Investigating serum factors promoting erythrocytic growth of Plasmodium falciparum

Experimental Parasitology 109 (2005) 7–15 www.elsevier.com/locate/yexpr

Investigating serum factors promoting erythrocytic growth of Plasmodium falciparum Hiroko Asahia,¤, Tamotsu Kanazawab, Nakami Hirayamac, Yousei Kajiharad a

b

Department of Parasitology, National Institute of Infectious Diseases, 23-1 Toyama 1-chome, Shinjuku-ku, Tokyo 162-8640, Japan Department of Parasitology and Tropical Medicine, University of Occupational and Environmental Health, Iseigaoka 1-1, Yahata-nishiku, Kitakyusyu 807-8555, Japan c Department of Immunology, National Institute of Infectious Diseases, 23-1 Toyama 1-chome, Shinjuku-ku, Tokyo 162-8640, Japan d Life Tech Division, Nihon Pharmaceutical Co. Ltd., Sumiyoshi-cho 26, Izumisano, Osaka 598-0061, Japan Received 11 May 2004; received in revised form 16 October 2004; accepted 19 October 2004 Available online 10 December 2004

Abstract The elucidation of factors inducing the growth of Plasmodium falciparum can provide critical information about the developmental mechanisms of this parasite and open the way to search for novel targets for malaria chemotherapy. The ability of components of a growth-promoting factor derived from bovine serum and various related substances to sustain growth of P. falciparum was characterized. A simple total lipid fraction (GFS-C) containing non-esteriWed fatty acids (NEFAs) as essential factors was noted to promote the parasite’s growth. Various proteins from a variety of animals were tested, indicating the importance not only of GFS-C, but also of speciWc proteins, such as bovine and human albumin, in the parasite growth. Several combinations of the NEFAs tested sustained low parasite growth. Among various phospholipids and lysophospholipids tested, lysophosphatidylcholine containing C-18 unsaturated fatty acids was found to sustain the complete development of the parasite in the presence of bovine albumin. Several other lysophospholipids can partially support growth of P. falciparum.  2004 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: ALB, albumin; ANOVA, multifactorial analysis of variance; BSAF, NEFA-free ALB from bovine; C16:0, hexadecanoic acid; C16:1, cis-9-hexadecenoic acid; C18:0, octadecanoic acid; C18:1, cis-9-octadecenoic acid; C18:2, cis,cis-9,12-octadecadienoic acid; C18:3, cis,cis,cis-6,9,12-octadecatrienoic acid; C20:4, cis-5,8,11,14-eicosatetraenoic acid; C20:5, cis-5,8,11,14,17-eicosapentaenoic acid; C22:6, cis4,7,10,13,16,19-docosahexaenoic acid; CDKs, cyclin-dependent protein kinases; CDPKs, calcium-dependent protein kinases; CHOL, cholesterol; CRPMI, basal medium; DGs, diglycerides; FBS, fetal bovine serum; GFS, a growth-promoting fraction derived from adult bovine serum; GFS-C, a total simple lipid fraction obtained from GFS; GFSRPMI, CRPMI containing 10% GFS; GFS-WM, a total complex lipid fraction obtained from GFS; Hepes, 4-(2-hydroxyethyl)-piperazine ethanesulfonic acid; HS, human serum; HSRPMI, CRPMI containing 10% HS; LPA, lysophosphatidic acid; 18:1 LPA, cis-9-oleoyl LPA; LPC, lysophosphatidylcholine; 12:0 LPC, lauroyl LPC; 14:0 LPC, myristoyl LPC; 16:0 LPC, palmitoyl LPC; 18:0 LPC, stearoyl LPC; 18:1 LPC, cis-9-oleoyl LPC; 18:2 LPC, cis-9-linoleoyl LPC; 18:U LPC, LPC containing primarily C-18 unsaturated fatty acids; LPC-brain, LPC from bovine brain; LPCRPMI, CRPMI containing 3 mg/ml BSAF and 40
g/ml 18:U LPC; LPE, lysophosphatidyl ethanolamine; LPI, lysophosphatidyl inositol; LPS, lysophosphatidyl serine; Lyso PAF, -O-alkyl LPC; MAPK, mitogen-activated protein kinase; MEK, MAPK/ extracellular signal-regulated kinase kinase; MGs, monoglycerides; NEFA, non-esteriWed fatty acid; OD650, absorbance at 650 nm; 8:0 PA, dioctanoyl phosphatidic acid; 18:1 PA, cis-9-dioleoyl phosphatidic acid; PAF, -acetyl--O-alkyl PC; PC, phosphatidylcholine; PE, phosphatidyl ethanolamine; PI, phosphatidyl inositol; PI-bovine, PI from bovine liver; PI-soybean, PI from soybean; PKC, protein kinase C; PLs, phospholipids; pLDH, parasite lactate dehydrogenase; PS, phosphatidyl serine; PTK, protein tyrosine kinase; RBCs, red blood cells; SD, standard deviation; SM, sphingomyelin; S-1-P, sphingosine 1-phosphate; SPC, sphingosylphosphorylcholine; sphingosine, D- sphingosine Keywords: Protozoan Plasmodium falciparum; Growth-promoting factor; Simple total lipids; Lysophospholipids

