Parasitol Res (1995) 81:320-323

9 Springer-Verlag 1995

J. Vargas-Villarreal - H. M a r t f n e z - R o d r f g u e z J. Castro-Garza - B . D . Mata-Cfirdenas M.T. G o n z f i l e z - G a r z a 9 S. Said-Ferngmdez

identification of Entamoeba histolytica intracellular phospholipase A and lysophospholipase L 1 activities

Received: 22 July 1994 / Accepted: 2 November 1994

Abstract Entamoeba histolytica phospholipase A and lysophospholipase activities from a vesicular subcellular fraction (P30) were analyzed. The products, obtained using specific substrates labeled with 14C or 3H, indicated the presence of phospholipase A 1 and A 2 as well as lysophospholipase L 1 activities. The enzymes detected could participate in phospholipid metabolism and the alkaline phospholipase A 2 may contribute to E. histolytica cytopathogenicity.

spectively. The alkaline one depends on Ca 2+ but the acidic one does not (Long-Krug et al. 1985). In previous studies we found that the major E. histolytica hemolytic activity, which is maximal at pH 8.0 with 1 mM Ca 2+, is located in a vesicular subcellular fraction called P30, and we showed that this is due to a phospholipase A activity (Said Fernandez and L6pez-Revilla 1982, 1983, 1988). In the present study we identified phospholipase A 2, (PLA A2), phospholipase A 1 (PLA A1) , and lysophospholipase L 1 (LPLA La) activities in P30.

Introduction Phospholipases A are responsible for phospholipid dig e s t i o n and membrane phospholipid turnover (for a review, see Van den Bosch 1980). In addition, these enzymes have been identified as part of the cytolytic mechanism of snake and insect venoms and of several parasitic protozoa (see Dennis 1983; Long-Krug and Ravdin 1988; Saffer et al. 1989). Lysophospholipases protect cells from accumulation of lytic lysophospholipids (cf. Van den Bosch 1980). Two phospholipases A 2 found on the surface of Entamoeba histolytica trophozoites have been implicated in the contact-mediated cytolytic mechanism of this parasite (Ravdin 1988); one is acidic and the other, alkaline, with maximal activity occurring at pH 4.5 and 7.5, re-

J. Vargas-Villarreal 9 J. Castro-Garza 9 B.D. Mata-Cfirdenas M.T. Gonzalez-Garza 9 S. Said-Fern~indez ( ~ ) Divisi6n de Biotogia Celular y Molecular, Centro de Investigacidn Biom6dica del Noreste, Instituto Mexicano del Seguro Social, Administraci6n de Correos No. 4 Apartado Postal 20, Colonia Independencia, Monterrey, C.R 64720, N.L. M6xico, Fax (52-8) 3-44-41-16 H. Mart/nez-Rodriguez Unidad de Laboratorios de Ingenier/a y Expresi6n Gen6ticas, Departamento de Bioqu/mica, Facultad de Medicina, Universidad Autdnoma de Nuevo Ledn, Calzada Madero y Dr. A. Aguirre Pequefio, Colonia Mitras Centro, Monterrey, C.E 64000, N.L. Mexico

Materials and methods Amebae HK9 strain Entamoeba histolytica trophozoites axenically cultured in PEHPS medium (Said-FernSndez et al. 1988) were used for all experiments. Obtaining P30 This subcellular fraction was prepared from freshly obtained trophozoite pellets as described elsewhere (Said-Fernandez and Ldpez-Revilla 1982). Briefly, after 3 days of incubation at 37~ amebae growing in the logarithmic phase were harvested by chilling of the cultures in ice water for 10 min, centrifuged for 15 min at 600 g and 4~ and washed two times with ice-cold phosphatebuffered saline (PBS). The pellet was resuspended in 2 vols. of cold balanced salt solution (BSS: 120 mM NaC1, 5 mM KC1, 1 mM MgSO4, 0.7 mM CaC12, 1 mM Trizma base, 5.5 mM glucose (pH 7.4) adjusted to 300 mosmol/kg with NaC1) and disrupted with an electrical motor-driven Elvehjem-Potter homogenizer at 1,000 rpm. The resultant homogenate was centrifuged at 135 g at 4~ for 15 min. The supernatant was saved and centrifuged at 30,000 g at 4~ for 15 min. The pellet (P30) was separated from its respective supernatant ($30), resuspended in 2 vols. of BSS, distributed in 0.5 ml, and stored in liquid nitrogen until use. Phospholipase A- and lysophospholipase-activity assays Amebic phospholipase activities were assayed by the method of Opperdoes and Van Roy (1982), with minor modifications: in 1.5ml borosilicate cone-bottom vials (Bellco Glass, Inc., Vineland,

