Structure of Leishmania mexicana phosphomannomutase highlights similarities with human isoforms

Phosphomannomutase (PMM) catalyses the conversion of mannose-6-phosphate to mannose-1-phosphate, an essential step in mannose activation and the biosynthesis of glycoconjugates in all eukaryotes. Deletion of PMM from Leishmania mexicana results in loss of virulence, suggesting that PMM is a promising drug target for the development of anti-leishmanial inhibitors. We report the crystallization and structure determination to 2.1 A of L. mexicana PMM alone and in complex with glucose-1,6-bisphosphate to 2.9 A. PMM is a member of the haloacid dehalogenase (HAD) family, but has a novel dimeric structure and a distinct cap domain of unique topology. Although the structure is novel within the HAD family, the leishmanial enzyme shows a high degree of similarity with its human isoforms. We have generated L. major PMM knockouts, which are avirulent. We expressed the human pmm2 gene in the Leishmania PMM knockout, but despite the similarity between Leishmania and human PMM, expression of the human gene did not restore virulence. Similarities in the structure of the parasite enzyme and its human isoforms suggest that the development of parasite-selective inhibitors will not be an easy task.     

Developmentally regulated sphingolipid synthesis in African trypanosomes

Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labelling confirmed stage-specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased more than threefold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.     

A novel antifolate resistance gene on the amplified H circle of Leishmania.

In several Leishmania spp., resistance to methotrexate and other drugs is often associated with amplification of the chromosomal H region in the form of extrachromosomal H circles. We report here that the H circle of Leishmania tarentolae contains an 867 bp open reading frame, ltdh, which mediates high levels of resistance to methotrexate and other antifolates, after transfection. The predicted amino acid sequence of the ltdh gene product has significant similarities to a family of short-chain dehydrogenases, enzymes that are involved in several oxido-reduction reactions in a wide range of organisms. To resist antifolates, Leishmania amplifies the ltdh gene as part of the H circle. We propose that LTDH might be involved in an alternative pathway for the synthesis of reduced folates and that ltdh overproduction represents a novel mechanism for resistance to antifolates. Our results support the hypothesis that the H region of the Leishmania genome contains several drug resistance genes and that preferential amplification of this region has evolved as a defense mechanism against cytotoxic drugs.     

Leishmania amazonensis: biosynthesis of polyprenols of 9 isoprene units by amastigotes

The isoprenoid metabolic pathway in protozoa of the Leishmania genus exhibits distinctive characteristics. These parasites, as well as other members of the Trypanosomatidae family, synthesize ergosterol, instead of cholesterol, as the main membrane sterol lipid. Leishmania has been shown to utilize leucine, instead of acetate as the main precursor for sterol biosynthesis. While mammalian dolichols are molecules containing 15-23 isoprene units, Leishmania amazonensis promastigotes synthesize dolichol of 11 and 12 units. In this paper, we show that the intracellular stages of L. amazonensis, amastigotes, synthesize mainly polyprenols of 9 isoprene units, instead of dolichol.     

Microtubule target for new antileishmanial drugs based on ethyl 3-haloacetamidobenzoates

A new family of antimicrotubule drugs named (3-haloacetamidobenzoyl) ureas and ethyl 3-haloacetamidobenzoates were found to be cytotoxic to the Leishmania parasite protozoa. While the benzoylureas were shown to strongly inhibit in vitro mammalian brain microtubule assembly, the ethyl ester derivatives were characterized as very poor inhibitors of this process. Ethyl 3-chloroacetamidobenzoate, MF29, was found to be the most efficient drug on the promastigote stage of three Leishmania species (IC50: 0.3-1.8 microM). MF29 maintained its activity against the clinical relevant intracellular stage of L. mexicana with IC50 value of 0.33 microM. It was the only compound that exhibits a high activity on all the Leishmania species tested. This compound appeared to alter parasite microtubule organisation as demonstrated by using antibodies directed against microtubule components and more precisely the class of microtubule decorated by the MAP2-like protein. It is interesting to notice that this MAP2-like protein was identified for the first time in a Leishmania parasite     

Characterisation of a developmentally regulated amino acid transporter gene from Leishmania amazonensis

The metabolism of protozoan parasites of the Leishmania genus is strongly based on amino acid consumption, but little is known about amino acid uptake in these organisms. In the present work, we identified a Leishmania amazonensis gene (La-PAT1) encoding a putative amino acid transporter that belongs to the amino acid/auxin permease family, a group of H(+)/amino acid symporters. This single copy gene is upregulated in amastigotes, the life cycle stage found in the mammalian host. La-PAT1 putative orthologous sequences were identified in Leishmania infantum, Leishmania donovani, Leishmania major and Trypanosoma.     

