A novel high-affinity arginine transporter from the human parasitic protozoan Leishmania donovani

We describe the first functional and molecular characterization of an amino acid permease (LdAAP3) from the human parasitic protozoan Leishmania donovani, the causative agent of visceral leishmaniasis in humans. This permease contains 480 amino acids with 11 predicted trans-membrane domains. Expressing LdAAP3 in Saccharomyces cerevisiae mutants revealed that LdAAP3 codes for a high-affinity arginine transporter (Km 1.9 microM). LdAAP3 is highly specific for arginine as its transport was not inhibited by other amino acids or arginine-related compounds. Using green fluorescence protein (GFP) fused to the N-terminus of LdAAP3, this transporter was localized to the surface membrane of promastigotes. The GFP-LdAAP3 chimera mediated a threefold increase in arginine transport in promastigotes, indicating that it is active and confirmed that LdAAP3 codes for an arginine transporter in parasite cells as well. LdAAP3 is novel as it shares a high level of homology with amino acid permeases from other trypanosomatidae but almost none with permeases from other phyla. The results of this work suggest that LdAAP3 might play a role in host-parasite interaction.     

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.     

Molecular cloning,biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue

The Src-homology 2 domain containing protein tyrosine phosphatase-1 (SHP-1) is involved in the pathogenesis of infection with Leishmania. Recently, we identified elongation factor-1 alpha (EF-1 alpha) from Leishmania donovani as a SHP-1 binding and activating protein [J. Biol. Chem. 277 (2002) 50190]. To characterize this apparent Leishmania virulence factor further, the cDNA encoding L. donovani EF-1 alpha was cloned and sequenced. Whereas nearly complete sequence conservation was observed amongst EF-1 alpha proteins from trypanosomatids, the deduced amino acid sequence of EF-1 alpha of L. donovani when compared to mammalian EF-1 alpha sequences showed a number of significant changes. Protein structure modeling-based upon the known crystal structure of EF-1 alpha for Saccharomyces cerevisiae-identified a hairpin loop present in mammalian EF-1 alpha and absent from the Leishmania protein which corresponded to a 12 amino acid deletion. Consistent with these structural differences, the sub-cellular distributions of L. donovani EF-1 alpha and host EF-1 alpha were strikingly different. Interestingly, infection of macrophages with L. donovani caused redistribution of host as well as pathogen EF-1 alpha. Since EF-1 alpha is essential for survival, the distinct biochemical and structural properties of Leishmania EF-1 alpha may provide a novel target for drug development.     

Leishmania aethiopica: strain identification and characterization of superoxide dismutase-B genes

This study was performed to characterize the genes that code for superoxide dismutase (SOD) in Leishmania aethiopica. It involved three main steps: specimen collection and parasite isolation, species identification, and molecular characterization of the SOD genes. Out of 20 skin slit specimens cultured and processed from suspected cutaneous leishmaniasis patients enrolled in the study, five (25%) were found to be positive for motile promastigotes. Isoenzyme electrophoresis and PCR-RFLP results confirmed that the isolates were L. aethiopica. Superoxide dismutase-B (SODB) genes were identified from L. aethiopica for the first time. Iron superoxide dismutase-B genes amplified from promastigotes of L. aethiopica (LaeFeSODB) were similar in size to the SODB genes of other Leishmania species. Nucleotide sequences of LaeFeSODB1 showed 95.4, 93.5, and 97.3% identity with L. donovani SODB1 (LdFeSODB1) L. major SODB1 (LmFeSODB1) and L. tropica SODB1 (LtrFeSODB1), respectively. Similarly, LaeFeSODB2 showed 95.9 and 94.1 and 97.6% identity with LdFeSODB2 and LmFeSODB2 and LtrFeSODB2, respectively. On the other hand, predicted amino acid sequence comparison indicated that LaeFeSODB1 had 91.3, 89.8, and 93.9% identity with LdFeSODB1, LmFeSODB1, and LtrFeSODB1, respectively. The difference in nucleic acid sequence of LaeFeSODB from that of LmFeSODB and LtrFeSODB can be utilized to develop specific molecular methods that help differentiate these species in places where there is an overlap in the distribution of these species. In addition, the data provide information about the situation of L. aethiopica with respect to SODB genes.     

