- Email: [email protected]
Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania
International Journal for Parasitology 32 (2002) 1693–1700 www.parasitology-online.com
Prostaglandin production from arachidonic acid and evidence for a 9,11endoperoxide prostaglandin H2 reductase in Leishmania q Zakayi Kabututu a,1, Samuel K. Martin b, Tomoyoshi Nozaki c, Shin-ichiro Kawazu d, Tetsuya Okada e, Craig Joe Munday a, Michael Duszenko f, Michael Lazarus a, Lucy W. Thuita g, Yoshihiro Urade a, and Bruno Kilunga Kubata a,* a
Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan b United States Army Medical Research Unit-Kenya, Unit 64109, APO AE 09831-64109, Kenya c Department of Parasitology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan d Research Institute, International Medical Center of Japan, Shinjuku-ku, Tokyo 162-8655, Japan e Department of Medical Science III, School of Health and Sport Sciences, Osaka University, Toyonaka, Osaka 560-0043, Japan f Physiologisch-chemisches Institut der Universita¨t Tu¨bingen, 72076 Tu¨bingen, Germany g Department of Biology, Georgetown University, Washington, DC 20057, USA Received 26 June 2002; received in revised form 8 August 2002; accepted 12 August 2002
Abstract Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F2a synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11endoperoxide PGH2 to PGF2a. Database search and sequence alignment alignment showed that L. major prostaglandin F2a synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F2a synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F2a synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Arachidonic acid metabolism; Prostaglandin production; Prostaglandin F2a synthase; Trypanosomatid; Kinetoplastid; Leishmania
1. Introduction Leishmania protozoa are the aetiologic agents of leishmaniases, a group of diseases that currently threaten 350 million people in 88 countries around the world, with greater than 15 million people known to be infected and about 1.5–2 million new cases estimated per year, many of which go unreported (World Health Organization, 2001). Infections with Leishmania parasites are associated with an overproduction of arachidonic acid metabolites, prostaglandins, and characterised by abnormalities of T-lymphoq Note: Nucleotide sequences for Leishmania donovani and Leishmania tropica PGFS are available from EMBL/Genbank/DDBJ under the accession numbers AB079545 and AB079546, respectively. * Corresponding author. Tel.: 181-6-6872-4851; fax: 181-6-6872-2841. E-mail address: [email protected] (B.K. Kubata). 1 Present address: Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871, Japan.
cyte function. For instance, humans and some strains of mice infected with Leishmania exhibit a number of immunological manifestations, such as the absence of response in parasite antigen-specific delayed hypersensitivity skin tests, deficiency in lymphokine production, and suppression of in vitro transformation of lymphocytes induced by parasite antigens and mitogens (Reiner, 1982; Reiner and Finke, 1983; Gutierrez et al., 1984; Murray et al., 1982), which indicate T cell unresponsiveness. All of these diverse functions of T-lymphocytes are regulated to some extent by arachidonic acid metabolites, believed to be solely produced by host macrophages (Humes et al., 1977; Bailey et al., 1982; Payan and Goetzl, 1983; Gemsa et al., 1979; Morley, 1981; Goetzl, 1981) and/or spleen cells (Farrell and Kirkpatrick, 1987). Leishmania can be divided into five major species, i.e. Leishmania donovani, Leishmania major, Leishmania tropica in the Old World and Leishmania mexicana and Leishmania amazonensis in the New World. These parasitic
0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(02)00160-1
1694
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
protozoa are transmitted as monoflagellated promastigotes to vertebrate hosts by the bite of sand flies of the genera Phlebotomine (in the Old World) and Lutzomyia (in the New World). In contact with normal human blood, these protozoan parasites invade phagocytic cells of their vertebrate hosts in which they replicate as non-flagellated amastigotes and can either lead to asymptomatic disease or present a wide variety of distinct clinical syndromes such as cutaneous, muco-cutaneous, and visceral leishmaniasis (Roberts et al., 2000; Ibrahim et al., 1999). The transformation from monoflagellated promastigotes to non-flagellated amastigotes of these parasites is accompanied by morphological and biochemical changes that are reflected by the overexpression of a number of stage-specific proteins (Chattopadhayay et al., 1996; Handman et al., 1984, 1995 ). One such protein whose gene was found to be highly expressed in L. major promastigotes is P100/11E (Kidane et al., 1989). Previous studies have identified P100/11E as an antigenic reductase in L. donovani (Jensen et al., 2001), in addition to the fact that the protein was found to be a member of the aldo-keto reductase superfamily with homology to 2,5-ketod-gluconic acid reductase, aldose reductase, and aldehyde reductase (Samaras and Spithill, 1989). However, the physiological substrates as well as the biochemical parameters of this reductase remain to be characterised. Earlier work with other parasitic protozoa in our laboratory has uncovered that the protozoan parasites Plasmodium falciparum (Kubata et al., 1998) and Trypanosoma brucei (Kubata et al., 2000) metabolise arachidonic acid to PGD2, PGE2, and PGF2a. To further gain insight into prostaglandin synthesis and function in trypanosomatids, we have extended our work to Leishmania and for the first time show the direct synthesis of immunoreactive prostaglandins and the presence of a downstream enzyme that converts PGH2 to PGF2a.
2. Materials and methods 2.1. Leishmania cells Isolates of L. donovani (WR 0130E) and L. tropica (WR 1063) were obtained from the cryobank of the Walter Reed Army Institute of Research and, maintained at 258C in culture medium (RPMI-1640 medium plus l-glutamine, HEPES buffer, sodium bicarbonate) supplemented with 10% heat-inactivated foetal bovine serum (FBS). Leishmania major (MHOM/SU/73/5ASKH) was maintained by serial passages in BALB/c mice. Draining lymph nodes from BALB/c mice infected with L. major were homogenised by glass slide in Schneider’s Drosophila medium (Life Technologies). Promastigotes were obtained by culturing amastigotes in Schneider’s medium supplemented with 20% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin for 3 days at 258C before harvesting by centrifugation at 1200 £ g for 10 min and washing them twice in Schneider’s
medium. Leishmania amazonensis (MRAT/BA/74/LV78) was maintained by serial passages of the promastigotes in Medium 199 (Life Technologies). The culture was expanded for 3 days at 258C in Medium 199 supplemented with 10% FBS, 25 mM HEPES (pH 7.4), 100 U/ml penicillin, and 100 mg/ml streptomycin. 2.2. Incubation of parasite lysates and enzyme assay Parallel cultures of L. donovani were prepared with and without 33 mM arachidonic acid, and monitored daily without a medium change. Log-phase promastigotes were obtained and washed twice, by centrifugation at 1200 £ g for 10 min, in the corresponding medium not supplemented with serum. Promastigotes (4 £ 10 9 cells) cultured with or without arachidonic acid were disrupted by hypotonic lysis using double-distilled water containing a cocktail of reversible and irreversible inhibitors (one tablet per 50 ml) of pancreas extract, pronase, thermolysin, chemotrypsin, trypsin, and papain (Completee; Roche Diagnostics). Prostaglandin production from arachidonic acid was measured by using the reaction mixture described by Kubata et al. (2000). Prostaglandins from the incubation of parasite lysates with arachidonic acid were extracted and separated by highperformance liquid chromatography (HPLC) as described previously (Kubata et al., 1998). The resulting PGD2, PGE2, and PGF2a were quantified by enzyme immunoassay (EIA) using their respective EIA kits (Cayman Chemical Co.). For PGF2a synthesis from PGH2, a standard reaction mixture containing 100 mM sodium phosphate (pH 7.0), 100 mM NADPH, and a diluted amount of enzyme in a final volume of 100 ml was used. The reaction was started by the addition of 1 ml of 500 mM 1-[ 14C]PGH2 (2.04 GBq/ mmol) and was carried out at 378C for 2 min and terminated by the addition of 250 ml of a stop solution (30:4:1 vol/vol/ vol diethyl ether/methanol/2 M citric acid). To test for the non-enzymatic formation of PGF2a, we incubated the reaction mixture containing all of the components in the absence of enzyme. The organic phase (50 ml) was applied to 20 £ 20 cm silica gel plates (Merck KGaA) at 48C, and the plates were developed in a solvent system of 90:2:1 vol/vol/vol diethyl ether/methanol/acetic acid at 2208C. The radioactivity on the plates was monitored and analysed by a Fluorescent Imaging Analyser FLA 2000 and Mac Bas V2.5 software (Fuji Photo Film Co.). For kinetic studies of PGF2a synthesis from PGH2 under different pH conditions, a reaction mixture containing a triple buffer system to allow the ionic strength to remain constant over a wide range of pH values (Ellis and Morrison, 1982), 100 mM NADPH, and a diluted amount of enzyme in a final volume of 100 ml was used. The triple buffer system contained 50 mM sodium phosphate, 50 mM sodium pyrophosphate, and 50 mM 3-[(1,1-dimethyl-2-hydroxyethyl) amino]-2hydroxypropane-sulphonic acid (AMPSO) and was adjusted to the appropriate pH with either sulphuric acid or sodium
