactate dehydrogenase-coupled assay system by measuring the decrease in absorbance at 340 nm resulting from the oxidation of NADH. Phosphofructokinase (EC ) was assayed according to Beutler (1975) by monitoring the oxidation of NADH at 340 nm. RESULTS AND DISCUSSION Trifluoperazine inhibited [ 14 C]-acetate incorporation into aflatoxins and activity of acetyl-coa carboxylase during aflatoxin production Since acetate is the primary precursor for the formation of various a atoxins, studies were conducted to ascertain whether tri uoperazine had any in uence on [ 14 C]-acetate incorporation into different a atoxins. The incorporation into a atoxins was decreased in a dose-dependent manner when the organism was cultured in presence of varying concentrations of tri uoperazine (Table 1). There was a 55% decrease in acetate incorporation into the rst formed a atoxin, AFB 1, a potent hepatocarcinogen, in presence of 014 mmol l 1 tri uoperazine (50% growth inhibitory concentration). At a concentration of 05 mmol l 1 and above, tri uoperazine completely inhibited acetate incorporation into AFB 2, AFG 1 and AFG 2. When included at 3 mmol l 1 concentration, tri uoperazine abolished the incorporation into AFB 1. The in vivo activity of acetyl-coa carboxylase was assayed in the organism cultured in presence or in absence of 014 mmol l 1 tri uoperazine both at trophophase and idiophase (Table 2). In cultures grown in absence of tri uoperazine the activity of acetyl-coa carboxylase increased (38-fold) at idiophase corresponding to maximal a atoxin production, indicating the requirement of the enzyme for a atoxin biosynthesis. Cultures grown in presence of

3 CALMODULIN ACTIVATES ACETYL-COA CARBOXYLASE IN AFLATOXIN PRODUCTION 279 Table 1 Incorporation of [ 14 C]-acetate into a atoxins during growth of Aspergillus parasiticus [ 14 C]-acetate incorporated 100 mg 1 dry weight of mycelia dpm 100 mg 1 dry weight mycelia Growth conditions AFB 1 AFB 2 AFG 1 AFG 2 Control TFP (mmol l 1 ) ND ND ND ND ND ND ND ND ND 300 ND ND ND ND Three day-old cultures (500 mg) were transferred to fresh basal medium (10 ml) containing 2 mci [ 14 C]-acetate and further grown for a period of 24 h in presence or in absence of varying concentrations of tri uoperazine (TFP). A atoxins resolved on TLC were scrapped and counted for incorporation. Values depicted are S.D. of three separate experiments. ND ± not detected. 014 mmol l 1 tri uoperazine had a decreased activity (30%) of acetyl-coa carboxylase at trophophase, which further decreased to an extent of 80% at idiophase. However, inclusion of tri uoperazine in the growth medium did not signi cantly alter acetyl-coa carboxylase activity during the transition from trophophase to idiophase. In order to further con rm this observed in vivo inhibition of acetyl-coa carboxylase by tri uoperazine, the enzyme was assayed in the presence of varying concentrations of tri uoperazine and calmodulin included in the reaction mixtures in vitro (Fig. 1). Tri uoperazine caused a dose-dependent inhibition of the activity of acetyl-coa carboxylase; activity of the enzyme was decreased twofold (55%) in presence of 01 mmol l 1 tri uoperazine. In contrast, inclusion of calmodulin in the reaction mixture enhanced its activity in a dose-dependent manner. Even at very low concentrations (03nmoll 1 ) calmodulin could enhance the enzyme activity by threefold. Table 2 In uence of tri uoperazine on the activity of regulatory enzymes Enzyme activity (Units) Trophophase Idiophase Enzyme Control TFP treated Control TFP treated Acetyl-CoA carboxylase (dpm mg 1 protein min 1 ) Glucose-6-phosphate dehydrogenase (mmol NADP reduced mg 1 protein min 1 ) Phosphofructokinase (mmol NADH oxidized mg 1 protein min 1 ) Pyruvate kinase (mmol NADH oxidized mg 1 protein min 1 ) The organism was grown either in presence or in absence of 014 mmol l 1 tri uoperazine (TFP) for a period of 2 or 3 days, corresponding to trophophase and idiophase, respectively, and the activity was assayed as described in Materials and methods. Values depicted are mean S.D. of three separate experiments.

