Identification of a Novel Prostaglandin F_2α Synthase in Trypanosoma brucei

Prostaglandins (PGs) of the 2 series are synthesized by the oxygenation of AA. In this pathway, AA is converted to PGH₂ by cyclooxygenases (COX-1 and -2). Subsequently, the resulting PGH₂ is converted in vivo and in vitro to various arachidonate metabolites, such as PGD₂, PGE₂, and PGF_2α (Fig. 1) by the action of their respective synthases 1.

PGs are actively produced and widely distributed in various tissues of mammals, where they are potent mediators of a large variety of physiological and pathological responses including regulation of vascular tone, miscarriage, ovarian dysfunction, infertility, sleepiness, inflammation, bronchoconstriction, pain, fever, immunosuppression, and other symptoms 2,3,4,5,6,7. However, PG production is not restricted to mammals. Studies have shown the production of PGs by parasites such as cestodes, trematodes, nematodes, and protozoa 8,9,10,11,12,13 in response to the addition of AA or calcium ionophore. In Schistosoma mansoni, PG production has been reported to be associated with the transformation of cercaria into schistosomules 14, whereas in the protozoan parasite Amoeba proteus, PGs may play a signal-coupling role during phagocytosis, because they elicit vacuole formation 15. PGF_2α and PGD₂ are also found to be consistently elevated in the plasma of animals experimentally infected with Trypanosoma congolense 16 and in the cerebrospinal fluid of humans with chronic infection of T. brucei gambiense 17, respectively. These results suggest an upregulation of PG production during African trypanosomiasis. However, the molecular basis for this upregulation has not yet been elucidated. In addition, despite the obvious importance of PGs in the pathogenesis of parasitic infections and of PG production in parasitic protozoa, little is known about the molecular mechanisms of PG production in these organisms. The results presented here identify for the first time a T. brucei protein that exhibits a PGF synthase activity capable of specifically converting PGH₂ to PGF_2α.

The nucleotide sequence data reported in this paper is available from EMBL/GenBank/DDBJ under accession number AB034727.

Bloodstream forms of T. brucei clone MITat 1.4 were isolated from infected rats as described previously 18. Trypanosome cells were cultivated in the presence or absence of 66 μM AA in a modified minimum essential medium supplemented with 10% FCS. The culture was incubated in a 5% CO₂ atmosphere at 37°C as described previously 18,19,20. Cells were harvested from the logarithmic growth phase or from the late stationary phase by centrifugation (1,500 g) at 4°C for 5 min. Cells were lysed and used as enzyme sources. After centrifugation, the supernatants were used for the determination of PGs secreted into the culture medium by live trypanosomes.

Lysates from logarithmic growth phase T. brucei (2.5 × 10⁸ cells) isolated from infected rats and from the logarithmic growth and late stationary phase (8.3 and 5.4 × 10⁷ cells, respectively) organisms isolated from bloodstream-form cultures were prepared by hypotonic lysis using double-distilled water containing a cocktail of reversible and irreversible inhibitors (one tablet in 25 ml) of pancreas extract, pronase, thermolysin, chemotrypsin, trypsin, and papain (Complete™; Roche Diagnostics). For PG production from AA, we used the reaction mixture described by Ujihara et al. 21 with the following modifications: 100 mM sodium phosphate, pH 7.0, 2 μM hematin, 5 mM tryptophan, 1 mM AA, and 300 μl of the respective T. brucei lysates in a final volume of 500 μl. The mixture was incubated at 37°C for 30 min, and then the reaction was stopped by addition of 100 μl of 1 M HCl and 6 vol of cold ethyl acetate.