321 N.J.) were mixed 1.0 ml of 100 mM TRIS-HC1 (pH 8.0), 2 mM CaC12, 0.2% Triton X-100, 0.27 mM phosphatidylcholine, and 4 gCi of 1,2 dipalmitoyl-[2- palmitoyl-l-14C]-phosphatidylcholine/ml (112 mCi/mmol; [2-14C- PA]-PC), of 1,2-dipalmitoylsn-glycero-3-pbosphoryl-[choline-methyl-3H]-choline/ml (60 Ci/ mmol; [3H-choline]-PC), or of 1-palmitoyl[1-~4C]-sn-glycero-3phosphatidylcholine/ml ([1- 14C]-LPC; 52 mCi/mmol). All three radioactive substrates were purchased from New England Nuclear (Boston, Mass.). The mixtures were sonicated with an Ultratip Labsonic System (Lab-Line Instrument Inc., Melrose Park, Ill.), which was operated at 40 W for 60 s. This emulsion was divided in 0.5-ml aliquots in vials and stored at -70~ until use. The assays were performed in 15- x 15-ram borosilicate test tubes in which 10 gl of one of the above-described substrate mixtures was poured, added and mixed with a vortex with 10 gl of a P30 suspension or bovine serum albumin (fraction V) containing 0-368 gg of total proteins. After 45 min of incubation at 37~ in a water bath, the phospholipid hydrolysis reaction was stopped by the addition of 25 gl of rat-liver total free fatty acids (FFA; final concentration, 1 mg/ml), 1.0 mg egg-yolk lysophosphatidylcholine/ml (LPC), and 0.75 mg egg-yolk phosphatidylcholine/ml (PC) in 5% trichloroacetic acid in n-butanol to bring the mixture to a final volume of 45 ~tl. The radioactivity in PC, LPC, and FFC from the assay mixtures was determined and separated by thin-layer chromatography (TLC) as follows: 25 gl of the above-described mixtures were put drop by drop in the origin of 20- x 20-cm silica-gel plates (0.25mm thickness, 60-mesh; Merck, Germany) and placed into a TLC developing tank with a solvent system of chloroform:methanol:acetic acid:water (140:40:16:8, by vol.). Lipid spots were developed by exposing the TLC plates to iodine vapor (Skipsky and Barclay 1969).

Calculations Disintegrations per minute (dpm) were calculated from the counts per minute (cpm) obtained in each vial by the single-label external-standard-ratio method recommended by the scintillation spectrometer manufacturer (Tri-Carb Liquid Scintillation Spectrometer, model 3255; Packard Instrument Company, Inc., Downers Grove, Ill.). Lipid mass, expressed in picomols of [3H]-LPC, [3H]-GPC, [214C-palmitic]-LPC, and [14C]-palmitic acid, was estimated according to the dpm values. All determinations were performed three times each in triplicate. The results are presented as mean average values+SE. Standard errors were calculated using the three mean values from each experiment.