The Leishmania donovani complex: genotypes of five metabolic enzymes (ICD, ME, MPI, G6PDH, and FH), new targets for multilocus sequence typing

Flagellates of the Leishmania donovani complex are causative agents of human cutaneous and visceral leishmaniasis. The complex is comprised of L. donovani, Leishmania infantum and Leishmania archibaldi, although the latter is not now considered to be a valid species. Morphological distinction of Leishmania species is impractical, so biochemical, immunological and DNA-based criteria were introduced. Multilocus enzyme electrophoresis (MLEE) is the present gold standard. We have sequenced the genes encoding five metabolic enzymes used for MLEE, both to resolve the DNA diversity underlying isoenzyme mobility differences and to explore the potential of these targets for higher resolution PCR-based multilocus sequence typing. The genes sequenced were isocitrate dehydrogenase, malic enzyme, mannose phosphate isomerase, glucose-6-phosphate dehydrogenase, and fumarate hydratase, for 17 strains of L. infantum, seven strains of L. donovani, and three strains of L. archibaldi. Protein mobilities predicted from amino acid sequences did not always accord precisely with reported MLEE profiles. A high number of heterozygous sites was detected. Heterozygosity was particularly frequent in some strains and indirectly supported the presence of genetic exchange in Leishmania. Phylogenetic analysis of a concatenated alignment based on a total of 263 kb protein-coding sequences showed strong correlation of genotype with geographical origin. Europe and Africa appear to represent independent evolutionary centres.     

A novel active DNA topoisomerase I in Leishmania donovani

A common feature shared by type I DNA topoisomerases is the presence of a "serine, lysine, X, X, tyrosine" motif as conventional enzyme active site. Preliminary data have shown that Leishmania donovani DNA topoisomerase I gene (LdTOP1A) lacked this conserved motif, giving rise to different theories about the reconstitution of an active DNA topoisomerase I in this parasite. We, herein, describe the molecular cloning of a new DNA topoisomerase I gene from L. donovani (LdTOP1B) containing the highly conserved serine, lysine, X, X, tyrosine motif. DNA topoisomerase I activity was detected only when both genes (LdTOP1A and LdTOP1B) were co-expressed in a yeast expression system, suggesting the existence of a dimeric DNA topoisomerase I in Leishmania parasites.     

Expression of calreticulin P-domain results in impairment of secretory pathway in Leishmania donovani and reduced parasite survival in macrophages

The secretory proteins of Leishmania are thought to be involved in the parasite survival inside the insect vector or mammalian host. It is clear from studies in higher eukaryotes that proper folding in the endoplasmic reticulum and targeting out of the endoplasmic reticulum is critical for the function of secretory proteins. The endoplasmic reticulum chaperones such as calreticulin play an important role in the quality control of secretory proteins. However, very little is known about the secretory pathway of trypanosomatid parasites such as Leishmania. In the present study, we show that overexpression of the P-domain of Leishmania donovani calreticulin in transfected L. donovani resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases. This effect is associated with an intracellular accumulation of active enzyme in these transfected parasites. In addition, parasites expressing the P-domain calreticulin showed a significant decrease in survival inside human macrophages. This study suggests that altering the function of an endoplasmic reticulum chaperone such as calreticulin in Leishmania may affect the targeting of proteins that are associated with the virulence of the parasite during their trafficking through the parasite secretory pathway.     

A proteogenomic approach to map the proteome of an unsequenced pathogen - Leishmania donovani

Visceral leishmaniasis or kala azar is the most severe form of leishmaniasis and is caused by the protozoan parasite Leishmania donovani. There is no published report on L. donovani genome sequence available till date, although the genome sequences of three related Leishmania species are already available. Thus, we took a proteogenomic approach to identify proteins from two different life stages of L. donovani. From our analysis of the promastigote (insect) and amastigote (human) stages of L. donovani, we identified a total of 22,322 unique peptides from a homology-based search against proteins from three Leishmania species. These peptides were assigned to 3711 proteins in L. infantum, 3287 proteins in L. major, and 2433 proteins in L. braziliensis. Of the 3711 L. donovani proteins that were identified, the expression of 1387 proteins was detectable in both life stages of the parasite, while 901 and 1423 proteins were identified only in promastigotes and amastigotes life stages, respectively. In addition, we also identified 13 N-terminally and one C-terminally extended proteins based on the proteomic data search against the six-frame translated genome of the three related Leishmania species. Here, we report results from proteomic profiling of L. donovani, an organism with an unsequenced genome.     