For PGF_2α synthesis from PGH₂, a standard reaction mixture that contained 100 mM sodium phosphate, pH 7.0, 20 μM NADP⁺, 100 μM glucose-6-phosphate, 1 U of glucose-6-phosphate dehydrogenase, and a diluted amount of enzyme in a final volume of 100 μl was used. The reaction was started by the addition of 1 μl of 500 μM 1-[¹⁴C]PGH₂ (2.04 Gbq/mmol) and was carried out at 37°C for 2 min and terminated by the addition of 250 μl of a stop solution (30:4:1 vol/vol/vol diethyl ether/methanol/2 M citric acid). To test for the nonenzymatic formation of PGF_2α, we incubated the reaction mixture containing all of the components in the absence of the enzyme. The organic phase (50 μl) was applied to 20 × 20-cm silica gel plates (Merck) at 4°C, and the plates were developed with a solvent system of 90:2:1 vol/vol/vol diethyl ether/methanol/acetic acid at −20°C. The radioactivity on the plates was monitored and analyzed by a Fluorescent Imaging Analyzer FLA 2000 and Mac Bas V2.5 software (Fuji Photo Film Co.). For assessment of substrate specificity, the reaction mixtures consisted of 100 mM sodium phosphate, pH 7.0, the purified recombinant enzyme, 100 μM NADPH, and substrates at various concentrations, as indicated in Table, in a total volume of 500 μl. Reactions were initiated by the addition of substrate, and the decrease in absorbance at 340 nm was monitored at 37°C. Blanks without enzyme or without substrate were included. The 9,10-phenanthrenequinone reductase activity was chosen to represent 100% activity. For 1-[¹⁴C]-PGD₂ and PGE₂ production, 40 μM 1-[¹⁴C]PGH₂ was incubated with either 250 μg of recombinant hematopoietic PGDS 22 or 500 μg of recombinant PGES 23. After incubation at 25°C for 5 min, the resulting PGs were extracted three times with cold ethyl acetate, dried at a low temperature under vacuum, and used as substrates for the T. brucei PGF_2α synthase (TbPGFS) specificity study.

After addition of [³H]PGD₂, [³H]PGE₂, and [³H]PGF_2α (60 Bq each per assay; NEN Life Science Products), used as tracers to determine the recovery during extractions, PGs recovered from the trypanosome culture medium (10 ml) and those from incubation of parasite lysates (300 μl) were extracted and separated by HPLC as described previously 9,21. The resulting PGD₂, PGE₂, and PGF_2α were quantified by enzyme immunoassay (EIA) with their respective EIA kits (Cayman Chemical).

Gas chromatography–selected ion monitoring (GC-SIM) analyses were run on a Hitachi M-80B double-focusing mass spectrometer equipped with a Van den Berg's solventless injector and a fused silica capillary column (Ultra no. 1; 25 m length, 0.32 mm internal diameter, 280°C column temperature). PGs recovered from culture media and those formed by the incubation of parasite lysates with AA were fractionated by HPLC and converted into their corresponding methyl ester (ME)-dimethylisopropylsilyl (DMiPS) ether or ME-methoxime (MO)-DMiPS ether derivatives according to the method described previously 24.

Bloodstream-form T. brucei cells (5 × 10¹¹) were isolated from infected rats and lysed by hypotonic lysis. Soluble proteins, obtained by differential centrifugation at 3,000 g for 15 min and then at 100,000 g for 1 h at 4°C, were fractionated with ammonium sulfate. The active fraction (40–100% saturation), resuspended in 0.1 M sodium phosphate, pH 7.0, was loaded onto a Hiload 16/60 Superdex 75 pg gel filtration column (Amersham Pharmacia Biotech) and eluted with 20 mM NaCl in 20 mM Tris/Cl, pH 8.0. Active fractions were pooled, dialyzed against 20 mM sodium phosphate, pH 7.0, and concentrated by use of Centricon centrifugal filters with a molecular mass cut-off of 3,000 daltons (Millipore). The concentrated active fraction was further loaded onto a Resource PHE Hydrophobic interaction column (Amersham Pharmacia Biotech), which had been equilibrated with 2 M ammonium sulfate in 20 mM sodium phosphate, pH 7.0, and eluted with a decreasing linear gradient of 2–0 M ammonium sulfate in the same buffer containing 1% (vol/vol) Tween 20. The active peak was dialyzed against 20 mM Tris/Cl, pH 8.0, and applied to a Mono Q HR 10/10 ion exchange column (Amersham Pharmacia Biotech) equilibrated with the same buffer. The elution was carried out with an increasing linear gradient of 0–400 mM NaCl in the same buffer. The active fraction was further purified by gel filtration on a Hiload 16/60 Superdex 200 pg column (Amersham Pharmacia Biotech).