Results

The hydrolysis products of [3H-choline]-PC in the presence of P30 were [3H]-LPC and [3H]-GPC. Levels of both increased linearly with the incubation period, during the first 15 min of incubation. Afterwards, until 30 min of incubation, levels of [3H]-LPC continued increasing linearly, but the accumulation of [3H]-GPC was at a plateau. No hydrolysis product was detected in assays containing bovine serum albumin instead of P30, nor was radioactive material recovered from the silica scraped from the interlipidic spot spaces of each lane (Fig. 1). After 45 min 0.013 pmol of [3H]-LPC and 0.004 pmol of [3H]-GPC were recovered from chromatography plates. When [1-palmitoyl-14C]-LPC served as Lipid identification the substrate, the [14C]-palmitic concentration raised PC, LPC, and L-o~-glycerylphosphorylcholine (GPC) spots from with P30 doses. The dose-response curve was an asympthe phospholipase- and lysophospholipase-activity assays were tote. No hydrolytic activity was observed in mixtures stained on the TLC plates with iodine vapor (for radioactivity determination) or with Dragendorf's reagent, which is specific for containing bovine serum albumin instead of P30. After choline-containing lipids (Skipsky and Barclay 1969). Their ap- 45 rain of incubation, 16 pmol of [t4C]-palmitic acid had pearance and relative migration coefficients (R/ PC, 0.43; LPC, accumulated in the assay mixtures (Fig. 2). 0.185; AGL, 0.908; and GPC, at the origin) were compared with When [2-14C-palmitic]-PC was used as the substrate, those of the corresponding standards (Sigma Chemical Co., St. [2-14C - palmitic]-LPC and [14C]-palmitic acid accumuLouis, Mo.) on the same plates. The lipid spots in each lane (developed with iodine vapor) were scraped and the material obtained lated as a function of P30 dose (Fig. 3). In assay mixfrom each spot was put into vials containing 5 ml of scintillation tures containing more than 100 gg total protein, the liquid (0.6% 2-5 diphenyloxazole in toluene), and its radioactivity was determined under conditions used for relatively unquenched samples with a model 3255 Tri-Carb liquid scintillation spectrometer equipped with an external standard source ([226Ra]) assembly (Packard Instrument Company, Inc., Downers Grove, Ill.).

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Effect of Ca 2+ on phospholipase A activity Experiments were performed as done to determine the time course of [3HI-PC hydrolysis except that reaction mixtures were supplemented with 1 mM ethylenediaminetetraacetic acid (EDTA) or with 0, 1, or 2 mM CaC12 and then incubated for 45 rain instead of using a constant CaC12 concentration and variable incubation periods.

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Time course of [3H]-PC hydrolysis Assay mixtures containing 368 gg of P30 total protein or bovine serum albumin and 4 gCi [3H-choline]-PC/ml dissolved in 0.020 ml of 50 mM TRIS-HC1 buffer (pH 8.0), 1 mM CaC12 and 0.1% Triton X-100 were incubated for variable periods ( 0 4 5 rnin) and the hydrolysis products ([3H]-LFC and [3H]-GFC) were separated and quantified as described above.

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Fig. 1 Time course of [3H]-LPC and [3H]-GPC accumulation by [3H-choline]-PC hydrolysis. Symbols correspond to average dpm values_+SE obtained for [3H]-LPC (O) or [3H]-GPC ([]) produced in the presence of variable P30 doses or for [3H]-LPC (0) or [3H]GPC (11) from bovine serum albumin assays

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Fig. 2 P30-dependent [l-]4C-palmitic]-LPC hydrolysis. Symbols correspond to dpm values___SE obtained for [14C]-palmitic acid produced in the presence of variable P30 (O) or bovine serum albumin (11) doses

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Fig. 3 P30 dose-dependent [2-14C-palmitic]-PC hydrolysis-product accumulation. Symbols correspond to dpm values_+SE obtained for [2-14C-palmitic]-LPC ((3) or []4C]-palmitic acid (0) produced in the presence of variable P30 doses or for [ 2 - 1 4 C palmitic]-LPC ([1) or []4C]-palmitic acid (11) detected after the reaction mixtures had been incubated in the presence of variable doses of bovine serum albumin

phospholipid hydrolysis appeared to be slightly more efficient than it was in those containing lower doses of the amebal fraction. After 45 min of incubation, 15 pmol of [2-14C-palmitic]-LPC and 7.6 pmol of [14C]-palmitic acid had accumulated. No hydrolysis product was detected in the presence of bovine serum albumin used instead of P30. The level of [3H]-LPC produced in P30 phospholipase A assays supplemented with no Ca 2+ (0.22+0.05 pmol) was, respectively, 31% (P<0.01) and 51% (P<0.001) lower than that produced in assays supplemented with t (0.32+0.03 pmol) or 2 mM Ca 2+ (0.45_+0.07 pmol), whereas no significant difference was found between data obtained in assays supplemented with no Ca 2+ versus those using 1 mM EDTA (0.26+0.05 pmol).