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
hydroxide. For inhibitor studies, 1 ml of various concentrations of PGH2 analogues U-44069 and U-46619 were incubated with the reaction mixture for 2 min prior to the addition of the substrate. 2.3. Polymerase chain reaction (PCR) amplification, cDNA cloning, and sequencing Total RNA was extracted from 10 9 cells of the promastigote form of L. major, L. donovani, and L. tropica with ISOGEN (a guanidine HCl/phenol procedure, Nippon Gene). First-strand cDNAs were synthesised by using avian myeloblastosis virus reverse transcriptase after annealing 1 mg of each total RNA with oligo dT adaptor primer (Takara Shuzo). To amplify PGF2a synthase cDNA for each species, we designed the gene-specific primers based on L. major P100/11E gene (accession number J04483) (Samaras and Spithill, 1989). The primers consisted of sense primer 5 0 -CAGAATTCATGGCTGGCGTTGATAAGGCA-3 0 and antisense primer 5 0 -CCGCTCGAGTTAGAACTGCGCCTCATCAGG-3 0 and carried EcoRI and XhoI restriction sites, respectively, at their 5 0 end. PCR was accomplished by using the first-strand cDNA of each species as a template, according to the following program: after initial denaturation at 958C for 1 min, the PCR proceeded at 948C for 1 min, 588C for 30 s, and 728C for 1 min for 30 cycles. For cloning of L. donovani and L. tropica PGF2a synthase cDNA, two pairs of PCR primer were used: L. donovani splice leader sequence (Lamontagne and Papadopoulou, 1999), i.e. 5 0 TAACGCTATATAAGTATCAGTTTCTGTACT-3 0 and L. major antisense primer for cloning the 5 0 portion of PGF2a synthase cDNA, and L. major sense and oligo dT adaptor primers for the 3 0 portion. PCR was carried out as described previously, but the annealing temperature was 638C for amplifying the 5 0 portion and 558C for the 3 0 portion. The PCR products were cloned into pGEM-T Easy vector (Promega, Madison), and the sequences were determined with a DNA sequencer Model 377 (Applied Biosystems). 2.4. Expression and purification of recombinant enzymes The amplified fragments containing the entire L. major PGF2a synthase, L. donovani PGF2a synthase, and L. tropica PGF2a synthase open reading frames (ORFs) were digested with EcoRI and XhoI restriction enzymes and then cloned into the corresponding sites of the pGEX-4T-1 expression vector (Amersham Pharmacia Biotech). The resultant expression vectors were used for transformation of Escherichia coli BL21(DE3). Transformed cells were cultured for 8–10 h in the presence of 0.5 mM isopropyl-b-d-thiogalactopyranoside (IPTG) at 308C. The E. coli BL21(DE3) culture was harvested by centrifugation, washed with phosphate buffered saline (PBS) containing inhibitor cocktail, suspended in the same buffer, and disrupted by sonication (6–10 bursts of 15 s each). After removal of debris by centrifugation (3000 £ g for 15 min), the recombinant
1695
glutathione-S-transferase (GST)-PGF2a synthase fusion proteins in the supernatant were purified by affinity chromatography on glutathione(GSH)-sepharose 4B resin (Amersham Pharmacia Biotech) according to the manufacturer’s protocol. The fusion proteins bound to GSH-Sepharose 4B gel were cleaved from GST by thrombin (10 units/mg of fusion protein) and eluted with PBS. The resulting recombinant PGF2a synthases were then dialysed against 20 mM Tris/Cl (pH 8.0) buffer and applied to a diethylaminoethyl (DEAE) anion-exchange column that had been equilibrated with the same buffer. The proteins were eluted with an increasing linear gradient of 0–500 mM NaCl in the same buffer. Protein concentration was determined by use of bicinchinonic acid reagent (PIERCE) with bovine serum albumin (BSA) as a standard following the manufacturer’s protocol. Protein purity was assessed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) on 14% (w/v) gels, and the gels were stained with Sypro Orange (BioRad Laboratories) or Coomassie Brilliant Blue (Daiichi Pure Chemicals). 2.5. Western blot analysis Appropriate concentrations of Leishmania promastigote cell lysates or membrane and cytosolic fractions of L. major were separated on 14% SDS-PAGE gels, and the proteins were transferred to Immobilone PVDF membranes (Millipore) at 200 mA for 1 h before blocking with BlockAce (Dainippon Seiyaku) for 1 h at 258C. The membranes were then first incubated overnight at 48C with rabbit antiT. brucei PGF2a synthase polyclonal antibody applied in 10% BlockAce and PBS containing 0.2% (vol/vol) Tween-20. This antibody was used as its immunogen (T. brucei PGF2a synthase) shares greater than 61% homology with L. major PGF2a synthase. After several washes in PBS containing 0.2% (vol/vol) Tween-20, the membranes were incubated in horseradish peroxidase conjugated donkey anti-rabbit IgG (Jackson Laboratories) at a concentration of 10 mg/ml for 1 h at room temperature. Following several more washes, the membranes were developed with reagents for ECL Western blotting detection following the manufacturer’s protocol (Amersham Pharmacia Biotech). 2.6. Immunofluorescence staining Cells used for (indirect) fluorescence microscopy were fixed in 4% paraformaldehyde in PBS, pH 7.4, at 48C for 45 min, and extensively washed with an excess volume of PBS. Fixed cells (10 8 cells) were then incubated with a 1:500 dilution of rabbit anti-T. brucei PGF2a synthase IgG, washed three times with PBS, and incubated with a 1:2,000 dilution of Alexa Fluor 488-conjugated goat antirabbit IgG (Molecular Probes, Inc.). These cells were stained with 4 0 ,6-diamidino-2-phenylindole (DAPI), washed three times with PBS, and visualised with a Zeiss model LSM 510 confocal microscope.
1696
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
of prostaglandins in L. donovani promastigotes and reported cloning, and molecular characterisation of leishmanial PGF2a synthase. 3.1. Production of prostaglandins by Leishmania
Fig. 1. Prostaglandin production by lysates of Leishmania donovani. Cell lysates from L. donovani cultured in the presence (1AA) or absence (2AA) of 33 mM arachidonic acid were assayed with or without 1 mM arachidonic acid. Resulting prostaglandins (PGs) were analysed by EIA as described in Section 2. Prostaglandin detection limits were less than 7.8, 7.8, and 3.6 pg/assay for PGD2, PGE2, and PGF2a, respectively. The values shown are the mean from three independent experiments along with standard error (SE).
3. Results and discussion In this study we have demonstrated the de novo synthesis
Leishmania donovani promastigotes were cultured in vitro in synthetic media in the presence or absence of 33 mM arachidonic acid. Lysates of these cells were assayed both with and without 1 mM arachidonic acid, and PGD2, PGE2 and PGF2a production was then determined by EIA. Figure 1 shows that lysates from L. donovani cultured in the presence of 33 mM arachidonic acid and assayed with 1 mM arachidonic acid produced 2.38 ^ 0.081, 1.07 ^ 0.078, and 1.8 ^ 0.059 ng/mg protein of PGD2, PGE2 and PGF2a, respectively. Assaying these lysates in the absence of 1 mM arachidonic acid resulted in an almost three- and twofold decrease in the amount of PGD2 and PGF2a, respectively, whereas no PGE2 production was observed. Cultivation of L. donovani promastigotes in the presence of 33 mM arachidonic acid had no big effect on either cell growth or de novo synthesis of prostaglandins by the parasite. Instead, lysates from L. donovani cultured in the absence of arachidonic acid, but assayed with 1 mM arachidonic acid, produced 2.18 ^ 0.102, 1.035 ^ 0.092, and 1.76 ^ 0.076 ng/mg protein of PGD2, PGE2, and PGF2a, respectively. A four- and twofold decrease in PGD2, and PGF2a synthesis, respectively, was observed in the absence
Fig. 2. Expression and enzymatic activity of the recombinant Leishmania major, Leishmania donovani, and Leishmania tropica PGF2a synthases, and effect of PGH2 analogues (U-46619 and U-44069) on L. major PGF2a synthase activity. (a) Leishmania major PGF2a synthase was expressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21(DE3) and purified as described in Section 2. Lane 1, lysate from E. coli BL21(DE3) transformed with pGEX-4T-1/L. major PGF2a synthase and treated with isopropyl-b-d-thiogalactopyranoside (IPTG) for 8–10 h; lane 2, partially purified L. major PGF2a synthase after GSH-Sepharose affinity chromatography and cleavage with thrombin to separate L. major PGF2a synthase from the fusion protein; lane 3, purified L. major PGF2a synthase after ion-exchange chromatography on a diethylaminoethyl (DEAE) column. (b) Thin layer chromatography (TLC) of the reduction of PGH2 by recombinant L. major PGF2a synthase. Lane A, substrate incubated in the absence of enzyme; lane B, substrate incubated in the presence of 15 mg of heat-treated (1008C for 5 min) pure L. major PGF2a synthase. Substrate incubated in the presence of 15 mg of pure recombinant L. major PGF2a synthase (lane C), pure recombinant L. donovani PGF2a synthase (lane D), and pure recombinant L. tropica PGF2a synthase (lane E). (c) Inhibitory effect of U46619 and U-44069 on L. major PGF2a synthase activity. Inhibition of L. major PGF2a synthase by U-46619 and U-44069 was carried out as described in Section 2. Residual enzymatic activity for the reduction of PGH2 was plotted against different concentrations of U-46619 and U-44069.
