First isolation of a new species of Leishmania responsible for human cutaneous leishmaniasis in Ghana and classification in the Leishmania enriettii complex.

An active case detection approach with PCR diagnosis was used in the Ho District of the Volta Region, Ghana that identified individuals with active cutaneous leishmaniasis. Three isolates were successfully cultured and DNA sequences from these were analysed (ribosomal RNA internal transcribed spacer 1; ribosomal protein L23a intergenic spacer; RNA polymerase II large subunit), showing them to be Leishmania, identical to each other but different from all other known Leishmania spp. Phylogenetic analysis showed the parasites to be new members of the Leishmania enriettii complex, which is emerging as a possible new subgenus of Leishmania parasites containing human pathogens.

First isolation of Leishmania from Northern Thailand: case report, identification as Leishmania martiniquensis and phylogenetic position within the Leishmania enriettii complex.

Since 1996, there have been several case reports of autochthonous visceral leishmaniasis in Thailand. Here we report a case in a 52-year-old Thai male from northern Thailand, who presented with subacute fever, huge splenomegaly and pancytopenia. Bone marrow aspiration revealed numerous amastigotes within macrophages. Isolation of Leishmania LSCM1 into culture and DNA sequence analysis (ribosomal RNA ITS-1 and large subunit of RNA polymerase II) revealed the parasites to be members of the Leishmania enriettii complex, and apparently identical to L. martiniquensis previously reported from the Caribbean island of Martinique. This is the first report of visceral leishmaniasis caused by L. martiniquensis from the region. Moreover, the majority of parasites previously identified as "L. siamensis" also appear to be L. martiniquensis.

Flavonoid dimers as bivalent modulators for pentamidine and sodium stiboglucanate resistance in leishmania.

Drug resistance by overexpression of ATP-binding cassette (ABC) transporters is an impediment in the treatment of leishmaniasis. Flavonoids are known to reverse multidrug resistance (MDR) in Leishmania and mammalian cancers by inhibiting ABC transporters. Here, we found that synthetic flavonoid dimers with three (compound 9c) or four (compound 9d) ethylene glycol units exhibited a significantly higher reversing activity than other shorter or longer ethylene glycol-ligated dimers, with approximately 3-fold sensitization of pentamidine and sodium stibogluconate (SSG) resistance in Leishmania, respectively. This modulatory effect was dosage dependent and not observed in apigenin monomers with the linker, suggesting that the modulatory effect is due to its bivalent nature. The mechanism of reversal activity was due to increased intracellular accumulation of pentamidine and total antimony in Leishmania. Compared to other MDR modulators such as verapamil, reserpine, quinine, quinacrine, and quinidine, compounds 9c and 9d were the only agents that can reverse SSG resistance. In terms of reversing pentamidine resistance, 9c and 9d have activities comparable to those of reserpine and quinacrine. Modulators 9c and 9d exhibited reversal activity on pentamidine resistance among LeMDR1(-/-), LeMDR1(+/+), and LeMDR1-overexpressed mutants, suggesting that these modulators are specific to a non-LeMDR1 pentamidine transporter. The LeMDR1 copy number is inversely related to pentamidine resistance, suggesting that it might be involved in importing pentamidine into the mitochondria. In summary, bivalency could be a useful strategy for the development of more potent ABC transporter modulators and flavonoid dimers represent a promising reversal agent for overcoming pentamidine and SSG resistance in parasite Leishmania.

The role of Leishmania enriettii multidrug resistance protein 1 (LeMDR1) in mediating drug resistance is iron-dependent.

In parasitic protozoan Leishmania enriettii, the role of a multidrug resistance (mdr) gene LeMDR1 (L. enriettii multidrug resistance 1) in mediating vinblastine resistance has been previously demonstrated by association, transfection and "gene knockout" studies. LeMDR1 has been shown to be located intracellularly and it was proposed to mediate drug resistance by sequestering drugs into intracellular organelles rather than by active efflux. Here we compared LeMDR1 overexpressed cell lines (Vint3 and V160), wild type (Le) and LeMDR1 "double knockout" mutant (LeMDR1-/-) and demonstrated that LeMDR1 gene copy number was associated with (1) higher level of intracellular iron, (2) increased sensitivity to an iron-dependent antibiotic, streptonigrin and (3) increased enzyme activity of an iron-sulfur-containing mitochondrial enzyme, aconitase. This result suggests that the normal function of LeMDR1 is related to mitochondrial iron homeostasis. To test such hypothesis, we have used the LeMDR1-overexpressing mutant V160 and LeMDR1-/- mutant to determine how iron level can affect its resistance level to drugs targeting either cytosol (vinblastine) or mitochondria (rhodamine 123 and pentamidine). It was found that the resistance level of V160 to vinblastine can be increased by iron whereas resistance to both rhodamine 123 and pentamidine can be increased by iron depletion and vice versa. Iron treatment can potentiate rhodamine 123 and pentamidine accumulation whereas iron deprivation can cause the reduction of rhodamine 123 accumulation. Our result highly suggests that LeMDR1's function in mediating drug resistance is iron-dependent.