Protein concentration was determined by use of bicinchoninic acid reagent (Pierce Chemical Co.) with BSA as a standard following the manufacturer's protocol. The purity of the protein was assessed by SDS-PAGE on 14% (wt/vol) gels, and the gels were stained with silver (Daiichi Pure Chemicals), sypro orange (Bio-Rad Laboratories), or Coomassie Brilliant Blue (Daiichi Pure Chemicals). Pure protein (220 μg) was subjected to in-gel digestion with lysyl-endopeptidase for the determination of the internal amino acid sequences according to Rosenfeld et al. 25.

Total RNA was extracted from bloodstream-form T. brucei cells (4 × 10⁸) by sour phenol, pH 4.5, saturated with TE buffer. First-strand cDNA was synthesized by avian myeloblastosis virus reverse transcriptase after annealing 1 μg of T. brucei total RNA with Oligo dT- Adaptor Primer (Takara Shuzo). For 3′ rapid amplification of cDNA ends (RACE), the first PCR amplification was carried out with gene-specific primers, i.e., sense primer 5′-AAGTTTATCGACACATGGAAGGCG-3′ and antisense primer M13 M4 (5′-GTTTTCCCAGTCACGAC-3′) and the first-strand cDNAs as the template by the following program: after initial denaturation at 95°C for 5 min, the PCR reaction proceeded at 94°C for 20 s, 55°C for 20 s, and 74°C for 30 s for 30 cycles. PCR products from the first PCR amplification were used as the templates for nested PCR amplification with other gene-specific primers, i.e., sense primer 5′-CTGTACGCCGATAAGAAGGTGCGCGCC-3′ and antisense primer M13 M4. For 5′ RACE, the resulting PCR product was amplified with a 23-mer sense primer from T. brucei spliced leader sequence, 5′-CGCTATTATTAGAACAGTTTCTG-3′ 26 and a gene-specific antisense primer, 5′-GTGTTACTACTCGAACACCGCA-3′. The amplified fragments were cloned into pGEM-T Easy vector (Promega). DNA sequences were determined from both strands with a SequiTherm cycle sequencing kit (Epicentre Technologies) and a LI-COR automated DNA sequencer (model 4000; LI-COR Inc.).

The sequences of 17 members of the aldo-keto reductase (AKR) superfamily were retrieved from public databases. The amino acid sequences were aligned with the TbPGFS sequence by using the CLUSTAL W program 27. A neighbor-joining method 28 of protein phylogeny was used to infer phylogenetic relationships among the sequences.

The coding region of TbPGFS cDNA was amplified by PCR using sense primer 5′-CGGAATTCATGGCTCTCACTCAATCCCTAA-3′ and antisense primer 5′-CGGTCGACCAGTGTTACTACTCGAACACCG-3′, which carried EcoRI and SalI restriction sites at their respective 5′ ends. The amplified fragments containing the entire open reading frame were digested with EcoRI and SalI and then cloned into the corresponding sites of pMAL-c2 vector (New England Biolabs, Inc.). The resultant expression vector was used for transformation of Escherichia coli DH5α. Transformed cells were cultured for 6–7 h in the presence of 0.5–1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and then harvested and sonicated. The recombinant protein in the soluble fraction was purified according to the manufacturer's protocol (New England Biolabs, Inc.).

Total RNA from 10⁷ cells of each growth stage was isolated as described above, separated on 1.2% (wt/vol) agarose–2% formaldehyde gels, and blotted onto a Hybond N⁺ membrane (Amersham Pharmacia Biotech). RNA was crosslinked by UV radiation for 6 min, and the membrane was then dried for 2 h at 80°C. After prehybridization, hybridization was performed for 3 h in 5× SSPE (saline sodium phosphate EDTA), 5× Denhardt's solution, 0.5% (wt/vol) SDS, and 0.5 mg sonicated salmon sperm DNA using the DIG system (Roche Diagnostics) according to the manufacturer's protocol. After high-stringency washing, the blot was pretreated with 0.1 M maleic acid, 0.15 M NaCl, pH 7.5, containing 1% blocking reagent before the antidigoxigenin–AP Fab fragment was added and was then developed with CDP-Star (all reagents from Roche Diagnostics). Chemiluminescence was detected by use of BioMax Light-1 film (Sigma-Aldrich).