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
1697
Table 1 Comparison of kinetic parameters of Leishmania major prostaglandin F2a synthase with those of Trypanosoma brucei prostaglandin F2a synthase Substrate
PGH2 NADPH 9,10-Phenanthrenequinone p-Nitrobenzaldehyde
Leishmania major prostaglandin F2a synthase
Trypanosoma brucei prostaglandin F2a synthase
Km (mM)
Vmax (nmol/min/mg)
Km (mM)
Vmax (nmol/min/mg)
15.0 3.0 12.5 12.8
270 390 4,780 7,280
1.30 5.70 0.42 0.80
2,000 15,000 48,000 22,000
of additional arachidonic acid; and no PGE2 formation was detected. The ability of the parasite lysates to synthesise prostaglandins from arachidonic acid was heat sensitive and not affected by 3 mM aspirin or 42 mM indomethacin (data not shown). As it has been observed previously (Kubata et al., 1998, 2000), our results clearly show that Leishmania parasites possess heat-sensitive molecule(s) that catalyses the conversion of arachidonic acid to prostaglandins. Lysates of L. donovani promastigotes produced high levels of prostaglandins in a way that is not dependent on an exogenous source of arachidonic acid in the culture medium, suggesting that either trace amount of arachidonic acid present in the serum would be sufficient to induce the catalyst within the promastigote cells or L. donovani promastigotes dispose of sufficient amount of available intracellular arachidonic acid. In addition, the disappearance of PGE2 synthesis upon assay of lysates in the absence of arachidonic acid indicates that L. donovani may possess a PGE2-degrading enzyme(s) that enables it to metabolise this prostanoid, since incubation of PGE2 with lysates of this parasite led to the detection of unidentified degradationproducts by HPLC (unpublished observations). Ongoing work in our laboratory will probably clarify the nature of those products and of the enzyme systems involved. 3.2. cDNA cloning and expression, and purification and characterisation of L. major PGF2a synthase Using the cDNA sequence of T. brucei PGF2a synthase (EMBL/GenBank/DDBJ, accession no. AB034727), we searched for homologous sequences in the database and identified a 1,423-bp cDNA sequence (GenBank/EMBL, accession no. J04483) of the developmentally regulated P100/11E gene from L. major (Samaras and Spithill, 1989) that exhibited the highest homology (61%) with TbPGFS. We then amplified a 855-bp fragment by PCR using P100/11E cDNA gene-specific primers and the cDNA product from reverse transcription of the L. major total RNA. The 855-bp fragment encoded an ORF that predicted a protein composed of 284 amino acid residues with a calculated Mr of 32,000. Cloning and sequencing of the 855-bp fragment revealed a sequence identical to that of the P100/11E ORF, indicating that the correct fragment had been isolated. To express heterologously the L. major PGF2a synthase,
we amplified the 855-bp ORF carrying EcoRI and XhoI restriction sites by PCR and cloned it into a pGEX expression vector. The recombinant protein was produced as a GST fusion protein in the cytosolic fraction of E. coli after IPTG induction and exhibited an Mr of ,60,000 on SDS-PAGE (Fig. 2a, lane 1). The fusion protein was purified by GSH-Sepharose affinity chromatography. Following cleavage of the fusion protein (Fig. 2a, lane 2) and subsequent purification by ion-exchange chromatography, we obtained L. major PGF2a synthase with an Mr of ,34,000 (Fig. 2a, lane 3). Lysates from crude extracts of E. coli BL21 expressing pGEX-L. major PGF2a synthase, but not those from the host cells alone or host cells containing only the vector (data not shown), reduced 9,11-endoperoxide PGH2 to PGF2a in the presence of NADPH. These results indicate that the PGF2a synthase activity depended on the L. major protein and not on any contaminating activity from the host cells or the vector. To show that the recombinant L. major PGF2a synthase was responsible for de novo synthesis of PGF2a, we boiled the pure protein at 1008C for 5 min and then incubated it with PGH2. The ability of recombinant L. major PGF2a synthase to synthesise PGF2a from PGH2 was abolished by the heat treatment (Fig. 2b, lane B), providing further evidence for the catalytic power of the leishmanial protein. We then determined some kinetic parameters of the recombinant pure protein. A comparison of the kinetic parameters between L. major PGF2a synthase and T. brucei PGF2a synthase (Kubata et al., 2000) is shown in Table 1. As compared with T. brucei PGF2a synthase, L. major PGF2a synthase showed higher Km values for PGH2 (almost 10-fold), 9,10-phenanthrenequinone (almost 30-fold), and p-nitrobenzaldehyde (almost 15-fold), while exhibiting a lower value for NADPH (almost twofold). Subsequently, L. major PGF2a synthase Vmax values were almost one order of magnitude lower than those of T. brucei PGF2a synthase. Leishmania major PGF2a synthase had also a broad range of temperature (30–458C) and pH (6–8) optima (data not shown). To further gain insight into the inhibitory effects of 9,11-endoperoxide PGH2 analogues on L. major PGF2a synthase catalytic power, we investigated the interference of U-44069 and U-46619 on the reduction of PGH2 by L. major PGF2a synthase. U-44069 and U-46619 are PGH2 receptor agonists with chemical structures similar to
1698
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
showed that U-44069 had no effect on the reduction of PGH2 by L. major PGF2a synthase as compared with the dose-dependent partial inactivation of PGH2 reduction produced by U-46619 (Fig. 2c). We have shown the direct evidence for the implication of L. major PGF2a synthase in the synthesis of PGF2a that is a biologically active molecule. The findings that Leishmania protozoa from the Old World produce prostaglandins and that they have an enzyme that catalyses PGF2a formation raise questions about the biology of Leishmania parasites and the interaction of these organisms with their mammalian host. Prostaglandins are potent mediators of physiological and pathological responses (Samuelsson, 1979–1980; Mathe et al., 1977; Oliw et al., 1983; Glew, 1992; Dubois et al., 1998; Hayaishi, 2000), some of which are observed in leishmaniases. It has also been reported that PGE2 and PGF2a may play a signal-coupling role during phagocytosis in the protozoan parasite Amoeba proteus, since they elicit vacuole formation (Prusch et al., 1989) whereas PGD2 was shown to play an important role in T. brucei cell growth regulation by inducing programmed cell death (unpublished observations). However, whether or not Leishmania-derived prostaglandins play similar roles remains to be established. In the present study, we have at least identified an enzyme that catalyses PGF2a synthesis in Leishmania parasite. Although the P100/11E gene of this enzyme is rather more abundantly expressed in promastigotes than in amastigotes (Samaras and Spithill, 1989), neither its function nor its specific reductive reaction or substrate had previously been identified. Fig. 3. Expression of PGF2a synthase in Leishmania. (A) Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of PGF2a synthase gene expression in Leishmania major (lane 1), Leishmania donovani (lane 2), Leishmania tropica (lane 3), Leishmania mexicana (lane 4), and Leishmania amazonensis (lane 5). Total RNA (1 mg) from logarithmic-phase promastigote cells of each species was extracted as described under Section 2 and used as a template for reverse transcription. The resulting cDNA product (1 ml) was then used in PCR amplification of the open reading frames of either PGF2a synthase (upper panel) or a-tubulin (lower panel) by using the L. major PGF2a synthase gene-specific sense and antisense primers described in Section 2 or a-tubulin gene-specific sense primer 5 0 CTGACGGAGTTCCAGACGAACCT-3 0 and antisense primer 5 0 -TTAGTACTCCTCGACGTCCTCCT-3 0 . The RT-PCR products were analysed on a 1.5% agarose gel. (B,C) Western blot analysis of PGF2a synthase in L. major promastigote (lanes B-1 and C-a), L. donovani (lane 2), L. tropica (lane 3), L. mexicana (lane 4), and L. amazonensis (lane 5). Lanes C-b and C-c indicate L. major membrane and cytosolic fractions, respectively. Total protein (100 ng) from lysates of each Leishmania species and from L. major membrane or cytosolic fractions was resolved by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and analysed by Western blotting as described under Section 2.
that of PGH2 with the exception that the oxygen atoms at C11 in U-44069 and C-9 in U-46619 are substituted with a CH2 group. Pre-incubation of L. major PGF2a synthase with various concentrations of either U-44069 or U-46619 followed by the incubation of the mixtures with PGH2
3.3. Distribution and cellular localisation of L. major PGF2a synthase in Leishmania The distribution of PGF2a synthase was investigated by detecting both the gene from the total RNAs and the protein in the lysates of L. major, L. donovani, L. tropica, L. mexicana, and L. amazonensis. An 855-bp fragment corresponding to the PGF2a synthase ORF (Fig. 3A, upper panel) was amplified and detected from the total RNAs of Old World species L. major (Fig. 3A, lane 1), L. donovani (Fig. 3A, lane 2), and L. tropica (Fig. 3A, lane 3) but not from those of New World species L. mexicana and L. amazonensis (Fig. 3A, lanes 4 and 5, respectively), whereas a 603-bp fragment of a-tubulin ORF (Fig. 3A, lower panel) was identified from RNAs of all Leishmania species. Sequence analysis of these PCR products revealed an almost 99% identity between L. major and L. donovani, and 100% identity between L. donovani and L. tropica (data not shown). The presence of PGF2a synthase in other subspecies of L. donovani, which are believed to occur in the Old (L. donovani, L. infantum) and New World (L. chagasi) species, may imply a role for this enzyme in vector competence (Sacks and Kamhawi, 2001). Interestingly, rabbit anti-T. brucei PGF2a synthase polyclonal antibody detected 34-kDa immunoreactive signals corresponding to L. major (Fig. 3B, lane 1), L. dono-
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
Fig. 4. Cellular localisation of Leishmania major PGF2a synthase by immunofluorescence. Leishmania major promastigotes were recovered from asynchronous cultures at the logarithmic-phase. Staining of DNA with 4 0 ,6-diamidino-2-phenylindole (DAPI) shows the location of the nucleus (blue) in the promastigote. Leishmania major PGF2a synthase is visualised by specific Trypanosoma brucei PGF2a synthase polyclonal antibody and anti-rabbit IgG-FITC (green). (A) Phase contrast, (B) DAPI and T. brucei PGF2a synthase polyclonal antibody double staining. Bar, 10 mm.
vani (Fig. 3B, lane 2), and L. tropica (Fig. 3B, lane 3). No signal was detected from lysates of L. mexicana and L. amazonensis (Fig. 3B, lanes 4 and 5, respectively). Moreover, immunoblot analysis of L. major whole lysate (Fig. 3C, lane a) and subcellular fractions (Fig. 3C, lanes b and c) for PGF2a synthase showed L. major PGF2a synthase to be localised in the cytosol (Fig. 3C, lane c). This localisation is in agreement with our previous finding that the orthologue T. brucei PGF2a synthase enzyme was also localised in the cytosol (Kubata et al., 2000). To further confirm this localisation, we stained whole promastigote cells of L. major. The immunofluorescence of fixed and permeabilised promastigotes obtained with FITC-conjugated anti-T. brucei PGF2a synthase IgG showed an abundance of stain localised in the reticular material throughout the cytoplasm (Fig. 4B), suggesting that L. major PGF2a synthase is a cytosolic protein. Pre-immune rabbit serum did not stain the parasites (data not shown). In the present study we have shown that Leishmania species convert arachidonic acid to PGD2, PGE2 and
1699
PGF2a. Furthermore we have characterised a recombinant PGF2a from L. major and demonstrated its distribution in Old World Leishmania species. However, this enzyme was not detected in New World Leishmania species although they do produce PGF2a (unpublished observations). It is most likely that in L. mexicana and L. amazonensis, another type of protein may be responsible for PGF2a synthesis since studies of prostaglandin synthesis in Trypanosoma from our laboratory found that the protein responsible for PGF2a synthesis in T. brucei (Kubata et al., 2000) is different from that for PGF2a synthesis in T. cruzi (unpublished observations). Alternatively, probably L. mexicana and L. amazonensis PGF2a synthase gene and protein were simply in insufficient amounts that did not allow their detection in the present study. Midgut environment conditions in sand fly species may have evolutionarily driven the expression of some Leishmania surface molecules such as lipophosphoglycans. Leishmania species-specific polymorphisms in lipophosphoglycans have been reported to control differences in the binding capacities for the midgut of different vectors, and the extent of binding predicts in which parasite/ sand fly combination the development of transmissible infections can occur (Sacks and Kamhawi, 2001; Pimenta et al., 1994; Sacks et al., 2000). Whether or not PGF2a synthase and/or PGF2a play some role in the vector competence of Leishmania infections remain to be investigated. Why PGF2a synthesis segregates between Old and New World parasites as well as why Leishmania produces prostaglandins is not clear to us at this time. It is, however, worth mentioning that the identification of prostaglandin-producing enzymes provides the basis for further molecular investigation of the physiological roles of these lipid mediators and that ongoing gene knockout experiments in our laboratory will probably elucidate the biological relevance of prostanoid production in parasitic protozoa.
