Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

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 F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) 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.     

Crystallization and preliminary X-ray crystallographic studies of Trypanosoma brucei prostaglandin F(2 alpha) synthase

Prostaglandin F(2 alpha) is a potent mediator of various physiological and pathological processes. Trypanosoma brucei prostaglandin F(2 alpha) synthase (TbPGFS) catalyzes the NADPH-dependent reduction of 9,11-endoperoxide PGH(2) to PGF(2 alpha), and could thus be involved in the elevation of the PGF(2 alpha) concentration during African trypanosomiasis. In the present report, the purification and crystallization of recombinant TbPGFS are described. The active recombinant enzyme was crystallized by the hanging-drop vapor-diffusion meth-od using ammonium sulfate as a precipitant. The crystal belonged to a tetragonal space group, P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters of a = b = 112.3 A, and c = 140.0 A. Native data up to 2.6 A resolution were collected from the crystal using our home facility.     

Kola acuminata proanthocyanidins: a class of anti-trypanosomal compounds effective against Trypanosoma brucei

Human African trypanosomiasis is undergoing an alarming rate of recrudescence in many parts of sub-Saharan Africa. Yet, there is no successful chemotherapy for the disease due to a limited number of useful drugs, side effects and drawbacks of the existing medication, as well as the development of drug resistance by the parasite. Here we describe a new lead anti-trypanosomal compound isolated from Kola acuminata (Makasu). We purified a proanthocyanidin by chromatographic procedures and confirmed its homogeneity and structure by Nuclear Magnetic Resonance and Matrix-Assisted Laser Desorption Ionisation Time-of-Flight mass spectrometry, respectively. In vitro, this compound potently induced growth arrest and lysis of bloodstream form trypanosomes in a dose- and time-dependent manner. In a mouse model, it exhibited a trypanostatic effect that extended the life of infected, treated animals up to 8 days post-infection against the 4 days for infected, untreated animals. The proanthocyanidin showed a low cytotoxicity against mammalian cells, whereas treated-BF showed massive enlargement of their flagellar pocket and lysosome-like structures caused by an intense formation of multivesicular bodies and vesicles within these organelles. The observed ultrastructural alterations caused rupture of plasma membranes and the release of cell contents, indicative of a necrotic process rather than a programmed cell death. Interestingly, the proanthocyanidin acted against BF but not procyclic form trypanosomes. This new anti-trypanosomal compound should be further studied to determine its efficacy and suitability as an anti-trypanosomal drug and may be used as a tool to define novel specific drug targets in BF trypanosomes.     

Structural and mutational analysis of Trypanosoma brucei prostaglandin H2 reductase provides insight into the catalytic mechanism of aldo-ketoreductases

Trypanosoma brucei prostaglandin F2alpha synthase is an aldo-ketoreductase that catalyzes the reduction of prostaglandin H2 to PGF2alpha in addition to that of 9,10-phenanthrenequinone. We report the crystal structure of TbPGFS.NADP+.citrate at 2.1 angstroms resolution. TbPGFS adopts a parallel (alpha/beta)8-barrel fold lacking the protrudent loops and possesses a hydrophobic core active site that contains a catalytic tetrad of tyrosine, lysine, histidine, and aspartate, which is highly conserved among AKRs. Site-directed mutagenesis of the catalytic tetrad residues revealed that a dyad of Lys77 and His110, and a triad of Tyr52, Lys77, and His110 are essential for the reduction of PGH2 and 9,10-PQ, respectively. Structural and kinetic analysis revealed that His110, acts as the general acid catalyst for PGH2 reduction and that Lys77 facilitates His110 protonation through a water molecule, while exerting an electrostatic repulsion against His110 that maintains the spatial arrangement which allows the formation of a hydrogen bond between His110 and C11 that carbonyl of PGH2. We also show Tyr52 acts as the general acid catalyst for 9,10-PQ reduction, and thus we not only elucidate the catalytic mechanism of a PGH2 reductase but also provide an insight into the catalytic specificity of AKRs.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

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 F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) 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.     

Insect Stage-Specific Receptor Adenylate Cyclases Are Localized to Distinct Subdomains of the Trypanosoma brucei Flagellar Membrane

Increasing evidence indicates that the Trypanosoma brucei flagellum (synonymous with cilium) plays important roles in host-parasite interactions. Several studies have identified virulence factors and signaling proteins in the flagellar membrane of bloodstream-stage T. brucei, but less is known about flagellar membrane proteins in procyclic, insect-stage parasites. Here we report on the identification of several receptor-type flagellar adenylate cyclases (ACs) that are specifically upregulated in procyclic T. brucei parasites. Identification of insect stage-specific ACs is novel, as previously studied ACs were constitutively expressed or confined to bloodstream-stage parasites. We show that procyclic stage-specific ACs are glycosylated, surface-exposed proteins that dimerize and possess catalytic activity. We used gene-specific tags to examine the distribution of individual AC isoforms. All ACs examined localized to the flagellum. Notably, however, while some ACs were distributed along the length of the flagellum, others specifically localized to the flagellum tip. These are the first transmembrane domain proteins to be localized specifically at the flagellum tip in T. brucei, emphasizing that the flagellum membrane is organized into specific subdomains. Deletion analysis reveals that C-terminal sequences are critical for targeting ACs to the flagellum, and sequence comparisons suggest that differential subflagellar localization might be specified by isoform-specific C termini. Our combined results suggest insect stage-specific roles for a subset of flagellar adenylate cyclases and support a microdomain model for flagellar cyclic AMP (cAMP) signaling in T. brucei. In this model, cAMP production is compartmentalized through differential localization of individual ACs, thereby allowing diverse cellular responses to be controlled by a common signaling molecule.