Glycoprotein 63 (gp63) genes show gene conversion and reveal the evolution of Old World Leishmania

Species of the subgenus Leishmania (Leishmania) cause the debilitating disease leishmaniasis on four continents. Species grouped within the Leishmania donovani complex cause visceral leishmaniasis, a life-threatening disease, often associated with poverty, and affecting some 0.5 million people each year. The Leishmania glycoprotein GP63, or major surface protease, is a metalloprotease involved in parasite survival, infectivity and virulence. Here, we show that evolution of the gp63 multigene family is influenced by mosaic or fragmental gene conversion. This is a major evolutionary force for both homogenisation and for generating diversity, even in the absence of sexual reproduction. We propose here that the high GC content at the third codon position in the gp63 family of Old World Leishmania may be higher in multicopy regions, under the biased gene conversion model, because increased copy numbers may lead to increased rates of recombination. We confirm that one class of gp63 genes with an extended 3'end signal, gp63(EXT), reveals genetic groups within the complex and gives insights into evolution and host associations. Gp63(EXT) genes can also provide the basis for rapid and reliable genotyping of strains in the L. donovani complex. Our results confirmed that a more stringent definition of Leishmania infantum is required and that the species Leishmania archibaldi should be suppressed.     

Characterization of quinonoid-dihydropteridine reductase (QDPR) from the lower eukaryote Leishmania major

Biopterin is required for growth of the protozoan parasite Leishmania and is salvaged from the host through the activities of a novel biopterin transporter (BT1) and broad-spectrum pteridine reductase (PTR1). Here we characterize Leishmania major quinonoid-dihydropteridine reductase (LmQDPR), the key enzyme required for regeneration and maintenance of H(4)biopterin pools. LmQDPR shows good homology to metazoan quinonoid-dihydropteridine reductase and conservation of domains implicated in catalysis and regulation. Unlike other organisms, LmQDPR is encoded by a tandemly repeated array of 8-9 copies containing LmQDPR plus two other genes. QDPR mRNA and enzymatic activity were expressed at similar levels throughout the infectious cycle. The pH optima, kinetic properties, and substrate specificity of purified LmQDPR were found to be similar to that of other qDPRs, although it lacked significant activity for non-quinonoid pteridines. These and other data suggest that LmQDPR is unlikely to encode the dihydrobiopterin reductase activity (PTR2) described previously. Similarly LmQDPR is not inhibited by a series of antifolates showing anti-leishmanial activity beyond that attributable to dihydrofolate reductase or PTR1 inhibition. qDPR activity was found in crude lysates of Trypanosoma brucei and Trypanosoma cruzi, further emphasizing the importance of H(4)biopterin throughout this family of human parasites.     

Leishmanicidal activities and cytotoxicities of bisnaphthoquinone analogues and naphthol derivatives from Burman Diospyros burmanica

A methanol extract of the wood of Diospyros burmanica, collected in Burma (Myanmar), was found to exhibit significant activity against Leishmania major. Subsequent chromatographically resolved fractionation led to the isolation of three novel bisnaphthoquinone analogues, burmanin A, B, and C (1-3), together with nine known compounds (4-12). The structure of 1 was confirmed by X-ray crystallography, and those of 2 and 3 by spectroscopic techniques, including 1D and 2D NMR. The inhibitory activities of the isolates were evaluated against the promastigote forms of Leishmania major and the murine macrophage-like cell line, RAW264.7.     

Isolation, characterization and molecular cloning of new temporins from the skin of the North African ranid Pelophylax saharica

Temporins are small antimicrobial peptides isolated from North American and Eurasian ranid frogs that are particularly active against Gram-positive bacteria. To date, no temporins have been characterized from North African frog species. We isolated three novel members of the temporin family, named temporin-1Sa (FLSGIVGMLGKLF(amide)), -1Sb (FLPIVTNLLSGLL(amide)), and -1Sc (FLSHIAGFLSNLF(amide)), from the skin of the Sahara frog Pelophylax (Rana) saharica originating from Tunisia. These temporins were identified by a combined mass spectrometry/molecular cloning approach. Temporin-1Sa was found to be highly active against Gram-positive and Gram-negative bacteria, yeasts and fungi (MIC=2-30muM). To our knowledge, this is the first 13-residue member of the temporin family with a net charge of +2 that shows such broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC=10muM) and Pseudomonas aeruginosa (MIC=31muM). Moreover, temporin-1Sa displays significant antiparasitic activity (IC(50) approximately 20muM) against the promastigote and amastigote stages of Leishmania infantum.     

Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.     

Functional identification of a Leishmania gene related to the peroxin 2 gene reveals common ancestry of glycosomes and peroxisomes.

Glycosomes are membrane-bounded microbody organelles that compartmentalize glycolysis as well as other important metabolic processes in trypanosomatids. The compartmentalization of these enzymatic reactions is hypothesized to play a crucial role in parasite physiology. Although the metabolic role of glycosomes differs substantially from that of the peroxisomes that are found in other eukaryotes, similarities in signals targeting proteins to these organelles suggest that glycosomes and peroxisomes may have evolved from a common ancestor. To examine this hypothesis, as well as gain insights into the function of the glycosome, we used a positive genetic selection procedure to isolate the first Leishmania mutant (gim1-1 [glycosome import] mutant) with a defect in the import of glycosomal proteins. The mutant retains glycosomes but mislocalizes a subset glycosomal proteins to the cytoplasm. Unexpectedly, the gim1-1 mutant lacks lipid bodies, suggesting a heretofore unknown role of the glycosome. We used genetic approaches to identify a gene, GIM1, that is able to restore import and lipid bodies. A nonsense mutation was found in one allele of this gene in the mutant line. The predicted Gim1 protein is related the peroxin 2 family of integral membrane proteins, which are required for peroxisome biogenesis. The similarities in sequence and function provide strong support for the common origin model of glycosomes and peroxisomes. The novel phenotype of gim1-1 and distinctive role of Leishmania glycosomes suggest that future studies of this system will provide a new perspective on microbody biogenesis and function.     

Leishmania donovani bisubunit topoisomerase I gene fusion leads to an active enzyme with conserved type IB enzyme function

All eukaryotic topoisomerase I enzymes are monomeric enzymes, whereas the kinetoplastid family (Trypanosoma and Leishmania) possess an unusual bisubunit topoisomerase I. To determine what happens to the enzyme architecture and catalytic property if the two subunits are fused, and to explore the functional relationship between the two subunits, we describe here in vitro gene fusion of Leishmania bisubunit topoisomerase I into a single ORF encoding a new monomeric topoisomerase I (LdTOPIL-fus-S). It was found that LdTOPIL-fus-S is active. Gene fusion leads to a significant modulation of in vitro topoisomerase I activity compared to the wild-type heterodimeric enzyme (LdTOPILS). Interestingly, an N-terminal truncation mutant (1-210 amino acids) of the small subunit, when fused to the intact large subunit [LdTOPIL-fus-Delta(1-210)S], showed reduced topoisomerase I activity and camptothecin sensitivity in comparison to LdTOPIL-fus-S. Investigation of the reduction in enzyme activity indicated that the nonconserved 1-210 residues of LdTOPIS probably act as a 'pseudolinker' domain between the core and catalytic domain of the fused Leishmania enzyme, whereas mutational analysis of conserved His453 in the core DNA-binding domain (LdTOPIL) strongly suggested that its role is to stabilize the enzyme-DNA transition state through hydrogen bonding to one of the nonbridging oxygens. Taken together, our findings provide an insight into the details of the unusual structure of bisubunit topoisomerase I of Leishmania donovani.     

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K(m) values for hypoxanthine, xanthine, adenine and guanine of 16.5 +/- 1.5, 8.5 +/- 0.6, 8.5 +/- 1.1, and 8.8 +/- 4.0 microM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.     

Leishmania proteins derived from recombinant DNA: current status and next steps

The parasite Leishmania is a major cause of disease worldwide. In the past 15 years, many groups have analysed DNA-derived proteins from Leishmania. Large amounts of data obtained by these groups can be collated to direct future research into Leishmania and to find novel immunological mechanisms and information about its metabolism. International coordination will increase both the basic knowledge about Leishmania and the capacity to apply this knowledge to combat leishmaniases.