To assess possible PG production and secretion by live trypanosomes, we cultured T. brucei in vitro in a chemically defined medium supplemented with 10% FCS. PGD₂, PGE₂, and PGF_2α secreted into the culture medium were quantified by EIA. As shown in Fig. 2 A, live trypanosomes secreted small amounts of PGs: 75 ± 10 pg/ml for PGD₂, 100 ± 9.4 pg/ml for PGE₂, and 156 ± 20 pg/ml for PGF_2α (n = 3). As with other parasitic protozoa, T. brucei does not synthesize AA de novo from acetate 29, but it does take up this fatty acid from its environment and does incorporate it into its total and separate classes of lipids 30,31. The presence of phospholipase A₂ 32,33, an enzyme that catalyzes the release of AA from phospholipids, in T. brucei indicates the existence of a free intracellular AA source in this parasitic protozoan. Thus, the small amounts of PGs, secreted by T. brucei in the absence of exogenously added AA, were most likely produced from the intracellular AA and from the trace amount of AA present in the serum used for the medium preparation. We then grew trypanosomes in the presence of 66 μM AA exogenously added and which had no effect on cell growth (our unpublished observations). PG accumulation in the media increased to 875 ± 16 pg/ml for PGD₂, 447 ± 11 pg/ml for PGE₂, and 211 ± 6 pg/ml for PGF_2α (n = 3), suggesting that, in vivo, T. brucei would rely on AA uptake from its environment (host tissues) to release significant amounts of PGs.

When the cell lysates were prepared from T. brucei grown in vitro in the presence or absence of AA and incubated with 1 mM AA, PGs were actively produced in a time- and dose-dependent manner (data not shown). PGF_2α was the major prostanoid synthesized in both cases, followed by PGE₂ and PGD₂ (Fig. 2 B). The addition of AA during the cultivation of trypanosomes increased PG formation by the lysates ∼12-fold for PGD₂, 11-fold for PGE₂, and 5-fold for PGF_2α.

The molecular masses and the characteristic fragment ions of PGs produced by trypanosomes were determined by GC-SIM analysis of the DMiPS ether derivatives. All of the materials eluted from HPLC had exactly the expected molecular masses: m/z 595 for PGD₂ (Fig. 2 C) and PGE₂ (Fig. 2 D) and m/z 668 for PGF_2α (Fig. 2 E). Relative ion intensity ratios of individual characteristic fragments of PG derivatives in the eluates were identical to those of authentic standards. Quantification by gas chromatography–mass spectrometry analysis (GC-MS) of PGs extracted from culture medium or trypanosome lysates incubated with AA gave values of the same order of magnitude as those determined by EIA, and finally PGs derived from trypanosomes coeluted with authentic standards on HPLC and showed the same titration curves as authentic PGs by EIA (data not shown).

To determine the nature of the protozoan catalyst and to evaluate whether or not the catalyst shares a kinship with COX-1 and COX-2 from mammalian tissues, we investigated the effects of heat and nonsteroidal antiinflammatory drugs (NSAIDs) on PG production by T. brucei lysates. Heating the lysates for 5 min at 100°C before incubation with 1 mM AA reduced the levels of PGD₂, PGE₂, and PGF_2α synthesis to <10% as compared with control values, whereas the addition of 3 mM aspirin or 42 μM indomethacin to the reaction mixture had no inhibitory effects on PG formation (Fig. 2 F). The results reported here show that PG-producing activities in T. brucei were heat sensitive and unaffected by aspirin or indomethacin. As NSAIDs are well established inhibitors of mammalian COX-1 and COX-2 34, the trypanosomal enzyme system involved in PG synthesis from AA is markedly different from its mammalian counterpart. Future investigations in our laboratory will probably unravel the identity of the enzymes involved in PG formation in this organism.