*

Corresponding author. Fax: +81 3 5285 1150. E-mail address: [email protected] (H. Asahi).

0014-4894/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2004.10.002

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H. Asahi et al. / Experimental Parasitology 109 (2005) 7–15

1. Introduction Malaria remains a devastating disease, particularly in the Tropics. The estimated incidence of malaria in the world is of the order of 300–500 million clinical cases annually. The annual estimates of malaria mortality, particularly those caused by the protozoan Plasmodium falciparum, vary from 1.5 to 2.7 million worldwide (WHO, 1997). The eYcacy of conventional antimalarial drugs and insecticides in controlling malaria outbreaks is declining, with increasing resistance of parasites and their vectors. This highlights the need to develop new chemotherapeutic approaches based on a better understanding of parasite biology and its interaction with the host. The continuous in vitro cultivation of P. falciparum erythrocytic stages represented a signiWcant advance in malaria research (Trager and Jenson, 1978). Despite considerable advances in understanding the molecular and genetic characteristics of this protozoan (PlasmoDB, http://www.plasmodb.org), the mechanisms responsible for P. falciparum growth remain largely unknown. It has been suggested that P. falciparum requires some factors present in human serum (HS), although the role of HS in the growth of this parasite is still unknown. When only dialyzable factors of HS (low-molecular-mass compounds) are present in the commonly employed basic medium, RPMI1640, the parasite fails to develop from rings to schizonts, but good growth is observed by adding non-dialyzable HS factors (Jensen, 1979). We previously reported a growth-promoting fraction derived from adult bovine serum (GFS) that yields good intraerythrocytic P. falciparum growth (Asahi and Kanazawa, 1994). In addition, a low-molecular-weight factor that is of importance in serum-free media was clearly shown to be a purine precursor, such as hypoxanthine, adenine or adenosine (Asahi et al., 1996; Divo and Jensen, 1982). It is essential but not suYcient for the optimum parasite growth (Asahi et al., 1996). GFS is a 55– 70% ammonium sulfate fraction of adult bovine serum, and contains lipid-rich albumin (ALB) as a major component (Kudo et al., 1987). Similarly, Cranmer et al. (1997) reported that commercially available lipidenriched bovine ALB (Albumax II; Gibco-BRL Life Technology, USA) can replace HS for the continuous in vitro cultivation of P. falciparum. Consequently, data are still too crude to allow direct identiWcation of the functional factors required for the growth of P. falciparum. The replacement of HS in a culture medium with chemically or functionally deWned substances would not only be advantageous for the culture of the parasite, but it also provides critical clues to a thorough understanding of the parasite’s proliferation at the erythrocyte stage, and may lead to the identiWcation of novel targets for malaria chemotherapy. Therefore, this study was undertaken to characterize the ability of the components

of GFS and various related substances to sustain parasite growth.

