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.

Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites.

Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 -/- and overexpressing cell lines. The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome. Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.

Characterization of a targeting motif for a flagellar membrane protein in Leishmania enriettii.

The surface membranes of eukaryotic flagella and cilia are contiguous with the plasma membrane. Despite the absence of obvious physical structures that could form a barrier between the two membrane domains, the lipid and protein compositions of flagella and cilia are distinct from the rest of the cell surface membrane. We have exploited a flagellar glucose transporter from the parasitic protozoan Leishmania enriettii as a model system to characterize the first targeting motif for a flagellar membrane protein in any eukaryotic organism. In this study, we demonstrate that the flagellar membrane-targeting motif is recognized by several species of Leishmania. Previously, we demonstrated that the 130 amino acid NH(2)-terminal cytoplasmic domain of isoform 1 glucose transporter was sufficient to target a nonflagellar integral membrane protein into the flagellar membrane. We have now determined that an essential flagellar targeting signal is located between amino acids 20 and 35 of the NH(2)-terminal domain. We have further analyzed the role of specific amino acids in this region by alanine replacement mutagenesis and determined that single amino acid substitutions did not abrogate targeting to the flagellar membrane. However, individual mutations located within a cluster of five contiguous amino acids, RTGTT, conferred differences in the degree of targeting to the flagellar membrane and the flagellar pocket, implying a role for these residues in the mechanism of flagellar trafficking.

Leishmania spp.: mechanisms of toxicity of nitrogen oxidation products.

Intracellular killing of Leishmania parasites within activated murine macrophages is thought to result from the toxic activities of nitrogen oxidation products (referred to as NO) released by the activated cells. In order to determine possible mechanisms of NO toxicity for these microorganisms, promastigotes of Leishmania major and Leishmania enriettii were exposed to NO generated chemically from acidified nitrite, S-nitrosocysteine, diethylamine NONOate, or nitroprusside. Treatment with these agents led to loss of viability (as determined from decreased motility and inhibition of [3H]TdR uptake upon reincubation in NO-free medium) with kinetics characteristic for each compound L. major was less sensitive to these effects than L. enriettii, and amastigotes displayed the same sensitivity as promastigotes of the same species. The early effects of NO toxicity could be detected within minutes of exposure to the NO donors; they included decreased respiration rate and inhibition of glucose, proline, and adenine incorporation. Inhibition of the activities of glyceraldehyde 3-phosphate dehydrogenase and of aconitase were also evidenced. In order to determine whether these phenomena reflected the mechanisms of toxicity of bona fide NO generated by macrophages, promastigotes were exposed to IFN-gamma + LPS-activated macrophages across permeable membranes. This resulted in marked inhibition of proline and adenine uptake in the parasites, which was restored, however, to control levels when macrophages were activated in the presence of the nitric oxide synthase inhibitor NGMMA. These results indicate that several cellular targets may be subject to NO toxicity in Leishmania parasites, including enzymes of glycolysis and respiratory metabolism as well as trans-membrane transport systems.

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.

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.

Transforming growth factor beta 1 regulation of macrophage activation depends on the triggering stimulus.

We have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the regulation of murine bone marrow-derived macrophage function. TGF-beta, added simultaneously with or up to 4 h before interferon-gamma (IFN-gamma) plus lipopolysaccharide (LPS), inhibited macrophage leishmanicidal activity, nitrite (NO2-) production, and secretion of prostaglandin E2. In contrast, no effect of TGF-beta could be demonstrated on macrophages stimulated with IFN-gamma plus tumor necrosis factor-alpha (TNF-alpha) under the same conditions. These results suggested that TGF-beta inhibited LPS-induced triggering of macrophage activation, which was confirmed by studies with IFN-gamma-primed cells. Interestingly, when macrophages were pretreated with TGF-beta for 24 h, NO2- production in response to IFN-gamma plus TNF-alpha was also inhibited. Although control and IFN-gamma/LPS-stimulated macrophages were found to secrete latent TGF-beta, only the IFN-gamma/LPS cultures produced biologically active TGF-beta. Significantly, active TGF-beta was present at concentrations shown earlier to inhibit macrophage function.