Another approach to demonstrate de novo synthesis of PGs in T. brucei was to use 1-[¹⁴C]PGH₂ as a substrate. As an initial attempt to identify PG-producing enzymes in T. brucei, we prepared membrane and cytosolic fractions from bloodstream-form trypanosomes grown in rats and incubated them with 1-[¹⁴C]PGH₂ in the presence or absence of various cofactors. Two major PG synthase activities were identified (Fig. 3A and Fig. B). Membrane proteins isomerized PGH₂ to PGD₂ (Fig. 3 A, lane 2). No PGD₂ was formed during incubation of PGH₂ with heat-inactivated membrane fractions (data not shown) or in the absence of membrane proteins, though a small nonenzymatic production of PGE₂ was observed under the same conditions (Fig. 3 A, lane 1).

On the other hand, in the presence of a NADPH-generating system, cytosolic proteins (Fig. 3 B, lane 2), but not heat-inactivated ones (Fig. 3 B, lane 3), synthesized PGF_2α from PGH₂. PGH₂ was converted nonenzymatically to PGE₂ but not to PGF_2α (Fig. 3 B, lane 1). Also, no PGF_2α formation was observed in the absence of the NADPH-generating system or in the presence of 5 μM of other cofactors such as dithiothreitol and glutathione (data not shown). The profile of PG production from PGH₂ is consistent with that of PG synthesis from AA. Our data show that PGH₂ may be used as a substrate for PG synthesis by a protozoan parasite. Earlier studies on cell–cell interactions in the eicosanoid pathway have demonstrated the enzymatic cooperation between cell types 35. It has been shown that platelets can transfer PGH₂ to cultured endothelial cells 36 and vascular tissue 37 for efficient conversion to PGI₂ and thromboxane, respectively, and that platelets can utilize AA released by endothelial cells for lipoxygenase metabolism 36. Additional evidence has also shown that such an exchange may occur in vivo 38,39. However, it is not yet clear whether T. brucei utilizes PGH₂ as a predominant pathway for PGF_2α generation in host tissues, because the nature of the trypanosomal catalyst that converts AA to PGs is not known. The results presented here show also that PGD₂ and PGF_2α synthases from PGH₂ are catalyzed by trypanosomal enzymes. PGH D-isomerase (PGD synthase) activity is localized in the membrane proteins, a fact that made its purification difficult, and PGH F_2α reductase (PGF_2α synthase) activity is in soluble proteins. As PGF_2α was the major prostanoid synthesized by trypanosome lysates, we started with the cytosolic activity to purify TbPGFS.

We purified TbPGFS from the soluble fraction of early logarithmic growth phase bloodstream trypanosomes by ammonium sulfate (40–100% saturation) fractionation and sequential column chromatographies. TbPGFS activity was monitored by the reduction of PGH₂ in the presence of a NADPH-generating system. Table summarizes the results of a typical purification procedure. Active fractions from the final gel filtration (Hiload 16/60 Superdex 200 pg) were pooled and analyzed by SDS-PAGE. Upon silver staining, a single band with a molecular mass of 33 kD was detected (Fig. 3 C, lane 2). Finally, TbPGFS was purified 1,075-fold with a recovery of ∼13% and a specific activity of 860 nmol/min/mg of protein.

We subjected the purified protein to partial amino acid sequencing. Although the NH₂ terminus was blocked, we identified two amino acid sequences, LWNSDQGYES and NIAVTAWSPL (Fig. 4 A), of two internal peptide fragments from in-gel digestion of the purified TbPGFS with lysyl-endopeptidase.

From a database search using the two amino acid sequences mentioned above, we identified the corresponding 538-bp gene fragment in public databases (available from EMBL/GenBank/DDBJ under accession number AQ945329) that encoded the amino acid sequence NIAVTAWSPL. As this genomic clone was truncated at the NH₂ terminus, we isolated a 1,164-bp full-length cDNA for TbPGFS by the RACE technique with Oligo dT- Adaptor Primer, T. brucei–specific spliced leader, and gene-specific primers. The cDNA encoded an open reading frame of 828 bp, which predicted a protein composed of 276 amino acid residues (Fig. 4 A) with a calculated molecular mass of 30,991 daltons. The deduced amino acid sequence of the cDNA contained both peptide sequences, LWNSDQGYES and NIAVTAWSPL, as identified in the isolated protein.