Acknowledgements We are grateful to Drs Hiroyuki Ishikawa of the Research Institute and International Medical Center of Japan and Yumiko Saito-Nakano and Osamu Fujita of the National Institute of Infectious Diseases, Tokyo, Japan, for their technical help. This work was supported in part by grants from programs Grants-in-Aid for Scientific Research (No. 14370087 to B.K.K.) and Grants-in-Aid for Scientific Research in Priority areas (No. 14021130 to B.K.K. and Y.U.) of the Ministry of Education, Culture, Sport, Science and Technology, Japan, and by a fellowship from the Takeda Science Foundation to C.J.M. It was also supported by grants from the following sources: the Deutsche Forschungsgesellschaft to M.D.; the Japan Science and Technology Corporation to B.K.K., M.L. (No. 199041) and to Y.U.; the Ministry of Education, Science, Culture, and Sports of Japan to Y.U. (Nos. 11877047 and 12558078), and the Japan Health Sciences Foundation (No. SA14706)
1700
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
and the Precursory Research for Embryonic Science and Technology (PRESTO) from the Japan Science and Technology Corporation to T.N. References Bailey, J.M., Bryant, R.W., Low, C.E., Pupillo, M.B., Vanderhoek, J.Y., 1982. Regulation of T-lymphocyte mitogenesis by the leucocyte product 15-hydroxyeicosatetraenoic acid (15-HETE). Cell. Immunol. 67, 112–20. Chattopadhayay, R., Kaur, S., Gangluy, N.K., Mahajan, R.C., 1996. Antigenic differences between axenic amastigotes and promastigotes of Leishmania donovani. Indian J. Med. Res. 104, 349–54. Dubois, R.N., Abramson, S.B., Crofford, L., Gupta, R.A., Simon, L.S., Van de Putte, L.B., Lipsky, P.E., 1998. Cyclooxygenase in biology and disease. FASEB J. 12, 1063–73. Ellis, K.J., Morrison, J.F., 1982. Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol. 87, 405–26. Farrell, J.P., Kirkpatrick, C.E., 1987. Experimental cutaneous leishmaniasis. II. A possible role for prostaglandins in exacerbation of disease in Leishmania major-infected BALB/c mice. J. Immunol. 138, 902–7. Gemsa, D., Seitz, M., Menzel, J., Grimm, W., Kramer, W., Till, G., 1979. Modulation of phagocytosis-induced prostaglandin release from macrophages. Adv. Exp. Med. Biol. 114, 421–6. Glew, R.H., 1992. Prostaglandins and thromboxanes. In: Devlin, T.M. (Ed.). Texbook of Biochemistry with Clinical Correlations, Wiley– Liss, New York, NY, pp. 461–6. Goetzl, E.J., 1981. Selective feedback of the 5-lipoxygenation of arachidonic acid in human T-lymphocytes. Biochem. Biophys. Res. Commun. 101, 344–50. Gutierrez, Y., Maksem, J.A., Reiner, N.E., 1984. Pathological changes in murine leishmaniasis (Leishmania donovani) with specific reference to the dynamics of granuloma formation in the liver. Am. J. Pathol. 114, 222–30. Handman, E., Jarvis, H.M., Michell, G.F., 1984. Leishmania major identification of stage-specific antigens and antigens shared by promastigotes and amastigotes. Parasite Immunol. 6, 223–33. Handman, E., Osborn, A.H., Symons, F.M., Van Driel, R., Cappai, R., 1995. The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle. Mol. Biochem. Parasitol. 74, 189–200. Hayaishi, O., 2000. Molecular mechanisms of sleep–wake regulation: a role of prostaglandin D2. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 355, 275– 80. Humes, J.L., Bonney, R.J., Pelus, L., Dahlgren, M.E., Sadowski, S.J., Kuehl, F.A., Davis, P., 1977. Macrophages synthesize and release prostaglandins in response to inflammatory stimuli. Nature 269, 149–51. Ibrahim, M.E., Lambson, B., Yousif, A.O., Deifalla, N.S., Alnaiem, D.A., Ismail, A., Yousif, H., Ghalib, H.W., Khalil, E.A., Kadaro, A., Barker, D.C., El Hassan, A.M., 1999. Kala-azar in a high transmission focus: an ethnic and geographic dimension. Am. J. Trop. Med. Hyg. 61, 941–4. Jensen, A.T., Kemp, K., Theander, T.G., Handman, E., 2001. Cloning, expression and antigenicity of the L. donovani reductase. APMIS 109, 461–8. Kidane, G.Z., Samaras, N., Spithill, T.W., 1989. Cloning of developmen-
tally regulated genes from Leishmania major and expression following heat induction. J. Biol. Chem. 264, 4244–50. Kubata, B.K., Eguchi, N., Urade, Y., Yamashita, K., Mitamura, T., Tai, K., Hayaishi, O., Horii, T., 1998. Plasmodium falciparum produces substances that are pyrogenic, somnogenic, and immunosuppressive in humans. J. Exp. Med. 188, 1197–202. Kubata, B.K., Duszenko, M., Kabututu, Z., Rawer, M., Szallies, A., Fujimori, K., Inui, T., Nozaki, T., Yamashita, K., Horii, T., Urade, Y., Hayaishi, O., 2000. Identification of a novel prostaglandin F2a synthase in Trypanosoma brucei. J. Exp. Med. 192, 1327–37. Lamontagne, J., Papadopoulou, B., 1999. Developmental regulation of spliced leader RNA gene in Leishmania donovani amastigotes is mediated by specific polyadenylation. J. Biol. Chem. 274, 6602–9. Mathe, A.A., Hedqvist, P., Strandberg, K., Leslie, C.A., 1977. Aspects of prostaglandin function in the lung (second of two parts). N. Engl. J. Med. 296, 910–4. Morley, J., 1981. Role of prostaglandins secreted by macrophages in the inflammatory process. Lymphokines 4, 377–83. Murray, H.W., Masur, H., Keithly, J.S., 1982. Cell-mediated immune response in experimental visceral leishmaniasis. I. Correlation between resistance to Leishmania donovani and lymphokine-generating capacity. J. Immunol. 129, 344–50. Oliw, E., Granstrom, E., Anggard, E., 1983. The prostaglandin and essential fatty acids. In: Pace-Asciak, C., Granstrom, E. (Eds.). Prostaglandins and Related Substances, Elsevier, Amsterdam, pp. 11–19. Payan, D.G., Goetzl, E.J., 1983. Specific suppression of human T-lymphocyte function by leukotriene B4. J. Immunol. 131, 551–3. Pimenta, P.F.P., Saraiva, E.M.B., Rowton, E., Modi, G.B., Garraway, L.A., Beverley, S.M., Turco, S.J., Sacks, D.L., 1994. Evidence that the vectorial competence of phlebotomine sand flies for different species of Leishmania is controlled by structural polymorphisms in the surface lipophosphoglycan. Proc. Natl Acad. Sci. USA 91, 9155–9. Prusch, R.D., Goette, S.M., Haberman, P., 1989. Prostaglandins may play a signal-coupling role during phagocytosis in Amoeba proteus. Cell Tissue Res. 255, 553–7. Reiner, N.E., 1982. Host-parasite relationship in murine leishmaniasis donovani: dynamics of pathophysiologic and immunologic changes. Infect. Immun. 38, 1223–30. Reiner, N.E., Finke, J.H., 1983. Interleukin 2 deficiency in murine leishmaniasis donovani and its relationship to depressed spleen cell responses to phytohemagglutinin. J. Immunol. 131, 1487–91. Roberts, L.J., Foote, S.J., Handman, E., 2000. A new standard for the assessment of disease progression in murine cutaneous leishmaniasis. Parasite Immunol. 22, 231–7. Sacks, D.L., Kamhawi, S., 2001. Molecular aspects of parasite-vector and vector-host interactions in leishmaniasis. Annu. Rev. Microbiol. 55, 453–83. Sacks, D.L., Modi, G., Rowton, E., Spath, G., Epstein, L., Turco, S., Beverley, S.M., 2000. The role of phosphoglycans in Leishmania-sand fly interactions. Proc. Natl Acad. Sci. USA 97, 406–11. Samaras, N., Spithill, T.W., 1989. The developmentally regulated P100/ 11E gene of Leishmania major shows homology to a superfamily of reductase genes. J. Biol. Chem. 264, 4251–4. Samuelsson, B., 1979–1980. Prostaglandins, thromboxans, and leukotrienes: formation and biological roles. Harvey Lect. 75, 1–40. World Health Organization, 2001. Leishmaniasis: the disease and its impact. WHO homepage available at http://www.who.int/
Prostaglandin production from arachidonic acid and evidence for a 9,11endoperoxide prostaglandin H2 reductase in Leishmania q Zakayi Kabututu a,1, Samuel K. Martin b, Tomoyoshi Nozaki c, Shin-ichiro Kawazu d, Tetsuya Okada e, Craig Joe Munday a, Michael Duszenko f, Michael Lazarus a, Lucy W. Thuita g, Yoshihiro Urade a, and Bruno Kilunga Kubata a,* a
Department of Molecular Behavioral Biology, Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan b United States Army Medical Research Unit-Kenya, Unit 64109, APO AE 09831-64109, Kenya c Department of Parasitology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162-8640, Japan d Research Institute, International Medical Center of Japan, Shinjuku-ku, Tokyo 162-8655, Japan e Department of Medical Science III, School of Health and Sport Sciences, Osaka University, Toyonaka, Osaka 560-0043, Japan f Physiologisch-chemisches Institut der Universita¨t Tu¨bingen, 72076 Tu¨bingen, Germany g Department of Biology, Georgetown University, Washington, DC 20057, USA Received 26 June 2002; received in revised form 8 August 2002; accepted 12 August 2002
Abstract Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F2a synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11endoperoxide PGH2 to PGF2a. Database search and sequence alignment alignment showed that L. major prostaglandin F2a synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F2a synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F2a synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol. q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Arachidonic acid metabolism; Prostaglandin production; Prostaglandin F2a synthase; Trypanosomatid; Kinetoplastid; Leishmania
1. Introduction Leishmania protozoa are the aetiologic agents of leishmaniases, a group of diseases that currently threaten 350 million people in 88 countries around the world, with greater than 15 million people known to be infected and about 1.5–2 million new cases estimated per year, many of which go unreported (World Health Organization, 2001). Infections with Leishmania parasites are associated with an overproduction of arachidonic acid metabolites, prostaglandins, and characterised by abnormalities of T-lymphoq Note: Nucleotide sequences for Leishmania donovani and Leishmania tropica PGFS are available from EMBL/Genbank/DDBJ under the accession numbers AB079545 and AB079546, respectively. * Corresponding author. Tel.: 181-6-6872-4851; fax: 181-6-6872-2841. E-mail address: [email protected] (B.K. Kubata). 1 Present address: Department of Molecular Protozoology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 5650871, Japan.