2. Materials and methods 2.1. Parasite, culture, and synchronization Cultures of the FCR/FMG (ATCC Catalogue No. 30932, Gambia) strain of P. falciparum were used in the experiments. Parasites were routinely maintained by in vitro culture techniques using culture medium free of whole serum, consisting of basal medium (CRPMI) supplemented with 10% GFS (Wako Pure Chemical Industries, Japan, under the trade name Daigo’s GF21), as previously reported (Asahi et al., 1996). This complete medium is termed GFSRPMI. CRPMI consists of RPMI1640 containing 2 mM glutamine, 25 mM 4-(2hydroxyethyl)-piperazine ethanesulfonic acid (Hepes), and 24 mM NaHCO3 (Gibco-BRL Life Technology, USA), 25
g/ml gentamycin (Sigma Chemical, USA), and 150
M hypoxanthine (Sigma). BrieXy, red blood cells (RBCs), which were preserved in Alsever’s solution (Asahi et al., 1996) for 3–30 days, were washed, dispensed in a 24-well culture plate at a hematocrit of 2% (1 ml of suspension/well), and cultured under a humidiWed atmosphere of 5% CO2, 5% O2, and 90% N2 at 37 °C. For subculture, 4 days after inoculation, infected and uninfected RBCs were washed with CRPMI. Parasitemia was adjusted to 0.1% (for subcultures) or 0.4% (for growth tests) by adding uninfected RBCs, and hematocrit adjusted to 2% by adding the appropriate volume of culture medium, either GFSRPMI or the medium to be tested. Cultures were occasionally synchronized by two successive exposures at 34–38-h intervals to 5% (w/v) D-sorbitol (Asahi and Kanazawa, 1994). 2.2. Growth-promoting activity experiments The growth experiments were performed by replacing GFSRPMI with CRPMI supplemented with the substances to be tested. The following were tested for their growth-promoting activity: 10% (v/v) heat-inactivated HS (O-type) (HSRPMI); 2.5–10% heat-inactivated fetal bovine serum (FBS); 3 mg/ml non-esteriWed fatty acid (NEFA)-free ALB from bovine (BSAF), human, porcine, ovine, guinea pig or equine serum; and 3 mg/ml ovalbumin, lactoglobulin, lysozyme, and casein (Merck, Germany). CRPMI containing BSAF at a Wnal concentration of 3 mg/ml, except when otherwise stated, was further supplemented with graded concentrations of: hexadecanoic acid (palmitic acid, C16:0); cis-9-hexadecenoic acid (palmitoleic acid, C16:1); octadecanoic acid (stearic acid, C18:0); cis-9-octadecenoic acid (oleic acid, C18:1); cis,cis9,12-octadecadienoic acid (linoleic acid, C18:2); cis,cis,cis-

H. Asahi et al. / Experimental Parasitology 109 (2005) 7–15

6,9,12-octadecatrienoic acid (-linolenic acid, C18:3); cis5,8,11,14-eicosatetraenoic acid (arachidonic acid, C20:4); cis- 5,8,11,14,17-eicosapentaenoic acid (C20:5); cis4,7,10,13,16,19-docosahexaenoic acid (C22:6); palmitoyl lysophosphatidyl serine (LPS), sodium salt; soybean lysophosphatidyl inositol (LPI) containing primarily C18:0 and C16:0; egg yolk lysophosphatidyl ethanolamine (LPE) containing primarily C18:0 and C16:0; lysophosphatidylcholine (LPC), -O-alkyl, from bovine heart (LysoPAF); lysophosphatidic acid (LPA), cis-9-oleoyl (18:1 LPA), sodium salt (Biomol Research Laboratory, USA); soybean LPC containing primarily C-18 unsaturated fatty acids (18:U LPC); lauroyl LPC (12:0 LPC); myristoyl LPC (14:0 LPC); palmitoyl LPC (16:0 LPC); stearoyl LPC (18:0 LPC); cis-9-oleoyl LPC (18:1 LPC); cis-9-linoleoyl LPC (18:2 LPC, Doosan Serdary Research Lab, USA); LPC from bovine brain (LPC-brain); sphingosylphosphorylcholine (lysosphingomyelin) (SPC); phosphatidylcholine (PC) from egg yolk; phosphatidyl serine (PS) from bovine brain; phosphatidyl ethanolamine (PE) containing primarily C18:1 and C16:0; phosphatidyl inositol (PI) containing primarily C18:1 and C16:0, from soybean (PI-soybean); bovine liver PI (PI-bovine), ammonium salt; -acetyl--O-alkyl PC (PAF); sphingosine 1-phosphate (S-1-P); D-sphingosine (sphingosine) from bovine brain sphingomyelin (SM); egg yolk SM containing primarily C16:0; dioctanoyl phosphatidic acid (8:0 PA), sodium salt; or cis-9-dioleoyl phosphatidic acid (18:1 PA), sodium salt. CRPMI supplemented with BSAF (3 mg/ml) and 18:U LPC (40
g/ml) is termed LPCRPMI. Unless otherwise stated, all compounds were obtained from Sigma. Parasites were cultured for 4 days after inoculation (two cycles of complete growth), except when otherwise stated. For the reconstitution of lipids, dried lipid precipitates were prepared by evaporating the organic solvent from lipid stock solutions in acetone or a mixture of chloroform and methanol by centrifugation under vacuum. CRPMI containing proteins, except when otherwise stated, was added to the dried lipid precipitates, and the mixtures were sonicated for 5 min and sterilized using a 0.45-
m Wlter. 2.3. Assessment of parasite growth Samples were obtained at the times indicated. Smears were prepared and stained with Giemsa. More than 10,000 RBCs were examined to determine parasitemia. The growth rate was Wrst estimated by dividing the parasitemia of the test sample 4 days after inoculation by the initial parasitemia, except when otherwise stated. Further measurement of growth rates was performed using the parasite lactate dehydrogenase (pLDH) assay (Makler and Hinrichs, 1993), in cultures containing growth promoters such as HS, GFS, GFS-C, 18:U LPC, and 18:1 LPA. The Malstat reagent (Flow, USA) was