The sterol-binding antibiotic nystatin inhibits entry of non-opsonized Leishmania donovani into macrophages

Leishmania donovani is an obligate intracellular parasite that infects macrophages of the vertebrate host resulting in visceral leishmaniasis in humans, a major public health problem worldwide. The molecular mechanisms involved in internalization of Leishmania are still poorly characterized. We report here that cholesterol sequestration by the sterol-binding antifungal polyene antibiotic nystatin markedly inhibits binding and entry of non-opsonized L. donovani promastigotes into macrophages. Interestingly, these effects are not observed when serum-opsonized L. donovani are used for infectivity studies thus pointing the essential role of cholesterol in mediating entry of the parasite via the non-opsonic pathway. Based on our earlier results where leishmanial infectivity was shown to be sensitive to physical depletion of cholesterol from macrophages, these results indicate that the mere sequestration of cholesterol in the host plasma membrane is sufficient to inhibit the binding and entry of non-opsonized L. donovani. These results represent the first report on the effect of a cholesterol-sequestering agent on the entry of Leishmania parasites to host macrophages. More importantly, these findings offer the possibility of reevaluating the mechanism behind the effectiveness of current therapeutic strategies to treat leishmaniasis.     

Towards multilocus sequence typing of the Leishmania donovani complex: resolving genotypes and haplotypes for five polymorphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD)

Multilocus enzyme electrophoresis is the gold standard for identification of Leishmania species and strains. Drawbacks include: only amino acid polymorphisms affecting electrophoretic mobility are detected; distinct allozymes can have coincident mobilities; few characters are available; and parasites must be cultured in bulk. So far, thousands of Leishmania strains have been phenotyped by multilocus enzyme electrophoresis. Here, we sequence enzyme-coding genes to provide a PCR-based higher resolution equivalent of multilocus enzyme electrophoresis, particularly for Leishmania infantum. Of 15 enzymes used for multilocus enzyme electrophoresis (MON typing) we have sequenced aspartate aminotransferase, glucose-6-phosphate isomerase, nucleoside hydrolase 1, nucleoside hydrolase 2 and 6-phosphogluconate dehydrogenase. Heterozygous alleles were common, with multiple heterozygous sites within a single locus for several of the genes. Haplotypes were resolved by allele-specific PCR and allele-specific sequencing. Heterozygous haplotypes conformed to the haplotypes of putative parents. One strain appeared to be hybrid across two genetic groups of the Leishmania donovani complex. In most cases, a single amino acid polymorphism was responsible for change in enzyme mobility. Some indistinguishable phenotypes were produced by distinct genotypes. Silent genetic polymorphisms provided enhanced discrimination over multilocus enzyme electrophoresis, for example, by subdividing the zymodeme MON-1. The PCR-based genotyping that we describe could be applied directly to clinical samples or to small volume cultures and in a multilocus sequence typing format. Furthermore, it can be used to detect recombination indirectly and for population genetics studies.     

Genomic organization and functional expression of differentially regulated cysteine protease genes of Leishmania donovani complex

For the first time, we report the genomic organization and characterization of Cathepsin L-like cysteine protease gene cluster from the members of Leishmania donovani complex. The cysteine protease gene cluster of Leishmania chagasi has five copies of tandemly arranged genes. The first gene (Ldccys1A) is identical to Ldccys1 cDNA and is predominantly expressed in promastigotes. The last gene (Ldccys1E) is identical to Ldccys1A with a 13 amino acids deletion in the mature domain, including one of the active site histidine residues and a truncated carboxyl terminal extension. It has a diverged 3' untranslated region and is also constitutively expressed in the parasite. Results from rapid amplification of cDNA ends (RACE) suggest that there are three different types of 3' untranslated regions, one of them is identical to that of Ldccys1A whereas another to Ldccys1E. The third one is also identical to Ldccys1A, but has a 154 nucleotides deletion near the polyA region and this gene is constitutively expressed. Gene organization and expression in L. donovani cluster is similar to that of L. chagasi. However, the last gene (Lddcys1F) is different from Ldccys1E as it lacks 13 amino acid deletions. Also, L. donovani possesses an additional copy of the gene (Lddcys1E), which is located away from the cluster. Furthermore, for the first time we have expressed full-length cysteine protease genes in an insect expression system. Ldccys1A and Ldccys1F cleaved gelatin whereas Ldccys1E was found to be inactive in gelatin assays.     

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.     