cyte function. For instance, humans and some strains of mice infected with Leishmania exhibit a number of immunological manifestations, such as the absence of response in parasite antigen-specific delayed hypersensitivity skin tests, deficiency in lymphokine production, and suppression of in vitro transformation of lymphocytes induced by parasite antigens and mitogens (Reiner, 1982; Reiner and Finke, 1983; Gutierrez et al., 1984; Murray et al., 1982), which indicate T cell unresponsiveness. All of these diverse functions of T-lymphocytes are regulated to some extent by arachidonic acid metabolites, believed to be solely produced by host macrophages (Humes et al., 1977; Bailey et al., 1982; Payan and Goetzl, 1983; Gemsa et al., 1979; Morley, 1981; Goetzl, 1981) and/or spleen cells (Farrell and Kirkpatrick, 1987). Leishmania can be divided into five major species, i.e. Leishmania donovani, Leishmania major, Leishmania tropica in the Old World and Leishmania mexicana and Leishmania amazonensis in the New World. These parasitic
0020-7519/02/$20.00 q 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0020-751 9(02)00160-1
1694
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
protozoa are transmitted as monoflagellated promastigotes to vertebrate hosts by the bite of sand flies of the genera Phlebotomine (in the Old World) and Lutzomyia (in the New World). In contact with normal human blood, these protozoan parasites invade phagocytic cells of their vertebrate hosts in which they replicate as non-flagellated amastigotes and can either lead to asymptomatic disease or present a wide variety of distinct clinical syndromes such as cutaneous, muco-cutaneous, and visceral leishmaniasis (Roberts et al., 2000; Ibrahim et al., 1999). The transformation from monoflagellated promastigotes to non-flagellated amastigotes of these parasites is accompanied by morphological and biochemical changes that are reflected by the overexpression of a number of stage-specific proteins (Chattopadhayay et al., 1996; Handman et al., 1984, 1995 ). One such protein whose gene was found to be highly expressed in L. major promastigotes is P100/11E (Kidane et al., 1989). Previous studies have identified P100/11E as an antigenic reductase in L. donovani (Jensen et al., 2001), in addition to the fact that the protein was found to be a member of the aldo-keto reductase superfamily with homology to 2,5-ketod-gluconic acid reductase, aldose reductase, and aldehyde reductase (Samaras and Spithill, 1989). However, the physiological substrates as well as the biochemical parameters of this reductase remain to be characterised. Earlier work with other parasitic protozoa in our laboratory has uncovered that the protozoan parasites Plasmodium falciparum (Kubata et al., 1998) and Trypanosoma brucei (Kubata et al., 2000) metabolise arachidonic acid to PGD2, PGE2, and PGF2a. To further gain insight into prostaglandin synthesis and function in trypanosomatids, we have extended our work to Leishmania and for the first time show the direct synthesis of immunoreactive prostaglandins and the presence of a downstream enzyme that converts PGH2 to PGF2a.
2. Materials and methods 2.1. Leishmania cells Isolates of L. donovani (WR 0130E) and L. tropica (WR 1063) were obtained from the cryobank of the Walter Reed Army Institute of Research and, maintained at 258C in culture medium (RPMI-1640 medium plus l-glutamine, HEPES buffer, sodium bicarbonate) supplemented with 10% heat-inactivated foetal bovine serum (FBS). Leishmania major (MHOM/SU/73/5ASKH) was maintained by serial passages in BALB/c mice. Draining lymph nodes from BALB/c mice infected with L. major were homogenised by glass slide in Schneider’s Drosophila medium (Life Technologies). Promastigotes were obtained by culturing amastigotes in Schneider’s medium supplemented with 20% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin for 3 days at 258C before harvesting by centrifugation at 1200 £ g for 10 min and washing them twice in Schneider’s
medium. Leishmania amazonensis (MRAT/BA/74/LV78) was maintained by serial passages of the promastigotes in Medium 199 (Life Technologies). The culture was expanded for 3 days at 258C in Medium 199 supplemented with 10% FBS, 25 mM HEPES (pH 7.4), 100 U/ml penicillin, and 100 mg/ml streptomycin. 2.2. Incubation of parasite lysates and enzyme assay Parallel cultures of L. donovani were prepared with and without 33 mM arachidonic acid, and monitored daily without a medium change. Log-phase promastigotes were obtained and washed twice, by centrifugation at 1200 £ g for 10 min, in the corresponding medium not supplemented with serum. Promastigotes (4 £ 10 9 cells) cultured with or without arachidonic acid were disrupted by hypotonic lysis using double-distilled water containing a cocktail of reversible and irreversible inhibitors (one tablet per 50 ml) of pancreas extract, pronase, thermolysin, chemotrypsin, trypsin, and papain (Completee; Roche Diagnostics). Prostaglandin production from arachidonic acid was measured by using the reaction mixture described by Kubata et al. (2000). Prostaglandins from the incubation of parasite lysates with arachidonic acid were extracted and separated by highperformance liquid chromatography (HPLC) as described previously (Kubata et al., 1998). The resulting PGD2, PGE2, and PGF2a were quantified by enzyme immunoassay (EIA) using their respective EIA kits (Cayman Chemical Co.). For PGF2a synthesis from PGH2, a standard reaction mixture containing 100 mM sodium phosphate (pH 7.0), 100 mM NADPH, and a diluted amount of enzyme in a final volume of 100 ml was used. The reaction was started by the addition of 1 ml of 500 mM 1-[ 14C]PGH2 (2.04 GBq/ mmol) and was carried out at 378C for 2 min and terminated by the addition of 250 ml of a stop solution (30:4:1 vol/vol/ vol diethyl ether/methanol/2 M citric acid). To test for the non-enzymatic formation of PGF2a, we incubated the reaction mixture containing all of the components in the absence of enzyme. The organic phase (50 ml) was applied to 20 £ 20 cm silica gel plates (Merck KGaA) at 48C, and the plates were developed in a solvent system of 90:2:1 vol/vol/vol diethyl ether/methanol/acetic acid at 2208C. The radioactivity on the plates was monitored and analysed by a Fluorescent Imaging Analyser FLA 2000 and Mac Bas V2.5 software (Fuji Photo Film Co.). For kinetic studies of PGF2a synthesis from PGH2 under different pH conditions, a reaction mixture containing a triple buffer system to allow the ionic strength to remain constant over a wide range of pH values (Ellis and Morrison, 1982), 100 mM NADPH, and a diluted amount of enzyme in a final volume of 100 ml was used. The triple buffer system contained 50 mM sodium phosphate, 50 mM sodium pyrophosphate, and 50 mM 3-[(1,1-dimethyl-2-hydroxyethyl) amino]-2hydroxypropane-sulphonic acid (AMPSO) and was adjusted to the appropriate pH with either sulphuric acid or sodium
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
