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. (+info)
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. (+info)
Quinacrine and a novel apigenin dimer can synergistically increase the pentamidine susceptibility of the protozoan parasite Leishmania.
Effect of increasing intravesicular pH on nitrite production and leishmanicidal activity of activated macrophages.
We examined the effect of bafilomycin A1 (BAF), an inhibitor of vacuolar-type H(+)-ATPases, on macrophages activation (measured as increased nitrite production and leishmanicidal activity) induced by interferon gamma alone or together with lipopolysaccharide or tumour necrosis factor alpha. BAF increased intravesicular pH and enhanced nitrite release by activated macrophages; however, the NO concentration necessary to kill parasites was higher in BAF-exposed than control macrophages, suggesting that microbicidal nitrogen derivatives were less active at alkaline pH. Antibody to tumour necrosis factor alpha inhibited BAF-induced nitrite production in interferon-activated cultures. To determine if enhanced NO synthesis was related to vesicular alkalinization, macrophages were incubated with the lysosomotropic bases NH4Cl and methylamine. These agents also increased intravesicular pH and nitrite production. Nitrite production was correlated with enhanced NO synthase activity in cytosolic extracts of the activated cells. (+info)
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. (+info)
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. (+info)
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. (+info)
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. (+info)