The time-dependent hydrolysis of pH-choline]-PC occurring in the presence of P30 with [3H]-LPC as the product confirmed our early observation that P30 has a phospholipase A (PLA) activity (Said-Fernfindez and Ldpez-Revilla 1988). In the same experiment, we detected [3H]-GPC as another hydrolysis product of the original [3H-choline]-PC substrate. According to our calculations, 0.016 pmol of [3H-choline]-PC was hydrolyzed, producing 0.013 pmol of [1-palmitic-3H-choline]-LPC and/or [2-palmitic-3H-choline]-LPC as the result of PLA A 1 and/or A 2 activity. Then, 28% of this lysophospholipid (LPL) was hydrolyzed, producing 0.004 pmol of [3Hcholine]-GFC. These results must be due to LPLA L 1 and/or LPLA L a (Dennis 1983). The presence in P30 of LPLA L 1 activity was confirmed in the analysis shown in Fig. 2, where [1-14C palmitic]-LPC was used as the substrate. In this experiment, 16 pmol of [14C]-palmitic acid was produced. LPLAs L 1 hydrolyze 1-acyl-lysophophatidyl derivates that are products of PLAs A 2. In another series of experiments we found that 23 pmol of [2-a4C-palmitic]-PC was hydrolyzed, of which 67% was detected as [2-14C palmitic]-LPC and 33%, as [a4C]-palmitic acid. Accordingly, at least 67% of the [2-14C-palmitic]-PC was hydrolyzed by a PLA A 1. The [14C]-palmitic acid could have been produced by an LPLA L: as a consequence of further hydrolysis of [2N4C-palmitic]-LPC, which was previously produced by the PLA A1; from the original [214C-palmitic]-PC substrate; or by the hydrolysis of [214C-palmitic]-PC by means of a PLA A e. At present, we cannot determine if an LPLA L e exists in P30, but ameba must have this activity, or a very efficient mechanism of phosphatidylcholine resynthesis, to avoid their autolysis via the PLA A 1 ([2J4C-palmitic]-LPC) product (see Shier 1979). In this study we found that 53% of the [3H]-LPC was produced by an amebic Cae+-independent PLA and that the remaining 47% of this product was due to a CaZ+-de pendent PLA. PLAs A e depend on Ca 2§ but, in general, PLAs A 1 and LPLAs do not (see Van den Bosch 1974; Avigad 1976; Dennis 1983). Thus P30 contains both a Ca2+-independent PLA A 1 and a Cae+-dependent PLA A: activity. The Entamoeba histolytica hemolytic activity (SaidFernfindez and L6pez-Revilla 1982) and the PLA A 2 detected in the present study have similar requirements for Ca 2+ and pH and are located in the same subcellular fraction (P30). The above-mentioned finding agrees with the previous suggestion (Said-Fern~mdez and L6pez-Revilla 1988; Long-Krug and Ravdin 1988) that an alkaline PLA A 2 is responsible, at least partially, for E. histolytica hemolytic and cytolytic activity. The alkaline PLA A 2 described by Long-Krug et al. (1985) could be the same as that currently detected. Nevertheless, at present we do not have enough information to determine if amebae have one or more alkaline, Ca2+-dependent PLAs A a, considering that several PLA A 2 forms with similar pH