hydroxide. For inhibitor studies, 1 ml of various concentrations of PGH2 analogues U-44069 and U-46619 were incubated with the reaction mixture for 2 min prior to the addition of the substrate. 2.3. Polymerase chain reaction (PCR) amplification, cDNA cloning, and sequencing Total RNA was extracted from 10 9 cells of the promastigote form of L. major, L. donovani, and L. tropica with ISOGEN (a guanidine HCl/phenol procedure, Nippon Gene). First-strand cDNAs were synthesised by using avian myeloblastosis virus reverse transcriptase after annealing 1 mg of each total RNA with oligo dT adaptor primer (Takara Shuzo). To amplify PGF2a synthase cDNA for each species, we designed the gene-specific primers based on L. major P100/11E gene (accession number J04483) (Samaras and Spithill, 1989). The primers consisted of sense primer 5 0 -CAGAATTCATGGCTGGCGTTGATAAGGCA-3 0 and antisense primer 5 0 -CCGCTCGAGTTAGAACTGCGCCTCATCAGG-3 0 and carried EcoRI and XhoI restriction sites, respectively, at their 5 0 end. PCR was accomplished by using the first-strand cDNA of each species as a template, according to the following program: after initial denaturation at 958C for 1 min, the PCR proceeded at 948C for 1 min, 588C for 30 s, and 728C for 1 min for 30 cycles. For cloning of L. donovani and L. tropica PGF2a synthase cDNA, two pairs of PCR primer were used: L. donovani splice leader sequence (Lamontagne and Papadopoulou, 1999), i.e. 5 0 TAACGCTATATAAGTATCAGTTTCTGTACT-3 0 and L. major antisense primer for cloning the 5 0 portion of PGF2a synthase cDNA, and L. major sense and oligo dT adaptor primers for the 3 0 portion. PCR was carried out as described previously, but the annealing temperature was 638C for amplifying the 5 0 portion and 558C for the 3 0 portion. The PCR products were cloned into pGEM-T Easy vector (Promega, Madison), and the sequences were determined with a DNA sequencer Model 377 (Applied Biosystems). 2.4. Expression and purification of recombinant enzymes The amplified fragments containing the entire L. major PGF2a synthase, L. donovani PGF2a synthase, and L. tropica PGF2a synthase open reading frames (ORFs) were digested with EcoRI and XhoI restriction enzymes and then cloned into the corresponding sites of the pGEX-4T-1 expression vector (Amersham Pharmacia Biotech). The resultant expression vectors were used for transformation of Escherichia coli BL21(DE3). Transformed cells were cultured for 8–10 h in the presence of 0.5 mM isopropyl-b-d-thiogalactopyranoside (IPTG) at 308C. The E. coli BL21(DE3) culture was harvested by centrifugation, washed with phosphate buffered saline (PBS) containing inhibitor cocktail, suspended in the same buffer, and disrupted by sonication (6–10 bursts of 15 s each). After removal of debris by centrifugation (3000 £ g for 15 min), the recombinant
1695
glutathione-S-transferase (GST)-PGF2a synthase fusion proteins in the supernatant were purified by affinity chromatography on glutathione(GSH)-sepharose 4B resin (Amersham Pharmacia Biotech) according to the manufacturer’s protocol. The fusion proteins bound to GSH-Sepharose 4B gel were cleaved from GST by thrombin (10 units/mg of fusion protein) and eluted with PBS. The resulting recombinant PGF2a synthases were then dialysed against 20 mM Tris/Cl (pH 8.0) buffer and applied to a diethylaminoethyl (DEAE) anion-exchange column that had been equilibrated with the same buffer. The proteins were eluted with an increasing linear gradient of 0–500 mM NaCl in the same buffer. Protein concentration was determined by use of bicinchinonic acid reagent (PIERCE) with bovine serum albumin (BSA) as a standard following the manufacturer’s protocol. Protein purity was assessed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) on 14% (w/v) gels, and the gels were stained with Sypro Orange (BioRad Laboratories) or Coomassie Brilliant Blue (Daiichi Pure Chemicals). 2.5. Western blot analysis Appropriate concentrations of Leishmania promastigote cell lysates or membrane and cytosolic fractions of L. major were separated on 14% SDS-PAGE gels, and the proteins were transferred to Immobilone PVDF membranes (Millipore) at 200 mA for 1 h before blocking with BlockAce (Dainippon Seiyaku) for 1 h at 258C. The membranes were then first incubated overnight at 48C with rabbit antiT. brucei PGF2a synthase polyclonal antibody applied in 10% BlockAce and PBS containing 0.2% (vol/vol) Tween-20. This antibody was used as its immunogen (T. brucei PGF2a synthase) shares greater than 61% homology with L. major PGF2a synthase. After several washes in PBS containing 0.2% (vol/vol) Tween-20, the membranes were incubated in horseradish peroxidase conjugated donkey anti-rabbit IgG (Jackson Laboratories) at a concentration of 10 mg/ml for 1 h at room temperature. Following several more washes, the membranes were developed with reagents for ECL Western blotting detection following the manufacturer’s protocol (Amersham Pharmacia Biotech). 2.6. Immunofluorescence staining Cells used for (indirect) fluorescence microscopy were fixed in 4% paraformaldehyde in PBS, pH 7.4, at 48C for 45 min, and extensively washed with an excess volume of PBS. Fixed cells (10 8 cells) were then incubated with a 1:500 dilution of rabbit anti-T. brucei PGF2a synthase IgG, washed three times with PBS, and incubated with a 1:2,000 dilution of Alexa Fluor 488-conjugated goat antirabbit IgG (Molecular Probes, Inc.). These cells were stained with 4 0 ,6-diamidino-2-phenylindole (DAPI), washed three times with PBS, and visualised with a Zeiss model LSM 510 confocal microscope.
1696
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
of prostaglandins in L. donovani promastigotes and reported cloning, and molecular characterisation of leishmanial PGF2a synthase. 3.1. Production of prostaglandins by Leishmania
Fig. 1. Prostaglandin production by lysates of Leishmania donovani. Cell lysates from L. donovani cultured in the presence (1AA) or absence (2AA) of 33 mM arachidonic acid were assayed with or without 1 mM arachidonic acid. Resulting prostaglandins (PGs) were analysed by EIA as described in Section 2. Prostaglandin detection limits were less than 7.8, 7.8, and 3.6 pg/assay for PGD2, PGE2, and PGF2a, respectively. The values shown are the mean from three independent experiments along with standard error (SE).
3. Results and discussion In this study we have demonstrated the de novo synthesis
Leishmania donovani promastigotes were cultured in vitro in synthetic media in the presence or absence of 33 mM arachidonic acid. Lysates of these cells were assayed both with and without 1 mM arachidonic acid, and PGD2, PGE2 and PGF2a production was then determined by EIA. Figure 1 shows that lysates from L. donovani cultured in the presence of 33 mM arachidonic acid and assayed with 1 mM arachidonic acid produced 2.38 ^ 0.081, 1.07 ^ 0.078, and 1.8 ^ 0.059 ng/mg protein of PGD2, PGE2 and PGF2a, respectively. Assaying these lysates in the absence of 1 mM arachidonic acid resulted in an almost three- and twofold decrease in the amount of PGD2 and PGF2a, respectively, whereas no PGE2 production was observed. Cultivation of L. donovani promastigotes in the presence of 33 mM arachidonic acid had no big effect on either cell growth or de novo synthesis of prostaglandins by the parasite. Instead, lysates from L. donovani cultured in the absence of arachidonic acid, but assayed with 1 mM arachidonic acid, produced 2.18 ^ 0.102, 1.035 ^ 0.092, and 1.76 ^ 0.076 ng/mg protein of PGD2, PGE2, and PGF2a, respectively. A four- and twofold decrease in PGD2, and PGF2a synthesis, respectively, was observed in the absence
Fig. 2. Expression and enzymatic activity of the recombinant Leishmania major, Leishmania donovani, and Leishmania tropica PGF2a synthases, and effect of PGH2 analogues (U-46619 and U-44069) on L. major PGF2a synthase activity. (a) Leishmania major PGF2a synthase was expressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21(DE3) and purified as described in Section 2. Lane 1, lysate from E. coli BL21(DE3) transformed with pGEX-4T-1/L. major PGF2a synthase and treated with isopropyl-b-d-thiogalactopyranoside (IPTG) for 8–10 h; lane 2, partially purified L. major PGF2a synthase after GSH-Sepharose affinity chromatography and cleavage with thrombin to separate L. major PGF2a synthase from the fusion protein; lane 3, purified L. major PGF2a synthase after ion-exchange chromatography on a diethylaminoethyl (DEAE) column. (b) Thin layer chromatography (TLC) of the reduction of PGH2 by recombinant L. major PGF2a synthase. Lane A, substrate incubated in the absence of enzyme; lane B, substrate incubated in the presence of 15 mg of heat-treated (1008C for 5 min) pure L. major PGF2a synthase. Substrate incubated in the presence of 15 mg of pure recombinant L. major PGF2a synthase (lane C), pure recombinant L. donovani PGF2a synthase (lane D), and pure recombinant L. tropica PGF2a synthase (lane E). (c) Inhibitory effect of U46619 and U-44069 on L. major PGF2a synthase activity. Inhibition of L. major PGF2a synthase by U-46619 and U-44069 was carried out as described in Section 2. Residual enzymatic activity for the reduction of PGH2 was plotted against different concentrations of U-46619 and U-44069.