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used as an indicator of parasite viability, and the pLDH assay was performed according to the manufacturer’s instruction. BrieXy, at the incubation times indicated, the RBCs in cultures were hemolyzed by three freeze–thaw cycles, and 10
l of infected erythrocyte was transferred to each well of a 96-well microtiter plate. Then 100
l of the Malstat reagent, 10
l of 1 mg/ml nitroblue tetrazolium (Wako) and 10
l of 1 mg/ml diaphorase (Wako) were added to each well. The plate was allowed to stand for 40 min at 37 °C, and the reaction was stopped by the addition of 50
l of 5% (v/v) acetic acid. The absorbance at 650 nm (OD650) was read on a plate reader. The initial OD650 value measured at the addition of assay reagents was subtracted from the endpoint reading. For each experiment, parasitized RBCs were divided into identical aliquots, and diVerent treatments were performed simultaneously. To make the results comparable across experiments, external untreated control wells, namely cultures in GFSRPMI and HSRPMI, were set up each time. In all the experiments, culture wells were run in triplicate. 2.4. Separation and analysis of lipids Known amounts of GFS were extracted by the method of Bligh and Dyer (Asahi et al., 1986). The fractions obtained, namely, the chloroform (total simple lipid fraction, GFS-C) and water–methanol (total complex lipid fraction, GFS-WM) fractions, were further evaporated. They were suspended in the original volume of the culture medium for assay of growth-promoting activity on the parasite. GFS-C was further separated by thin-layer chromatography using petroleum ether–diethylether–acetic acid (80:30:1, by volume) as a developing solvent system, as described previously (Asahi et al., 1986). Gas–liquid chromatography of NEFAs was performed as described previously (Asahi et al., 1986). 2.5. Statistical analysis Statistical signiWcance for diVerences was evaluated using multifactorial analysis of variance (ANOVA). All calculations were performed using StatView 5.0J software (SAS Institute, USA). The P value for signiWcance was 0.05, and all pairwise comparisons were made post hoc with Bonferroni/Dunn’s test. For graphical representation of the data, y-axis error bars indicate the standard deviation (SD) of the data for each point on the Wgure.

3. Results 3.1. Essential GFS factors for P. falciparum growth After extraction of lipids from GFS, GFS-C, and GFS-WM and a mixture of the two fractions were tested