Human mixed infections of Leishmania spp. and Leishmania-Trypanosoma cruzi in a sub Andean Bolivian area: identification by polymerase chain reaction/hybridization and isoenzyme

Parasites belonging to Leishmania braziliensis, Leishmania donovani, Leishmania mexicana complexes and Trypanosoma cruzi (clones 20 and 39) were searched in blood, lesions and strains collected from 28 patients with active cutaneous leishmaniasis and one patient with visceral leishmaniasis. PCR-hybridization with specific probes of Leishmania complexes (L. braziliensis, L. donovani and L. mexicana) and T. cruzi clones was applied to the different DNA samples. Over 29 patients, 8 (27.6%) presented a mixed infection Leishmania complex species, 17 (58.6%) a mixed infection Leishmania-T. cruzi, and 4 (13.8%) a multi Leishmania-T. cruzi infection. Several patients were infected by the two Bolivian major clones 20 and 39 of T. cruzi (44.8%). The L. braziliensis complex was more frequently detected in lesions than in blood and a reverse result was observed for L. mexicana complex. The polymerase chain reaction-hybridization design offers new arguments supporting the idea of an underestimated rate of visceral leishmanisis in Bolivia. Parasites were isolated by culture from the blood of two patients and lesions of 10 patients. The UPGMA (unweighted pair-group method with arithmetic averages) dendrogram computed from Jaccard's distances obtained from 11 isoenzyme loci data confirmed the presence of the three Leishmania complexes and undoubtedly identified human infections by L. (V.) braziliensis, L. (L.) chagasi and L. (L.) mexicana species. Additional evidence of parasite mixtures was visualized through mixed isoenzyme profiles, L. (V.) braziliensis-L. (L.) mexicana and Leishmania spp.-T. cruzi.The epidemiological profile in the studied area appeared more complex than currently known. This is the first report of parasitological evidence of Bolivian patients with trypanosomatidae multi infections and consequences on the diseases' control and patient treatments are discussed.     

Comparative multivariate analysis of codon and amino acid usage in three Leishmania genomes

Multivariate analysis of codon and amino acid usage was performed for three Leishmania species, including L. donovani, L. infantum and L. major. It was revealed that all three species are under mutational bias and translational selection. Lower GC 12 and higher GC 3S in all three parasites suggests that the ancestral highly expressed genes (HEGs), compared to lowly expressed genes (LEGs), might have been rich in AT-content. This also suggests that there must have been a faster rate of evolution under GC-bias in LEGs. It was observed from the estimation of synonymous/non-synonymous substitutions in HEGs that the HEG dataset of L. donovani is much closer to L. major evolutionarily. This is also supported by the higher d N value as compared to d S between L. donovani and L. major, suggesting the conservation of synonymous codon positions between these two species and the role of translational selection in shaping the composition of protein-coding genes.     

Degradation of pteridine reductase 1 (PTR1) enzyme during growth phase in the protozoan parasite Leishmania donovani

Pteridine reductase 1 (PTR1) is an essential enzyme of pterin and folate metabolism in the protozoan parasite Leishmania. The present work is focused on the degradation of PTR1 during growth phase in Leishmania donovani. Western blot analysis with PTR1-GFP transfected promastigotes revealed that PTR1 protein was degraded in the stationary phase of growth at the time when the parasites were undergoing metacyclogenesis. Fluorescence microscopy revealed cytoplasmic localization of GFP tagged protein extending to the flagellum in these stationary phase promastigotes, implying that degradation of the protein was not by the usual multivesicular tubule lysosome (MVT) pathway. A probable destruction box of nine amino acids Q63ADLSNVAK71 and possible lysine residue K156 was identified in L. donovani PTR1 to be the site for ubiquitin conjugation. This suggests that PTR1 degradation during the stationary phase of growth is mediated by the proteasome. This leads to lower levels of H4-biopterin, which favors metacyclogenesis, and subsequently results in a highly infective stage of the parasite. Therefore, this finding has importance to identify new target molecule like the proteasome for therapeutic intervention.     