Sequences required for the flagellar targeting of an integral membrane protein.

Previous studies have established that the ISO1 glucose transporter of Leishmania enriettii resides primarily in the flagellar membrane, whereas the ISO2 glucose transporter is located in the pellicular plasma membrane surrounding the cell body. This pronounced difference in subcellular targeting is conferred by the NH2-terminal domain of the transporters, since this is the only region of the two permeases that differs in sequence. Analysis of the 130 residue NH2-terminal domain of ISO1 using multiple terminal deletion mutants and various internal deletion mutants established that a sequence located between amino acids 84 and 100 of this domain is required for flagellar trafficking. In addition, chimeras between ISO1 and ISO2 indicated that the region between residues 110 and 118 of ISO1 is also required for flagellar targeting. These results imply that flagellar targeting information for this integral membrane protein does not constitute a simple linear sequence of amino acids but is at least bipartite in structure.

Cytoskeletal association is important for differential targeting of glucose transporter isoforms in Leishmania.

The major glucose transporter of the parasitic protozoan Leishmania enriettii exists in two isoforms, one of which (iso-1) localizes to the flagellar membrane, while the other (iso-2) localizes to the plasma membrane of the cell body, the pellicular membrane. These two isoforms differ only in their cytosolic NH2-terminal domains. Using immunoblots and immunofluorescence microscopy of detergent-extracted cytoskeletons, we have demonstrated that iso-2 associates with the microtubular cytoskeleton that underlies the cell body membrane, whereas the flagellar membrane isoform iso-1 does not associate with the cytoskeleton. Deletion mutants that remove the first 25 or more amino acids from iso-1 are retargeted from the flagellum to the pellicular membrane, suggesting that these deletions remove a signal required for flagellar targeting. Unlike the full-length iso-1 protein, these deletion mutants associate with the cytoskeleton. Our results suggest that cytoskeletal binding serves as an anchor to localize the iso-2 transporter within the pellicular membrane, and that the flagellar targeting signal of iso-1 diverts this transporter into the flagellar membrane and away from the pellicular microtubules.

Partial protection against malaria by immunization with Leishmania enriettii expressing the Plasmodium yoelii circumsporozoite protein.

Since infection with Leishmania species induces CD4+ and CD8+ anti-leishmania T cells, we assessed protection against malaria by immunization with Leishmania enriettii transfected with the gene encoding the Plasmodium yoelii circumsporozoite protein (PyCSP). The recombinant plasmid appeared to be a circular episome in the host cells. Reverse transcription PCR showed that the PyCSP was trans-spliced by the addition of the 39-bp spliced leader of L. enriettii at its 5' end. The transfectant expressed a protein in a pattern similar to that found in the sporozoite itself. Immunofluorescence and immunoelectron microscopy indicated that PyCSP was abundantly expressed on the surface of the parasite. Mice immunized with the transfectant produced antibodies to sporozoites, had a delay in onset of parasitemia after challenge, and 4 of 22 (18%) were completely protected. The protected mice had cytotoxic T lymphocytes against the PyCSP. Immunization with recombinant vaccinia, Salmonella typhimurium, and pseudorabies virus expressing the PyCSP induces excellent immune responses, but has not been shown to protect against challenge. Thus, the modest protection found in these initial studies represents a step forward. After further work Leishmania may prove to be an important live vector vaccine system for induction of protective immune responses.

Differential targeting of two glucose transporters from Leishmania enriettii is mediated by an NH2-terminal domain.

Leishmania are parasitic protozoa with two major stages in their life cycle: flagellated promastigotes that live in the gut of the insect vector and nonflagellated amastigotes that live inside the lysosomes of the vertebrate host macrophages. The Pro-1 glucose transporter of L. enriettii exists as two isoforms, iso-1 and iso-2, which are both expressed primarily in the promastigote stage of the life cycle. These two isoforms constitute modular structures: they differ exclusively and extensively in their NH2-terminal hydrophilic domains, but the remainder of each isoform sequence is identical to that of the other. We have localized these glucose transporters within promastigotes by two approaches. In the first method, we have raised a polyclonal antibody against the COOH-terminal hydrophilic domain shared by both iso-1 and iso-2, and we have used this antibody to detect the transporters by confocal immunofluorescence microscopy and immunoelectron microscopy. The staining observed with this antibody occurs primarily on the plasma membrane and the membrane of the flagellar pocket, but there is also light staining on the flagellum. We have also localized each isoform separately by introducing an epitope tag into each protein sequence. These experiments demonstrate that iso-1, the minor isoform, resides primarily on the flagellar membrane, while iso-2, the major isoform, is located on the plasma membrane and the flagellar pocket. Hence, each isoform is differentially sorted, and the structural information for targeting each transporter isoform to its correct membrane address resides within the NH2-terminal hydrophilic domain.