4 280 J. PRAVEEN RAO AND C. SUBRAMANYAM Fig. 1 The activity of acetyl-coa carboxylase was assayed in vitro in absence (1) or presence of tri uoperazine (2a ± 1 mmol l 1,2b± 10 mmol l 1, 2c ± 100 mmol l 1 ) or calmodulin (3a ± 006 nmol l 1, 3b ± 012 nmol l 1,3c±03 nmol l 1 ) as described in Materials and methods. Values depicted are mean S.D. of three separate experiments Influence of trifluoperazine on the activity of enzymes of primary metabolism involved in aflatoxin production Studies were also conducted to ascertain whether the antia atoxigenic property of tri uoperazine was accompanied with alterations in the activities of enzymes involved in primary metabolism. In absence of tri uoperazine, it was noted that the activity of pyruvate kinase was increased twofold at idiophase corresponding to maximal a atoxin production (3 d) when compared to its activity at trophophase (2 d). In addition, glucose-6-phosphate dehydrogenase activity was enhanced (14-fold) during the idiophase of the organism (Table 2). In comparison with the above results, culturing the organism in presence of tri uoperazine (014 mmol l 1 ) resulted in decreased activity of glucose-6-phosphate dehydrogenase to an extent of 30 and 35% at trophophase and idiophase, respectively. In such conditions there was also increase in the activities of phosphofructokinase (18% at trophophase; 30% at idiophase) and pyruvate kinase (70% at trophophase; 40% at idiophase) at both phases of growth. The channelling of acetate into various metabolic pathways, including the primary energy-yielding pathways as well as towards formation of secondary metabolites, generally depends upon the genetic organization and the metabolic status of an organism. Primary events of metabolism are also likely to determine the onset of secondary metabolic pathways by providing necessary energy as well as essential precursors for the synthesis of secondary metabolites. In this regard, the importance of glycolysis for a atoxin biosynthesis has been suggested (Tyagi and Venkitasubramanian 1981). A atoxins are known to be synthesized after active growth has ceased, using acetyl- CoA derived from extramitochondrial pool (Berg 1956). In view of the obtained results it was thus hypothesized that the activity of acetyl-coa carboxylase, the enzyme involved in acetate channelling into various pathways including the formation of polyketides leading to a atoxins, would be regulated by intracellular signalling molecules such as calcium and calmodulin. Adopting such an approach yielded valuable information not only on the metabolic status of the organism at times corresponding to trophophase and idiophase but also on the probable regulatory roles of Ca 2 and calmodulin on a atoxin production. In addition, the decreased activity of glucose-6-phosphate dehydrogenase noted in cultures grown in presence of tri uoperazine signi ed that under such conditions there may be a probable limitation of reduction potential (reduced NADP) which is essentially required for the biosynthesis of a atoxins (Shih and Marth 1974). However, the enhanced activity of phosphofructokinase (30%) and pyruvate kinase (40%) in cultures grown in presence of tri uoperazine also suggested higher glycolytic activity both during trophophase and idiophase. Interestingly, while enhanced activity of acetyl-coa carboxylase was observed at idiophase under control conditions, no signi cant variations were noted at trophophase and idiophase in cultures grown in presence of tri uoperazine. On the contrary, in comparison with control cultures the activity was reduced to an extent of 80% at idiophase, in cultures grown in presence of tri uoperazine. This result suggested a decreased formation of malonyl-coa during tri uoperazine-mediated inhibition of a atoxin formation. The present studies also yielded valuable information on the regulation of acetyl-coa carboxylase by calmodulin in vitro. It was noted that the addition of tri uoperazine (01 mmol l 1 ) in the reaction mixture inhibited the activity of acetyl-coa carboxylase twofold and calmodulin (03 nmol l 1 ) -stimulated the activity threefold. The observed requirement of calmodulin for the activity of acetyl-coa carboxylase is in agreement with earlier studies wherein Ca 2 -calmodulin-dependent phosphorylation of the enzyme was implicated in the regulation of the enzyme (Hardie et al. 1986). In view of our earlier studies implicating the importance of calmodulin for a atoxin production, a putative role for Ca 2 -calmodulin in phosphorylation/