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for their ability to sustain parasite growth. GFS-C and the GFS-C + GFS-WM mixture exhibited growth-promoting activity only in the presence of BSAF (Fig. 1A). The parasites retained their normal morphology and gametocytes were rarely recognized. Neither BSAF nor GFS-C alone sustained parasite growth (Fig. 1A). GFS-C was further separated by thin-layer chromatography. The fractions including phospholipids (PLs), diglycerides (DGs), cholesterol (CHOL), monoglycerides (MGs), NEFAs, and CHOL-ester, and their mixtures were assayed for their growth-promoting ability in the presence of BSAF. The parasite growth was promoted only in the presence of NEFAs or mixtures containing NEFAs, although the extent of growth was much less than that for GFS-C + BSAF, GFS-C + GFS-WM + BSAF, GFSRPMI, and HSRPMI (Figs. 1A and B). These results imply that GFS-C containing NEFAs as essential factors was the functional factor inducing parasite growth. However, other factors contributing to the growth-promoting activity of GFS should not be ruled out. To determine whether proteins other than BSAF can sustain parasite growth, P. falciparum was cultured with GFS-C and various proteins. The parasite growth in the culture medium enriched with human ALB was similar to that of medium supplemented with BSAF. Guinea pig, ovine, equine, and porcine ALB also supported parasite growth, but to a lesser extent (Fig. 2A). The addi-

tion of ovalbumin, lactoglobulin, lysozyme, and casein to the culture medium enriched with GFS-C did not promote parasite growth. To determine the optimum protein concentration for growth of P. falciparum, BSAF or human ALB at diVerent concentrations was added to the culture medium enriched with GFS-C. Relatively high concentrations of protein (0.75–3 mg/ml BSAF and 3 mg/ml human ALB) were necessary for the optimum parasite growth (Fig. 2B). These results indicate the importance not only of lipids (GFS-C), but also of proteins in promoting and sustaining parasite growth. The components of the NEFA fraction of GFS-C were further analyzed by gas–liquid chromatography to identify the acids involved. The NEFA fraction was shown to contain mainly C18:1 (43%), C16:0 (21%), C18:0 (14%), C18:2, C16:1, C20:4, C20:5, and C22:6, which are commonly found in human and bovine sera (Manku et al., 1983). Each of the commercially available NEFAs enriched with BSAF was tested, either individually or in mixtures, for their ability to promote parasite growth. Individual NEFAs combined with BSAF did not promote parasite growth. In contrast, mixtures of NEFAs, such as C16:0 + C18:1, C16:0 + C18:1 + C22:6, and C16:0 + C18:0 + C18:1 + C18:2, promoted parasite growth, although the growth rate was much lower than that for GFSC + BSAF, GFSRPMI, and HSRPMI (Fig. 3).

Fig. 1. Ability of: (A) fractions derived from GFS and (B) simple-lipid fractions derived from GFS-C to sustain growth of P. falciparum. The culture media contained 3 mg/ml BSAF, except for GFS-C, GFS-C + GFS-WM, GFSRPMI, and HSRPMI, which are shown for comparison. Growth rate was determined as described in Materials and methods. *SigniWcant diVerence (P < 0.001) versus GFS-C + BSAF; versus GFS-C + GFSWM + BSAF; versus GFSRPMI; versus HSRPMI. #No signiWcant diVerence. These experiments were repeated twice, with similar results.

H. Asahi et al. / Experimental Parasitology 109 (2005) 7–15

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Fig. 2. (A) EVect of various proteins on the ability of GFS-C to sustain growth of P. falciparum. Each protein was added at 3 mg/ml in the presence (+) or absence (¡) of GFS-C. Also shown for comparison is the growth in 2.5–10% FBS, GFSRPMI, and HSRPMI. (B) Growth rate obtained in the GFS-C-containing medium supplemented with diVerent concentrations of BSAF and human ALB. *SigniWcant diVerence (P < 0.001) versus BSAF + GFS-C; versus human ALB + GFS-C; versus GFSRPMI; versus HSRPMI. #No signiWcant diVerence. Each protein in the presence of GFSC was compared, except for FBS, GFSRPMI, and HSRPMI. Growth rate was determined as described in Materials and methods. These experiments were repeated at least twice, with similar results.

Fig. 3. Growth of P. falciparum in the presence of various NEFAs and combinations of NEFAs at graded concentrations. Also shown for comparison is the growth of the parasite in CRPMI enriched with GFS-C + BSAF, GFSRPMI, and HSRPMI. The culture media contained 3 mg/ml BSAF, except for GFSRPMI and HSRPMI. Growth rate was determined as described in Materials and methods. *SigniWcant diVerence (P < 0.001) versus GFS-C + BSAF; versus GFSRPMI; versus HSRPMI. #No signiWcant diVerence. The highest growth rate obtained with each NEFA or each mixture of NEFAs was compared. Each NEFA was mixed at ratios of C16:0 + C18:1 (1:2), C16:0 + C18:1 + C22:6 (1:2:0.1–2.5), and C16:0 + C18:0 + C18:1 + C18:2 (1:1:1:1). This experiment was repeated at least twice, with similar results.