Identification of a cis-acting gene regulatory element from the lemdr1 locus of Leishmania enriettii.

The goal of this work is to identify those elements which control gene expression in the parasitic protozoan, Leishmania enriettii. To date there has been no report of a transcriptional controlling element identified in this parasite. In the present study, we have found that a region of the lemdr1 locus of L. enriettii can down-regulate the steady state mRNA level of the reporter neomycin phosphotransferase gene when it is placed in an extrachromosomal expression vector, pALTneo. Through deletion analysis, we have mapped a putative regulatory sequence to the flanking region upstream of the lemdr1 gene. This cis regulatory element is shown to control gene expression in an orientation-dependent and position independent manner. By performing nuclear run-on analysis, we have demonstrated that this element mainly exerts its effect at the transcriptional level by determining the strandedness of transcription on the pALTneo vector. The identification of this novel cis regulatory element should further our understanding of the transcriptional process in Leishmania and other related trypanosomatids.

Functional expression of two glucose transporter isoforms from the parasitic protozoan Leishmania enriettii.

The parasitic protozoan Leishmania enriettii contains a family of tandemly repeated genes, designated Pro-1, that encode proteins with significant sequence similarity to mammalian facilitative glucose transporters. Pro-1 mRNAs are expressed almost exclusively in the promastigote or insect stage of the parasite life cycle. The Pro-1 tandem repeat encodes two isoforms of the putative transporter, iso-1 and iso-2, which have identical predicted amino acid sequences except for their NH2-terminal hydrophilic domains. We have now expressed both iso-1 and iso-2 by microinjecting their RNAs into Xenopus oocytes and assaying these oocytes for transport of various radiolabeled ligands. Both iso-1 and iso-2 transport [3H]2-deoxy-D-glucose, confirming that each protein is a bona fide glucose transporter. Each isoform also transports fructose and, to a much lesser degree, mannose. Compounds which inhibit 2-deoxy-D-glucose transport in L. enriettii promastigotes also inhibit transport in the microinjected oocytes expressing each isoform, indicating that the substrate specificities and pharmacological properties of each isoform are similar to those measured for 2-deoxy-D-glucose transport in intact parasites. The Km for transport of 2-deoxyglucose in oocytes expressing iso-1 is similar to that for oocytes expressing iso-2. These results reveal that both transporter isoforms have closely related functional properties and that the difference in their structures may serve some other purpose such as differential subcellular localization.

Cloning and functional analysis of an extrachromosomally amplified multidrug resistance-like gene in Leishmania enriettii.

The goal of this work was to investigate the mechanism of drug resistance in Leishmania enriettii as a model system for drug resistance both in human leishmaniasis and on other parasitic diseases. Parasites were selected in increasing concentrations of vinblastine, an inhibitor of microtubule assembly, and resistant clones were isolated which grew in concentrations 5-30 times the IC50 (30 micrograms ml-1) of parental cells. The vinblastine-resistant parasites were also resistant to puromycin, an unrelated drug which inhibits protein synthesis. This cross-resistance to unrelated drugs had previously been observed in mammalian cells and recently in L. donovani. The proposed mechanism for this cross-resistance is drug efflux mediated by increased expression of a P-glycoprotein molecule encoded by a multidrug resistance (mdr) gene. Here we report the identification, cloning and sequencing of an mdr-like gene from L. enriettii, lemdr1, and demonstrate that this gene is amplified on an extrachromosomal circle of 35-40 kb in vinblastine-resistant L. enriettii. The longest open reading frame in the cloned gene is 1280 amino acids with a predicted protein of 140 kDa. The predicted protein has a structure similar to that for all other reported P-glycoproteins namely 12 transmembrane domains and 2 ATP binding sites, arranged in 2 similar half-molecules. Comparison of the primary amino acid sequence with other known mdr gene products demonstrates a significant homology with 37% amino acid identity with human mdr1 and 83% identity with the L. donovani ldmdr1 gene. The lemdr1 gene was cloned in the expression vector pALTNEO and transfected into wild-type L. enriettii and the resulting transfected cells were resistant to vinblastine but at lower levels than in the selected mutant cells.