5 CALMODULIN ACTIVATES ACETYL-COA CARBOXYLASE IN AFLATOXIN PRODUCTION 281 dephosphorylation of acetyl-coa carboxylase is being proposed during a atoxin production. Designing suitable antagonists for Ca 2 -calmodulin-mediated processes involved in regulating the activity of acetyl-coa carboxylase may thus be considered as a favourable strategy in combating a atoxin production. ACKNOWLEDGEMENTS The authors thank Ms T. Jayashree for technical help and valuable suggestions during the preparation of the manuscript. JPR thanks the University Grants Commission for the award of a senior research fellowship. REFERENCES Adye, J. and Mateles, R.I. (1964) Incorporation of labelled compounds into a atoxins. Biochemica Biophysica Acta 86, 418±420. Berg, P. (1956) Acyl adenylates: an enzymatic mechanism of acetate activation. Journal of Biological Chemistry 222, 991±1013. Bucher, T. and P eiderer, G. (1955) Pyruvate kinase from muscle. Methods in Enzymology 1, 435±440. Beutler, E. (1975) Phosphofructokinase. In Red Cell Metabolism: a Manual of Biochemical Methods ed. Beutler, E. pp. 42±44. New York, NY: Grune and Stratton. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248±254. De Moss, R.D. (1955) Glucose-6-phosphate and 6-phosphogluconic dehydrogenase from Leuconostoc mesenteroides. Methods in Enzymology 1, 435±440. Hardie, D.G., Carling, D., Ferrari, S., Guy, P.S. and Aitken, A. (1986) Characterization of the phosphorylation of rat mammary ATP-citrate lyase and acetyl-coa carboxylase by Ca 2 and calmodulin-dependent multiprotein kinase and Ca 2 and phospholipid-dependent protein kinase. European Journal of Biochemistry 157, 553±561. Jayashree, T. and Subramanyam, C. (1999) Antia atoxigenic activity of eugenol is due to inhibition of lipid peroxidation. Letters in Applied Microbiology 28, 179±183. Maggon, K.K., Gupta, S.K. and Venkitasubramanian, T.A. (1977) Biosynthesis of a atoxins. Bacteriological Reviews 41, 822±855. Praveen Rao, J. and Subramanyam, C. (1999) Requirement of Ca 2 for a atoxin production: inhibitory effect of Ca 2 channel blockers on a atoxin production by Aspergillus parasiticus NRRL Letters in Applied Microbiology 28, 55±58. Praveen Rao, J., Sashidhar, R.B. and Subramanyam, C. (1998) Inhibition of a atoxin production by tri uoperazine in Aspergillus parasiticus NRRL World Journal of Microbiology and Biotechnology 14, 71±75. Shih, C.-N. and Marth, E.H. (1974) A atoxin formation, lipid synthesis and glucose metabolism by Aspergillus parasiticus during incubation with and without agitation. Biochemica Biophysica Acta 338, 286±296. Smith, J.E., Solomons, G., Lewis, C. and Anderson, J.G. (1995) Role of mycotoxins in human and animal nutrition and health. Natural Toxins 3, 187±192. Steyn, P.S. (1995) Mycotoxins, general view, chemistry and structure. Toxicology Letters 82±83, 843±851. Sumper, M. (1981) Acetyl CoA carboxylase from yeast. Methods in Enzymology 71, 34±37. Tyagi, J.S. and Venkitasubramanian, T.A. (1981) The role of glycolysis in a atoxin biosynthesis. Canadian Journal of Microbiology 27, 1276±1282. Yao, R.C. and Hsieh, D.P.H. (1974) Step of dichlorvos inhibition in the pathway of a atoxin biosynthesis. Applied Microbiology 28, 52±57. Zaika, L.L. and Buchanan, R.L. (1987) Review of compounds affecting the biosynthesis or bioregulation of a atoxins. Journal of Food Protection 50, 691±708.