3.2. EVect of lysophospholipids and phospholipids on parasite growth Although phospholipid fractions from GFS-C separated by thin-layer chromatography were detected at

various concentrations and showed no growth-promoting eVect on the parasite, various commercially available phospholipids and lysophospholipids were tested for their possible eYcacy in sustaining the growth of P. falciparum. Among LPCs, 18:U LPC was found to sus-

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Fig. 4. EVects of various lysophospholipids at graded concentrations on growth of P. falciparum. Also shown for comparison is the parasite growth in GFSRPMI and HSRPMI. The culture media contained 3 mg/ml BSAF, except for GFSRPMI and HSRPMI. Growth rate was determined as described in Materials and methods. *SigniWcant diVerence (P < 0.001) versus 18:U LPC at a concentration of 40
g/ml; versus GFSRPMI; versus HSRPMI. #No signiWcant diVerence. The highest growth rate obtained with each lysophospholipid, except for 18:U LPC, was compared. 18:1 LPC and 18:2 LPC were mixed at the ratio of 1:1. This experiment was repeated at least twice, with similar results.

tain parasite growth in the presence of BSAF, in a dose-dependent manner, similar to GFSRPMI and HSRPMI (Fig. 4). The highest growth rate was obtained at 40
g/ml, with a decline at 80
g/ml. The parasite retained normal morphology and gametocytes were rarely recognized. The addition of 18:1 LPA, 18:1 LPC, 18:2 LPC, and a combination of 18:1 LPC and 18:2 LPC was also beneWcial to a much lesser extent to parasite growth. The parasite cultured in the presence of LPS, LPE, LPI, LysoPAF, 12:0 LPC, 14:0 LPC, 16:0 LPC, 18:0 LPC, LPC-brain, and SPC failed to develop (Fig. 4). The supplementation of CRPMI containing BSAF by adding phospholipids, such as PC, PS, PE, PI-soybean, PI-bovine, PAF, S-1-P, sphingosine, SM, 8:0 PA, and 18:1 PA, did not show a growth-promoting eVect on the parasite (data not shown). 3.3. Growth of the parasite cultured in media containing various growth promoters, measured by a parasite-speciWc pLDH assay Infected RBCs were maintained in the culture media containing growth promoters detected here for 2–5 days, and parasite growth was assessed by measuring pLDH activity. Development of the parasite in the CRPMI enriched with 18:U LPC + BSAF (LPCRPMI), or with GFS-C + BSAF was similar to that in GFSRPMI and HSRPMI (Fig. 5). Less growth was evident in the CRPMI enriched with 18:1 LPA + BSAF (Fig. 5). Para-

Fig. 5. Growth of P. falciparum cultured in the presence of various growth promoters. RBCs infected with the parasite were maintained asynchronously in culture media: CRPMI enriched with 18:U LPC (40
g/ml) and BSAF (䊊); GFS-C and BSAF (£); 18:1 LPA (40
g/ml) and BSAF (I); GFSRPMI (䉱); and HSRPMI (䊏). Also shown for comparison is the parasite growth in CRPMI supplemented with BSAF alone (䊐). The growth of the second subculture is shown, except for the Wrst subculture in CRPMI supplemented with 18:1 LPA + BSAF, and with BSAF alone. BSAF was added at the concentration of 3 mg/ml. The parasite growth was determined by pLDHbased Malstat assay, as described in Materials and methods. This experiment was repeated twice, with similar results.

site in the CRPMI supplemented with BSAF alone failed to develop. These results are similar to those assessed by determining the growth rate based on microscopic observation of parasitemia.