Sequence analysis, database search, and alignment of the TbPGFS amino acid sequence (Fig. 4 A) revealed that the TbPGFS is a member of subfamily 5A of the AKR superfamily 40. The TbPGFS amino acid sequence showed 61% identity to that of a Leishmania major putative reductase and between 48 and 55% identity to bacterial protein sequences. In contrast, TbPGFS showed rather low identities to AKR from mammals and plants (39–40%). Furthermore, phylogenetic analysis (Fig. 4 B) confirmed that TbPGFS formed a clade together with L. major putative reductase and Bacillus subtilis putative morphine dehydrogenase. However, bootstrap proportions of the clades forming Trypanosomatid-Bacillus and Leishmania-Trypanosoma were relatively low (500–600), and thus the statistical support for any particular topology linking TbPGFS to a mammal, plant, yeast, or bacteria AKR cluster was rather weak. These results demonstrate that TbPGFS is different not only from other AKRs of mammals, including mammalian PGFS, but from AKRs of plants, yeast, and prokaryotes, indicating that it is a novel enzyme. The fact that this enzyme forms a distinct and distant clade from mammalian PGF synthases demonstrates that the process of PG formation existed early in the evolution of the animal kingdom.

TbPGFS protein was heterologously expressed in a pMAL-c2 expression system. The recombinant enzyme was produced as a fusion protein with maltose-binding protein (MBP) in the cytosolic fraction of E. coli after IPTG induction (Fig. 5 A, lane 3). Upon SDS-PAGE, the fusion protein, MBP–TbPGFS, exhibited a molecular mass of ∼76 kD (Fig. 5 A, lane 3). Crude proteins from E. coli expressing MBP–TbPGFS (Fig. 5 B, lane 4), but not lysates from E. coli host or from E. coli transformed with empty pMAL-c2 vector (Fig. 5 B, lanes 2 and 3), converted PGH₂ to PGF_2α only in the presence of a NADPH-generating system, indicating that the detected synthase activity depended on T. brucei protein and not on any contaminating activity from the host or from the vector. This is in good agreement with early results demonstrating de novo synthesis of PGF_2α from PGH₂ by the isolated enzyme (Fig. 3 B and 5 B), and these findings show that the cloned T. brucei cDNA encoded a bona fide PGF synthase and provide conclusive evidence for the existence of specific PG biosynthetic pathways in a pathogenic parasite. The fusion protein was purified by amylose affinity chromatography (Fig. 5 A, lane 4), gel filtration after factor Xa cleavage to separate TbPGFS from MBP (Fig. 5 A, lane 5), and ion exchange chromatography (Fig. 5 A, lane 6). The purified recombinant TbPGFS had an apparent molecular mass of 33 kD (Fig. 5 A, lane 6), identical to that of the wild-type protein (Fig. 3 C, lane 2).

Characterizing the purified recombinant enzyme and comparing its molecular properties with those of a number of mammalian PGF synthases purified and characterized from our laboratory 41,42,43,44,45, we found that TbPGFS exhibited a high and specific PGFS activity toward PGH₂ (2 μmol/min/mg), a broad range of temperatures (25–40°C), and pH 6,7,8,9 optima, the lowest K_m (1.3 μM) for PGH₂, and the highest k_cat value of 63 min⁻¹ (Table). As a member of the AKR superfamily, the purified TbPGFS showed AKR activity toward other substrates as well. It exhibited significantly high activity toward 9,10-phenanthrenequinone, p-nitrobenzaldehyde (the common substrates for this superfamily), and PGH₂ but remarkably weak activity toward progesterone, sodium glucuronate, or testosterone (Table). However, the AKR substrates did not compete with PGH₂ when added simultaneously in excess (Fig. 5 C), suggesting the existence of different catalytic sites for both types of substrate.