Effect of pH and temperature on protein kinase release by Leishmania donovani

During their life cycle Leishmania are exposed to environments that differ markedly in pH and temperature. The effect of these factors on protein kinase release into the surrounding environment by Leishmania donovani promastigotes was examined. Promastigotes release protein kinase activity both constitutively and following induction by incubation with an exogenous substrate, phosvitin. The substrate specificity of the constitutive and induced activities was similar, unlike that previously described for Leishmania major promastigotes. The Leishmania donovani enzymes phosphorylate phosvitin, but not casein, mixed histones or protamine sulphate, and both activities are shed over a wide pH range from 6 to 9. Transfer of promastigotes from pH 7.4/30 degrees C to pH 5.0-5.5/37 degrees C, conditions that mimic those encountered by parasites following transmission from sandflies to a mammalian host and uptake by macrophages, inhibited release of the constitutive activity. Identical conditions had only a minor effect on induced protein kinase release. Both types of protein kinase activities released at pH 7.4 were still active when assayed at pH 5.0. Characterisation of the constitutive and induced promastigote protein kinases showed that casein kinase 1- and casein kinase 2-like activities are released by Leishmania donovani. Constitutive enzyme release decreased over time, however, the addition of phosvitin to these "casein kinase-depleted" promastigotes induced elevated casein kinase 1 and casein kinase 2 shedding. These results suggest that shed protein kinase might play a role in parasite survival and adaptation to host environments.     

Over-expression of Leishmania major MAP kinases reveals stage-specific induction of phosphotransferase activity

During the infectious cycle, protozoan parasites undergo various developmental transitions and switch virulence factors in response to extracellular signals in insect vectors and human hosts. Despite the importance of environmental sensing in parasite pathogenicity, little is known about the pathways that transduce extracellular signals into stage-specific gene expression. Here, we used a transgenic approach to gain insight into localisation and activity of three green fluorescence protein (GFP)-tagged Leishmania major mitogen-activated protein kinases, LmaMPK4, 7 and 10. The GFP-LmaMPKs in both L. major and Leishmania donovani transgenic lines showed predominant cytoplasmic localisation and the over-expression had no effect on promastigote morphology, growth and the ability to differentiate into stationary-phase metacyclics for L. major and axenic amastigotes for L. donovani. We isolated the GFP-tagged MPKs from parasite extracts and tested their phosphotransferase activity across various culture conditions. For all three GFP-LmaMPKs, kinase activity was low or absent in promastigote extracts but significantly increased in L. major promastigotes after exposure to pH 5.5 and 34 degrees C, and in axenic L. donovani amastigotes. Enhanced activity correlated with increased GFP-LmaMPK phosphorylation as judged by phospho-specific fluorescent staining of the immuno-precipitated kinases. We could extend these findings to the endogenous LmaMPK10, which accumulated in the phospho-protein fraction of axenic amastigotes but not promastigotes, and thus follows the stage-specific phosphorylation profile of episomally expressed GFP-LmaMPK10. These results provide evidence for the functional conservation of Leishmania MAP kinases in parasite environmental sensing and underscore the potential of transgenic approaches to gain insight into signaling events during the Leishmania life cycle.     

A unique modification of the eukaryotic initiation factor 5A shows the presence of the complete hypusine pathway in Leishmania donovani

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of an unusual amino acid hypusine (N(€)-(4-amino-2-hydroxybutyl) lysine), which is present on only one cellular protein, eukaryotic initiation factor 5A (eIF5A). We present here the molecular and structural basis of the function of DOHH from the protozoan parasite, Leishmania donovani, which causes visceral leishmaniasis. The L. donovani DOHH gene is 981 bp and encodes a putative polypeptide of 326 amino acids. DOHH is a HEAT-repeat protein with eight tandem repeats of α-helical pairs. Four conserved histidine-glutamate sequences have been identified that may act as metal coordination sites. A ~42 kDa recombinant protein with a His-tag was obtained by heterologous expression of DOHH in Escherichia coli. Purified recombinant DOHH effectively catalyzed the hydroxylation of the intermediate, eIF5A-deoxyhypusine (eIF5A-Dhp), in vitro. L. donovani DOHH (LdDOHH) showed ~40.6% sequence identity with its human homolog. The alignment of L. donovani DOHH with the human homolog shows that there are two significant insertions in the former, corresponding to the alignment positions 159-162 (four amino acid residues) and 174-183 (ten amino acid residues) which are present in the variable loop connecting the N- and C-terminal halves of the protein, the latter being present near the substrate binding site. Deletion of the ten-amino-acid-long insertion decreased LdDOHH activity to 14% of the wild type recombinant LdDOHH. Metal chelators like ciclopirox olamine (CPX) and mimosine significantly inhibited the growth of L. donovani and DOHH activity in vitro. These inhibitors were more effective against the parasite enzyme than the human enzyme. This report, for the first time, confirms the presence of a complete hypusine pathway in a kinetoplastid unlike eubacteria and archaea. The structural differences between the L. donovani DOHH and the human homolog may be exploited for structure based design of selective inhibitors against the parasite.