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4. Discussion To provide a deWnitive list of substances that are necessary and suYcient for P. falciparum in erythrocytic stages, the ability of components of GFS and various related substances to sustain parasite growth was characterized. It was noted that GFS-C containing NEFAs as essential factors promoted parasite growth. As well as lipids (GFS-C), it was observed that proteins were also important in promoting and sustaining parasite growth. Various chemically deWned substances that are known to exhibit biological functions against various cells were tested for their ability to promote parasite growth. It was noted that 18:U LPC is suYcient for the complete development of the parasite in the presence of BSAF, and several other supplements, including 18:1 LPA, were also partially beneWcial to parasite growth. As assessed by the pLDH-based Malstat assay, good development of the parasite was also evident in cultures in LPCRPMI and a medium enriched with GFS-C and BSAF, similar to cultures in GFSRPMI and HSRPMI. A growth-promoting factor similar to GFS is known to be present in the sera of various animals, such as horses and goats, when tested using the growth of myeloma cells as an index (Asahi and Kanazawa, 1994). Thus, it was speculated that a growth-promoting factor for P. falciparum exists not only in bovine serum, but also in the sera of certain animal species (Asahi and Kanazawa, 1994). However, BSAF and human ALB supported the ability of GFS-C to promote parasite growth, followed by guinea pig, ovine, equine and porcine ALB to a substantially lesser extent at the same concentration of GFS-C. This suggests that carrier proteins regulate the growth-promoting activity of GFS-C on the parasite. Mitamura et al. (2000) reported that NEFAs obtained from GFS and HS are the key factors for promoting parasite growth, and that the NEFAs involved have to be in pairs (one saturated and one unsaturated), with C16:0 and C18:1 as the best combination. They showed that the maximum eYcacy of the C16:0 and C18:1 mixture for controlling parasite growth is 81%. However, we observed that the eYcacy of the C16:0 and C18:1 mixture is only 27% (at 80
g/ml); mixtures of three or four NEFAs were better than mixtures of two, and GFS-C was essential for the optimum parasite growth. Nevertheless, GFS-C needs to con tain NEFAs as essential factors to exert growth-promoting ability on the parasite. NEFAs may be required for the growth of various cells for three purposes: (1) as a source of nutrition; (2) as a precursor for prostaglandin synthesis; and (3) for incorporation into the lipids of cell membranes. If the parasites require NEFAs merely for nutrition, any single NEFA, such as C18:1 or C16:0, or any combinations of several saturated or unsaturated NEFAs may have a similar eVect, although it has been speculated

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that malaria parasites are incapable of generating fatty acids or phospholipids via de novo biosynthesis of fatty acids and apparently require an external source (Holz, 1977; Vial and Ancelin, 1998). For malaria parasites, the second possibility can also be excluded because C18:1 and C16:0 are not used for prostaglandin synthesis. This led us to consider another function of NEFAs. For example, unsaturated fatty acids including C18:1, C18:2, and C20:4 have been shown to activate protein kinase C (PKC) dependently on or independently of Ca2+ and phospholipid (Murakami et al., 1986). It has been suggested that PKC activation by NEFAs is speciWc to their cis-form and not due to their detergent-like action (Murakami et al., 1986). Activation of parasite protein kinases and/or RBCs with GFS-C and NEFAs might be involved in sustaining parasite growth. LPC is a major component of oxidized low-density lipoproteins. Indeed, a high-density lipoprotein, one of the plasma lipoproteins, has been shown to support the intraerythrocytic growth of P. falciparum without the occurrence of endocytosis in parasitized RBCs (Grellier et al., 1991). Among the various lysophospholipids tested, only 18:U LPC was found to be entirely active in sustaining parasite growth. 18:1 LPA, 18:1 LPC, and 18:2 LPC were much less active, and other lysophospholipids were practically inactive. This suggests that stimulation with 18:U LPC mimics the GFS and HS eVects of promoting parasite growth and that certain structural parameters are important for the growthpromoting activity. The diVerence in metabolic lability may underlie the marked diVerence in speciWc activity between the various lysophospholipids tested, as shown here. In fact, lysophospholipase (Zidovetzki et al., 1994) and a novel phospholipase that hydrolyzes SM and lysocholinephospholipids such as LPCs and lysoPAF (Hanada et al., 2002) have been identiWed in P. falciparum. Alternatively, it is also possible that the diVerent biological eVects of lysophospholipids on parasite growth result from distinct lysophospholipid receptors in the parasite, similar to those on other cells (Kabarowski et al., 2001; Zhu et al., 2001). LPC, although not as well studied as LPA, has been shown to be involved in numerous biological processes, including cell proliferation, tumor cell invasiveness, and inXammation (Bassa et al., 1999; Kume and Gimbrone, 1994). Based on current understanding, lysophospholipids such as LPC and LPA are now widely recognized as intracellular signaling molecules, despite the fact that the precise underlying molecular mechanism of the action of LPC and LPA and the relationships between the structure and various cellular activities are yet unclear. The activation of protein kinases, including PKC, p38 mitogen-activated protein kinase (MAPK), p42MAPK, jun kinase and protein tyrosine kinase (PTK), has been the primary mechanism implicated in