PGF_2α is synthesized either by the 9,11-endoperoxide reduction of PGH₂ or by the 9-keto reduction of PGE₂ (Fig. 1; reference 46). These reactions are catalyzed by PGH 9,11-endoperoxide reductase 41 and PGE₂ 9-keto reductase 46,47 in mammals. A third pathway, the 11-keto reduction of PGD₂ catalyzed by PGD 11-keto reductase, does not produce PGF_2α as initially thought. Instead, it leads to the formation of 9α,11β-PGF₂, a stereoisomer of PGF_2α 48. PGFSs described to date are enzymes that exhibit two activities on the same molecule, i.e., PGH 9,11-endoperoxide reductase activity and PGD 11-keto reductase activity 41,48. To elucidate the catalytic properties of TbPGFS, we incubated the recombinant TbPGFS with either 1-[¹⁴C]PGD₂ (Fig. 5 D, lanes 1 and 2) or 1-[¹⁴C]PGE₂ (Fig. 5 D, lanes 3 and 4). No 9α,11β-PGF₂ or PGF_2α formation was observed with either substrate. This finding demonstrates that TbPGFS is a rather specific PGH 9,11-endoperoxide reductase devoid of any PGD 11-keto reductase or PGE 9-keto reductase activity on the same molecule.

In the protozoan parasites Trypanosoma and Leishmania, several enzymes involved in citric acid cycle 49, respiratory chain activities 50,51,52,53,54, lysosomal function, or in the metabolism of protein, carbohydrate, and purine are developmentally regulated. To investigate PG production and the expression of TbPGFS mRNA during the T. brucei life cycle, we cultured trypanosome cells in the presence of 66 μM exogenous AA and harvested them at 19 h for early logarithmic phase or at 44 h for late stationary phase. We then used EIA to quantify PGs secreted by live trypanosomes into the media or produced by trypanosome lysates. Trypanosome cells from both the early logarithmic and late stationary phases released PGD₂, PGE₂, and PGF_2α (Fig. 6 A). No significant difference was observed between the levels of PGD₂ and PGE₂ secreted by the two growth stages. However, early logarithmic phase trypanosomes released PGF_2α fourfold more than those in late stationary phase. On the other hand, lysates from early logarithmic and late stationary phase trypanosomes produced different levels of all three PGs (Fig. 6 B). Early logarithmic phase trypanosomes produced a significantly high level of PGF_2α as compared with lysates from late stationary phase organisms (Fig. 6 B).

Northern blot analysis revealed that TbPGFS mRNA was abundantly expressed during the early logarithmic phase of trypanosomes grown in rats (Fig. 6 C) or in vitro (Fig. 6 C, 19 h) and was downregulated during the late stationary growth stage of these organisms (Fig. 6 C, 44 h). The expression profile, shown in Fig. 6 C, was found to be in good agreement with the TbPGFS activity in the respective cells (Fig. 6 D), which was higher during the early logarithmic growth stage of cells from rats (1.00 ± 0.07 nmol/min/mg) and during the early logarithmic growth stage of cells from in vitro culture (0.42 ± 0.02 nmol/min/mg) but lower during the late stationary growth stage of these organisms (0.04 ± 0.01 nmol/min/mg). Taken together, our data show that PG production as well as TbPGFS activity and TbPGFS mRNA expression are developmentally regulated.

Infection of mammals by African trypanosomes results in the release of high levels of PGs 16,17 that may, in part, be involved in the pathogenesis of the disease. It is generally believed and accepted that the PGs are produced by host cells after their stimulation with trypanosome products 55,56. We have shown that the parasite itself produces PGs and may contribute directly to the production of these mediators in mammals. However, the physiological relevance of PG production in T. brucei remains unknown. The findings reported here open up ways to investigate the role of TbPGFS in T. brucei replication and development as well as provide a new tool to study the role of parasite-derived PGs in the pathogenesis of African trypanosomiasis.

We thank E. Melnikow, D. Irikura, and T. Okada for technical assistance and Dr. H. Toh for assistance in database search.

This work was supported in part by a Science and Technology Agency fellowship (no. 298141) to B.K. Kubata and by grants from the Deutsche Forschungsgemeinschaft to M. Duszenko, the Japan Science and Technology Corporation to B.K. Kubata and Y. Urade, the Ministry of Education, Science, Culture, and Sports of Japan to Y. Urade (nos. 11877047 and 12558078) and O. Hayaishi (no. 12877044), and the Ministry of Health and Welfare of Japan to O. Hayaishi (no. 100107).