323 and Ca 2+ requirements have been purified from the same source (see Dennis 1983). Thus far, two E. histolytica PLA A 2 activities have been described, one being alkaline and the other, acidic (Long-Krug et al. 1985), plus the PLA A 1 and L P L A L I activities detected in the present study. At least one alkaline PLA A 2 could be related to the E. histolytica cytopathogenic mechanism. The other three enzymatic activities could be related to this parasite's phospholipid metabolism, as has been described for most of the known acidic PLAs A 2, PLAs A1, and LPLAs (cf. Van den Bosch 1980). Phospholipid metabolism of E. histolytica is poorly understood. In the particular case of PLAs and LPLAs, it is not yet known (1) how many PLAs A t and A~ are produced by E. histoIytica trophozoites, (2) what the specific role of each of these might be, or (3) the nature of the relationship between amebic PLAs AI, PLAs A 2, and LPLAs in the lipid metabolism. In addition, the following issues need to be examined: which of the enzymes are virulence factors, what their particular mode of action might be, and which PLAs are produced by pathogenic strains and which others, by nonpathogenic strains. Possibly the first step that should be taken to investigate these questions should be to search for, to purify, and to characterize the PLAs and LPLAs from pathogenic and nonpathogenic E. histolytica isolates. With this idea, the purification of P30 PLAs A 2 is in progress. Acknowledgements We thank Leticia Navarro Marmolejo, Alejandro Olvera Rodrfguez, and Antonio Luna de la Rosa for their technical assistance and artwork, respectively. Javier Vargas Villarreal received a Master in Science Fellowship from the Instituto Mexicano del Seguro Social. This project was partially supported by CONACYT, M6xico, grant F328-M9212.

References Avigad G (1976) Microbial phospholipases. In: Bernheimer AW (ed) Mechanisms in bacterial toxinology, ch 5. John Wiley & Sons, New York, pp 100-167 Dennis EA (1983) Phospholipases. In: Boyer P (ed) The enzymes, vol XVI, ch 9. Academic Press, New York, pp 307-353 Long-Krug SA, Ravdin JI (1988) The role of amebic phospholipases in cytolysis of target cells by Entamoeba histolytica. In: Ravdin JI (ed) Amebiasis. Human infection by Entamoeba histolytica, ch 15. John Wiley & Sons, New York, pp 232-250 Long-Kurg SA, Fisher KJ, Hygmitb RM, Ravdin JI (1985) Phospholipase A enzymes of Entamoeba histoIytica: description and subcelullar localization. J Infect Dis 152:536-541 Opperdoes FR, Van Roy J (1982) The phospholipases of Trypanosoma brucei bloodstream forms and cultured procyclics. Mol Biochem Parasitol 5:309-319 Ravdin JI (1988) Pathogenesis of amebiasis: an overview. In: Raydin JI (ed) Amebiasis. Human infection by Entamoeba histolytica, ch 10. John Wiley & Sons, New York, pp 166-176 Said-Fernfindez S, L6pez-Revilla R (1982) Subcellular distribution and stability of the major hemolytic activity of Entamoeba histolytica trophozoites. Z Parasitenkd 67:249-254 Said-Fernfindez S, L6pez-Revilla R (1983) Latency and heterogeneity of Entamoeba histolytica hemolysins. Z Parasitenkd 69: 435-438 Said-Fern~indez S, Ldpez-Revilla R (1988) Free fatty acids released from phospholipids are the major heat-stable hemolytic factor of Entamoeba histolytica trophozoites. Infect Immun 56:874-879 Said-Fernfindez S, Vargas-Villarreal J, Castro-Garza J, Mata Cfirdenas BD, Navarro-Marmolejo L, Lozano-Garza G, Martfnez- Rodrfguez H (1988) PEHPS medium: an alternative for axenic cultivation of Entamoeba histolytica and E. invadens. Trans R Soc Trop Med Hyg 82:249-253 Saffer LD, Long-Krug SA, Schwartzman JD (1989) The role of phospholipase in host cell penetration by Toxoplasma gondii. Am J Trop Med Hyg 40:145-149 Shier WT (1979) Activation of high levels of endogenous phospholipase A 2 in cultured cells. Proc Natl Acad Sci USA 76: 195-199 Skipsky VR Barclay M (1969) Thin-layer chromatography of lipids. In: Lowenstein JM (ed) Methods in enzymology. Academic Press, New York, pp 530-599 Van den Bosch H (1974) Phosphoglycerides metabolism. Annu Rev Biochem 43:243-277 Van den Bosch H (1980) Intracellular phospholipases A. Biochim Biophys Acta 604:191-246