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
1697
Table 1 Comparison of kinetic parameters of Leishmania major prostaglandin F2a synthase with those of Trypanosoma brucei prostaglandin F2a synthase Substrate
PGH2 NADPH 9,10-Phenanthrenequinone p-Nitrobenzaldehyde
Leishmania major prostaglandin F2a synthase
Trypanosoma brucei prostaglandin F2a synthase
Km (mM)
Vmax (nmol/min/mg)
Km (mM)
Vmax (nmol/min/mg)
15.0 3.0 12.5 12.8
270 390 4,780 7,280
1.30 5.70 0.42 0.80
2,000 15,000 48,000 22,000
of additional arachidonic acid; and no PGE2 formation was detected. The ability of the parasite lysates to synthesise prostaglandins from arachidonic acid was heat sensitive and not affected by 3 mM aspirin or 42 mM indomethacin (data not shown). As it has been observed previously (Kubata et al., 1998, 2000), our results clearly show that Leishmania parasites possess heat-sensitive molecule(s) that catalyses the conversion of arachidonic acid to prostaglandins. Lysates of L. donovani promastigotes produced high levels of prostaglandins in a way that is not dependent on an exogenous source of arachidonic acid in the culture medium, suggesting that either trace amount of arachidonic acid present in the serum would be sufficient to induce the catalyst within the promastigote cells or L. donovani promastigotes dispose of sufficient amount of available intracellular arachidonic acid. In addition, the disappearance of PGE2 synthesis upon assay of lysates in the absence of arachidonic acid indicates that L. donovani may possess a PGE2-degrading enzyme(s) that enables it to metabolise this prostanoid, since incubation of PGE2 with lysates of this parasite led to the detection of unidentified degradationproducts by HPLC (unpublished observations). Ongoing work in our laboratory will probably clarify the nature of those products and of the enzyme systems involved. 3.2. cDNA cloning and expression, and purification and characterisation of L. major PGF2a synthase Using the cDNA sequence of T. brucei PGF2a synthase (EMBL/GenBank/DDBJ, accession no. AB034727), we searched for homologous sequences in the database and identified a 1,423-bp cDNA sequence (GenBank/EMBL, accession no. J04483) of the developmentally regulated P100/11E gene from L. major (Samaras and Spithill, 1989) that exhibited the highest homology (61%) with TbPGFS. We then amplified a 855-bp fragment by PCR using P100/11E cDNA gene-specific primers and the cDNA product from reverse transcription of the L. major total RNA. The 855-bp fragment encoded an ORF that predicted a protein composed of 284 amino acid residues with a calculated Mr of 32,000. Cloning and sequencing of the 855-bp fragment revealed a sequence identical to that of the P100/11E ORF, indicating that the correct fragment had been isolated. To express heterologously the L. major PGF2a synthase,
we amplified the 855-bp ORF carrying EcoRI and XhoI restriction sites by PCR and cloned it into a pGEX expression vector. The recombinant protein was produced as a GST fusion protein in the cytosolic fraction of E. coli after IPTG induction and exhibited an Mr of ,60,000 on SDS-PAGE (Fig. 2a, lane 1). The fusion protein was purified by GSH-Sepharose affinity chromatography. Following cleavage of the fusion protein (Fig. 2a, lane 2) and subsequent purification by ion-exchange chromatography, we obtained L. major PGF2a synthase with an Mr of ,34,000 (Fig. 2a, lane 3). Lysates from crude extracts of E. coli BL21 expressing pGEX-L. major PGF2a synthase, but not those from the host cells alone or host cells containing only the vector (data not shown), reduced 9,11-endoperoxide PGH2 to PGF2a in the presence of NADPH. These results indicate that the PGF2a synthase activity depended on the L. major protein and not on any contaminating activity from the host cells or the vector. To show that the recombinant L. major PGF2a synthase was responsible for de novo synthesis of PGF2a, we boiled the pure protein at 1008C for 5 min and then incubated it with PGH2. The ability of recombinant L. major PGF2a synthase to synthesise PGF2a from PGH2 was abolished by the heat treatment (Fig. 2b, lane B), providing further evidence for the catalytic power of the leishmanial protein. We then determined some kinetic parameters of the recombinant pure protein. A comparison of the kinetic parameters between L. major PGF2a synthase and T. brucei PGF2a synthase (Kubata et al., 2000) is shown in Table 1. As compared with T. brucei PGF2a synthase, L. major PGF2a synthase showed higher Km values for PGH2 (almost 10-fold), 9,10-phenanthrenequinone (almost 30-fold), and p-nitrobenzaldehyde (almost 15-fold), while exhibiting a lower value for NADPH (almost twofold). Subsequently, L. major PGF2a synthase Vmax values were almost one order of magnitude lower than those of T. brucei PGF2a synthase. Leishmania major PGF2a synthase had also a broad range of temperature (30–458C) and pH (6–8) optima (data not shown). To further gain insight into the inhibitory effects of 9,11-endoperoxide PGH2 analogues on L. major PGF2a synthase catalytic power, we investigated the interference of U-44069 and U-46619 on the reduction of PGH2 by L. major PGF2a synthase. U-44069 and U-46619 are PGH2 receptor agonists with chemical structures similar to
1698
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
showed that U-44069 had no effect on the reduction of PGH2 by L. major PGF2a synthase as compared with the dose-dependent partial inactivation of PGH2 reduction produced by U-46619 (Fig. 2c). We have shown the direct evidence for the implication of L. major PGF2a synthase in the synthesis of PGF2a that is a biologically active molecule. The findings that Leishmania protozoa from the Old World produce prostaglandins and that they have an enzyme that catalyses PGF2a formation raise questions about the biology of Leishmania parasites and the interaction of these organisms with their mammalian host. Prostaglandins are potent mediators of physiological and pathological responses (Samuelsson, 1979–1980; Mathe et al., 1977; Oliw et al., 1983; Glew, 1992; Dubois et al., 1998; Hayaishi, 2000), some of which are observed in leishmaniases. It has also been reported that PGE2 and PGF2a may play a signal-coupling role during phagocytosis in the protozoan parasite Amoeba proteus, since they elicit vacuole formation (Prusch et al., 1989) whereas PGD2 was shown to play an important role in T. brucei cell growth regulation by inducing programmed cell death (unpublished observations). However, whether or not Leishmania-derived prostaglandins play similar roles remains to be established. In the present study, we have at least identified an enzyme that catalyses PGF2a synthesis in Leishmania parasite. Although the P100/11E gene of this enzyme is rather more abundantly expressed in promastigotes than in amastigotes (Samaras and Spithill, 1989), neither its function nor its specific reductive reaction or substrate had previously been identified. Fig. 3. Expression of PGF2a synthase in Leishmania. (A) Reverse transcriptase polymerase chain reaction (RT-PCR) analysis of PGF2a synthase gene expression in Leishmania major (lane 1), Leishmania donovani (lane 2), Leishmania tropica (lane 3), Leishmania mexicana (lane 4), and Leishmania amazonensis (lane 5). Total RNA (1 mg) from logarithmic-phase promastigote cells of each species was extracted as described under Section 2 and used as a template for reverse transcription. The resulting cDNA product (1 ml) was then used in PCR amplification of the open reading frames of either PGF2a synthase (upper panel) or a-tubulin (lower panel) by using the L. major PGF2a synthase gene-specific sense and antisense primers described in Section 2 or a-tubulin gene-specific sense primer 5 0 CTGACGGAGTTCCAGACGAACCT-3 0 and antisense primer 5 0 -TTAGTACTCCTCGACGTCCTCCT-3 0 . The RT-PCR products were analysed on a 1.5% agarose gel. (B,C) Western blot analysis of PGF2a synthase in L. major promastigote (lanes B-1 and C-a), L. donovani (lane 2), L. tropica (lane 3), L. mexicana (lane 4), and L. amazonensis (lane 5). Lanes C-b and C-c indicate L. major membrane and cytosolic fractions, respectively. Total protein (100 ng) from lysates of each Leishmania species and from L. major membrane or cytosolic fractions was resolved by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and analysed by Western blotting as described under Section 2.
that of PGH2 with the exception that the oxygen atoms at C11 in U-44069 and C-9 in U-46619 are substituted with a CH2 group. Pre-incubation of L. major PGF2a synthase with various concentrations of either U-44069 or U-46619 followed by the incubation of the mixtures with PGH2
3.3. Distribution and cellular localisation of L. major PGF2a synthase in Leishmania The distribution of PGF2a synthase was investigated by detecting both the gene from the total RNAs and the protein in the lysates of L. major, L. donovani, L. tropica, L. mexicana, and L. amazonensis. An 855-bp fragment corresponding to the PGF2a synthase ORF (Fig. 3A, upper panel) was amplified and detected from the total RNAs of Old World species L. major (Fig. 3A, lane 1), L. donovani (Fig. 3A, lane 2), and L. tropica (Fig. 3A, lane 3) but not from those of New World species L. mexicana and L. amazonensis (Fig. 3A, lanes 4 and 5, respectively), whereas a 603-bp fragment of a-tubulin ORF (Fig. 3A, lower panel) was identified from RNAs of all Leishmania species. Sequence analysis of these PCR products revealed an almost 99% identity between L. major and L. donovani, and 100% identity between L. donovani and L. tropica (data not shown). The presence of PGF2a synthase in other subspecies of L. donovani, which are believed to occur in the Old (L. donovani, L. infantum) and New World (L. chagasi) species, may imply a role for this enzyme in vector competence (Sacks and Kamhawi, 2001). Interestingly, rabbit anti-T. brucei PGF2a synthase polyclonal antibody detected 34-kDa immunoreactive signals corresponding to L. major (Fig. 3B, lane 1), L. dono-
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
Fig. 4. Cellular localisation of Leishmania major PGF2a synthase by immunofluorescence. Leishmania major promastigotes were recovered from asynchronous cultures at the logarithmic-phase. Staining of DNA with 4 0 ,6-diamidino-2-phenylindole (DAPI) shows the location of the nucleus (blue) in the promastigote. Leishmania major PGF2a synthase is visualised by specific Trypanosoma brucei PGF2a synthase polyclonal antibody and anti-rabbit IgG-FITC (green). (A) Phase contrast, (B) DAPI and T. brucei PGF2a synthase polyclonal antibody double staining. Bar, 10 mm.
vani (Fig. 3B, lane 2), and L. tropica (Fig. 3B, lane 3). No signal was detected from lysates of L. mexicana and L. amazonensis (Fig. 3B, lanes 4 and 5, respectively). Moreover, immunoblot analysis of L. major whole lysate (Fig. 3C, lane a) and subcellular fractions (Fig. 3C, lanes b and c) for PGF2a synthase showed L. major PGF2a synthase to be localised in the cytosol (Fig. 3C, lane c). This localisation is in agreement with our previous finding that the orthologue T. brucei PGF2a synthase enzyme was also localised in the cytosol (Kubata et al., 2000). To further confirm this localisation, we stained whole promastigote cells of L. major. The immunofluorescence of fixed and permeabilised promastigotes obtained with FITC-conjugated anti-T. brucei PGF2a synthase IgG showed an abundance of stain localised in the reticular material throughout the cytoplasm (Fig. 4B), suggesting that L. major PGF2a synthase is a cytosolic protein. Pre-immune rabbit serum did not stain the parasites (data not shown). In the present study we have shown that Leishmania species convert arachidonic acid to PGD2, PGE2 and
1699
PGF2a. Furthermore we have characterised a recombinant PGF2a from L. major and demonstrated its distribution in Old World Leishmania species. However, this enzyme was not detected in New World Leishmania species although they do produce PGF2a (unpublished observations). It is most likely that in L. mexicana and L. amazonensis, another type of protein may be responsible for PGF2a synthesis since studies of prostaglandin synthesis in Trypanosoma from our laboratory found that the protein responsible for PGF2a synthesis in T. brucei (Kubata et al., 2000) is different from that for PGF2a synthesis in T. cruzi (unpublished observations). Alternatively, probably L. mexicana and L. amazonensis PGF2a synthase gene and protein were simply in insufficient amounts that did not allow their detection in the present study. Midgut environment conditions in sand fly species may have evolutionarily driven the expression of some Leishmania surface molecules such as lipophosphoglycans. Leishmania species-specific polymorphisms in lipophosphoglycans have been reported to control differences in the binding capacities for the midgut of different vectors, and the extent of binding predicts in which parasite/ sand fly combination the development of transmissible infections can occur (Sacks and Kamhawi, 2001; Pimenta et al., 1994; Sacks et al., 2000). Whether or not PGF2a synthase and/or PGF2a play some role in the vector competence of Leishmania infections remain to be investigated. Why PGF2a synthesis segregates between Old and New World parasites as well as why Leishmania produces prostaglandins is not clear to us at this time. It is, however, worth mentioning that the identification of prostaglandin-producing enzymes provides the basis for further molecular investigation of the physiological roles of these lipid mediators and that ongoing gene knockout experiments in our laboratory will probably elucidate the biological relevance of prostanoid production in parasitic protozoa.