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several actions of LPC (Bassa et al., 1998, 1999; Jing et al., 2000). The activation of upstream membrane/ cytoplasmic PTK has also been shown to be an early principal event in LPC-mediated signaling processes (Bassa et al., 1999; Ozaki et al., 1999). On the other hand, extensive phosphorylation by the parasite has been shown during the invasion of RBCs by the malaria parasite and the growth of an internalized parasite (Suetterlin et al., 1991). Various protein kinases belonging to the superfamily of serine/threonine protein kinases, including Ca2+-dependent protein kinases (CDPKs), MAPKs, and cyclin-dependent protein kinases (CDKs), as well as phosphatase, have been identiWed in P. falciparum (Doerig, 2004; Graeser et al., 1997; Kappes et al., 1999). However, Read and Mikkelsen (1990) suggested that P. falciparum does not possess a PKC homologue, and CDPKs are functional homologues of PKC in the parasite. So far, no members of the conventional PTK subfamily have been reported from the various sequence databases (Doerig, 2004; PlasmoDB), although tyrosine phosphorylation has been observed in P. falciparum (Sharma, 2000). Staurosporine, a protein kinase inhibitor with a broad target spectrum, including PKC, MAPK, and PTK, and PTK inhibitors have been shown to inhibit the invasion of P. falciparum, Plasmodium knowlesi, and Plasmodium chaboudi (Dluzewski and Garcia, 1996; Gazarini and Garcia, 2003; Ward et al., 1994). In fact, on the basis of previous Wndings, we tested the eVects of staurosporine on growth of the parasite cultured in diVerent media. This kinase inhibitor strongly aVects the growth of P. falciparum cultured in LPCRPMI, as well as in GFSRPMI and HSRPMI at markedly diVerent levels (Asahi et al., unpublished). A series of more speciWc inhibitors that aVect PKC, MAPK, and MAPK/extracellular signal-regulated kinase kinase (MEK) also inhibited parasite growth at signiWcantly diVerent concentrations (Asahi et al., unpublished). Namely, the growth-promoting ability of LPCRPMI and GFSRPMI was consistently more susceptible to disruption by the protein kinase inhibitors than that of HSRPMI. The regulation of signaling by lysophospholipids in association with protein kinase activation of the parasite or/and host RBCs may play a critical role in growth of P. falciparum, similar to the cases with other cells (Bassa et al., 1999; Jing et al., 2000). However, the target protein kinases of the kinase inhibitors used in the tests are yet to be identiWed, since, as pointed out (Bain et al., 2003; Davies et al., 2000), many kinase inhibitors exhibit cross- or loose speciWcity, and many malarial protein kinases are known to display atypical structural and functional properties of many of their protein kinase homologues (Doerig, 2004). A further study is necessary to determine the mechanisms underlying GFS-C, NEFAs, LPC or LPA action. It is particularly noteworthy that the single biologically

functional phospholipid, 18:U LPC, can replace HS or GFS and induces complete growth of the parasite. Elucidation of the factors interacting with the growth-promoting agents at the molecular level may provide novel crucial targets for malaria chemotherapy, including vaccine development.

Acknowledgments This work was supported, in part, by a Grant-in-Aid for ScientiWc Research from the Ministry of Education, Culture, Sports Science and Technology, Japan (13670259 to H.A.). We thank Dr. A. Moribayashi of the National Institute of Infectious Diseases, Tokyo, Japan, for her advice and for performing gas chromatography, and Dr. M. Makler of Flow Inc., USA, for his invaluable advice in performing the Malstat assay.

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