Acknowledgements We are grateful to Drs Hiroyuki Ishikawa of the Research Institute and International Medical Center of Japan and Yumiko Saito-Nakano and Osamu Fujita of the National Institute of Infectious Diseases, Tokyo, Japan, for their technical help. This work was supported in part by grants from programs Grants-in-Aid for Scientific Research (No. 14370087 to B.K.K.) and Grants-in-Aid for Scientific Research in Priority areas (No. 14021130 to B.K.K. and Y.U.) of the Ministry of Education, Culture, Sport, Science and Technology, Japan, and by a fellowship from the Takeda Science Foundation to C.J.M. It was also supported by grants from the following sources: the Deutsche Forschungsgesellschaft to M.D.; the Japan Science and Technology Corporation to B.K.K., M.L. (No. 199041) and to Y.U.; the Ministry of Education, Science, Culture, and Sports of Japan to Y.U. (Nos. 11877047 and 12558078), and the Japan Health Sciences Foundation (No. SA14706)
1700
Z. Kabututu et al. / International Journal for Parasitology 32 (2002) 1693–1700
and the Precursory Research for Embryonic Science and Technology (PRESTO) from the Japan Science and Technology Corporation to T.N. References Bailey, J.M., Bryant, R.W., Low, C.E., Pupillo, M.B., Vanderhoek, J.Y., 1982. Regulation of T-lymphocyte mitogenesis by the leucocyte product 15-hydroxyeicosatetraenoic acid (15-HETE). Cell. Immunol. 67, 112–20. Chattopadhayay, R., Kaur, S., Gangluy, N.K., Mahajan, R.C., 1996. Antigenic differences between axenic amastigotes and promastigotes of Leishmania donovani. Indian J. Med. Res. 104, 349–54. Dubois, R.N., Abramson, S.B., Crofford, L., Gupta, R.A., Simon, L.S., Van de Putte, L.B., Lipsky, P.E., 1998. Cyclooxygenase in biology and disease. FASEB J. 12, 1063–73. Ellis, K.J., Morrison, J.F., 1982. Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol. 87, 405–26. Farrell, J.P., Kirkpatrick, C.E., 1987. Experimental cutaneous leishmaniasis. II. A possible role for prostaglandins in exacerbation of disease in Leishmania major-infected BALB/c mice. J. Immunol. 138, 902–7. Gemsa, D., Seitz, M., Menzel, J., Grimm, W., Kramer, W., Till, G., 1979. Modulation of phagocytosis-induced prostaglandin release from macrophages. Adv. Exp. Med. Biol. 114, 421–6. Glew, R.H., 1992. Prostaglandins and thromboxanes. In: Devlin, T.M. (Ed.). Texbook of Biochemistry with Clinical Correlations, Wiley– Liss, New York, NY, pp. 461–6. Goetzl, E.J., 1981. Selective feedback of the 5-lipoxygenation of arachidonic acid in human T-lymphocytes. Biochem. Biophys. Res. Commun. 101, 344–50. Gutierrez, Y., Maksem, J.A., Reiner, N.E., 1984. Pathological changes in murine leishmaniasis (Leishmania donovani) with specific reference to the dynamics of granuloma formation in the liver. Am. J. Pathol. 114, 222–30. Handman, E., Jarvis, H.M., Michell, G.F., 1984. Leishmania major identification of stage-specific antigens and antigens shared by promastigotes and amastigotes. Parasite Immunol. 6, 223–33. Handman, E., Osborn, A.H., Symons, F.M., Van Driel, R., Cappai, R., 1995. The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle. Mol. Biochem. Parasitol. 74, 189–200. Hayaishi, O., 2000. Molecular mechanisms of sleep–wake regulation: a role of prostaglandin D2. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 355, 275– 80. Humes, J.L., Bonney, R.J., Pelus, L., Dahlgren, M.E., Sadowski, S.J., Kuehl, F.A., Davis, P., 1977. Macrophages synthesize and release prostaglandins in response to inflammatory stimuli. Nature 269, 149–51. Ibrahim, M.E., Lambson, B., Yousif, A.O., Deifalla, N.S., Alnaiem, D.A., Ismail, A., Yousif, H., Ghalib, H.W., Khalil, E.A., Kadaro, A., Barker, D.C., El Hassan, A.M., 1999. Kala-azar in a high transmission focus: an ethnic and geographic dimension. Am. J. Trop. Med. Hyg. 61, 941–4. Jensen, A.T., Kemp, K., Theander, T.G., Handman, E., 2001. Cloning, expression and antigenicity of the L. donovani reductase. APMIS 109, 461–8. Kidane, G.Z., Samaras, N., Spithill, T.W., 1989. Cloning of developmen-
tally regulated genes from Leishmania major and expression following heat induction. J. Biol. Chem. 264, 4244–50. Kubata, B.K., Eguchi, N., Urade, Y., Yamashita, K., Mitamura, T., Tai, K., Hayaishi, O., Horii, T., 1998. Plasmodium falciparum produces substances that are pyrogenic, somnogenic, and immunosuppressive in humans. J. Exp. Med. 188, 1197–202. Kubata, B.K., Duszenko, M., Kabututu, Z., Rawer, M., Szallies, A., Fujimori, K., Inui, T., Nozaki, T., Yamashita, K., Horii, T., Urade, Y., Hayaishi, O., 2000. Identification of a novel prostaglandin F2a synthase in Trypanosoma brucei. J. Exp. Med. 192, 1327–37. Lamontagne, J., Papadopoulou, B., 1999. Developmental regulation of spliced leader RNA gene in Leishmania donovani amastigotes is mediated by specific polyadenylation. J. Biol. Chem. 274, 6602–9. Mathe, A.A., Hedqvist, P., Strandberg, K., Leslie, C.A., 1977. Aspects of prostaglandin function in the lung (second of two parts). N. Engl. J. Med. 296, 910–4. Morley, J., 1981. Role of prostaglandins secreted by macrophages in the inflammatory process. Lymphokines 4, 377–83. Murray, H.W., Masur, H., Keithly, J.S., 1982. Cell-mediated immune response in experimental visceral leishmaniasis. I. Correlation between resistance to Leishmania donovani and lymphokine-generating capacity. J. Immunol. 129, 344–50. Oliw, E., Granstrom, E., Anggard, E., 1983. The prostaglandin and essential fatty acids. In: Pace-Asciak, C., Granstrom, E. (Eds.). Prostaglandins and Related Substances, Elsevier, Amsterdam, pp. 11–19. Payan, D.G., Goetzl, E.J., 1983. Specific suppression of human T-lymphocyte function by leukotriene B4. J. Immunol. 131, 551–3. Pimenta, P.F.P., Saraiva, E.M.B., Rowton, E., Modi, G.B., Garraway, L.A., Beverley, S.M., Turco, S.J., Sacks, D.L., 1994. Evidence that the vectorial competence of phlebotomine sand flies for different species of Leishmania is controlled by structural polymorphisms in the surface lipophosphoglycan. Proc. Natl Acad. Sci. USA 91, 9155–9. Prusch, R.D., Goette, S.M., Haberman, P., 1989. Prostaglandins may play a signal-coupling role during phagocytosis in Amoeba proteus. Cell Tissue Res. 255, 553–7. Reiner, N.E., 1982. Host-parasite relationship in murine leishmaniasis donovani: dynamics of pathophysiologic and immunologic changes. Infect. Immun. 38, 1223–30. Reiner, N.E., Finke, J.H., 1983. Interleukin 2 deficiency in murine leishmaniasis donovani and its relationship to depressed spleen cell responses to phytohemagglutinin. J. Immunol. 131, 1487–91. Roberts, L.J., Foote, S.J., Handman, E., 2000. A new standard for the assessment of disease progression in murine cutaneous leishmaniasis. Parasite Immunol. 22, 231–7. Sacks, D.L., Kamhawi, S., 2001. Molecular aspects of parasite-vector and vector-host interactions in leishmaniasis. Annu. Rev. Microbiol. 55, 453–83. Sacks, D.L., Modi, G., Rowton, E., Spath, G., Epstein, L., Turco, S., Beverley, S.M., 2000. The role of phosphoglycans in Leishmania-sand fly interactions. Proc. Natl Acad. Sci. USA 97, 406–11. Samaras, N., Spithill, T.W., 1989. The developmentally regulated P100/ 11E gene of Leishmania major shows homology to a superfamily of reductase genes. J. Biol. Chem. 264, 4251–4. Samuelsson, B., 1979–1980. Prostaglandins, thromboxans, and leukotrienes: formation and biological roles. Harvey Lect. 75, 1–40. World Health Organization, 2001. Leishmaniasis: the disease and its impact. WHO homepage available at http://www.who.int/