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.

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.

Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

Structural isoforms of a membrane transport protein from Leishmania enriettii.

A membrane transport protein of the glucose transporter superfamily from Leishmania enriettii is encoded by a family of tandemly repeated genes. The first gene in this tandem repeat codes for a structural isoform that contains a unique amino-terminal hydrophilic domain, probably located in the cytoplasm; the remainder of the protein is identical to the polypeptide encoded by the internal genes in the tandem repeat. The unique isoform is represented by a distinct stable RNA.

Structure and regulation of histone H2B mRNAs from Leishmania enriettii.

We have studied the structure and expression of histone H2B mRNA and genes in the parasitic protozoan Leishmania enrietti. A genomic clone containing three tandemly repeated genes has been sequenced and shown to encode three identical histone proteins and two types of closely related mRNA sequence. We have also sequenced three independent cDNA clones and demonstrated that the Leishmania H2B mRNAs are polyadenylated, similar to the basal histone mRNAs of higher eucaryotes and the histone mRNAs of yeast. In addition, the Leishmania mRNAs contain inverted repeats near the poly(A) tail which could form stem-loops similar in secondary structure, but not in sequence, to the 3' stem-loops of nonpolyadenylated replication-dependent histones of higher eucaryotes. Unlike the replication-dependent histones, the Leishmania histone H2B mRNAs do not decrease in abundance following treatment with inhibitors of DNA synthesis. The histone mRNAs are differentially expressed during the parasite life cycle and accumulate to a higher level in the extracellular promastigotes (the form which in nature lives within the gut of the insect vector) than in the intracellular amastigotes (the form that lives within the mammalian host macrophages).

Transcriptional control of gene expression during development of Dictyostelium discoideum.

During development of the cellular slime mold Dictyostelium discoideum, approximately 2,000 to 3,000 regulated mRNAs are induced when amoebae enter multicellular aggregates. We used in vitro transcription in isolated nuclei to follow the synthesis of individual mRNA precursors during development; these were quantitated by hybridization to cloned cDNAs or genomic DNAs. Those RNAs that are present at all stages of development--the common RNAs--were transcribed by nuclei from cells at all stages of development. By contrast, those RNAs that are present only after cells begin to aggregate--here called aggregation stage RNAs--were transcribed only by nuclei from cells at the aggregation and postaggregation stages of development. The temporal pattern of in vitro transcription correlated well with the time course of accumulation of different aggregation stage mRNAs. Continued expression of aggregation stage genes normally depends upon cell-to-cell contact or cyclic AMP (cAMP); when cells are disaggregated, the regulated mRNAs are rapidly and specifically degraded. When cAMP is subsequently added to the disaggregated cells, most of the mRNAs reaccumulate. We show here that disaggregation reduced 2- to 10-fold the relative transcription of several aggregation stage RNAs, whereas addition of cAMP to disaggregated cells reinduced the level of regulated gene transcription to values approximating those found in normal postaggregation cells. These results indicate that a representative set of Dictyostelium aggregation stage genes are under transcriptional control; both the transcription and the stability of these mRNAs require either continued cell-to-cell interactions or cAMP.

Functional expression of a myo-inositol/H+ symporter from Leishmania donovani.

The vast majority of surface molecules in such kinetoplastid protozoa as members of the genus Leishmania contain inositol and are either glycosyl inositol phospholipids or glycoproteins that are tethered to the external surface of the plasma membrane by glycosylphosphatidylinositol anchors. We have shown that the biosynthetic precursor for these abundant glycolipids, myo-inositol, is translocated across the parasite plasma membrane by a specific transporter that is structurally related to mammalian facilitative glucose transporters. This myo-inositol transporter has been expressed and characterized in Xenopus laevis oocytes. Two-electrode voltage clamp experiments demonstrate that this protein is a sodium-independent electrogenic symporter that appears to utilize a proton gradient to concentrate myo-inositol within the cell. Immunolocalization experiments with a transporter-specific polyclonal antibody reveal the presence of this protein in the parasite plasma membrane.

Genetics and biochemistry of Leishmania membrane transporters.

The ability to clone and functionally express genes encoding membrane transporters in Leishmania and related parasitic protozoa has illuminated the processes whereby these parasites acquire nutrients from their hosts. It is now possible to probe the physiological functions of these permeases and investigate their role in drug delivery and resistance.

Nucleoside transporters of parasitic protozoa.

Protozoan parasites are incapable of synthesizing purine nucleotides de novo and so must salvage preformed purines from their hosts. This process of purine acquisition is initiated by the translocation of preformed host purines across parasite or host membranes. Here, we report upon the identification and isolation of DNAs encoding parasite nucleoside transporters and the functional characterization of these proteins in various expression systems. These potential approaches provide a powerful approach for a thorough molecular and biochemical dissection of nucleoside transport in protozoan parasites.

Structure of mRNA encoded by tubulin genes in Leishmania enriettii.

Both the alpha- and beta-tubulin genes of Leishmania enriettii are encoded by mRNA of 2.0-2.2 kb in length. We have shown previously that the alpha- and beta-tubulin genes are arranged in separate, tandem repeats of 2 and 4 kb, respectively, and now report the mapping of mature mRNA onto these cloned genes. Here we show that the alpha-tubulin gene contains a very short intergenic region (100-200 bases) whereas the larger, tandemly repeated beta-tubulin gene contains a 1.8-2.0 kb region not found in mature mRNA. Comparison of S-1 mapping and primer extension results indicates that the messenger RNAs for both alpha- and beta-tubulin contain a sequence of about 35 base pairs located at the 5' end that is not encoded contiguously with the rest of the mRNA. This short 5' sequence may be added to the body of the tubulin mRNAs either through splicing of a precursor RNA molecule or by a novel post-transcriptional processing reaction. The alpha- and beta-tubulin genes are arranged identically in the two developmental stages of the parasite life cycle and are present in equal copy number.

The flagellum and flagellar pocket of trypanosomatids.

The flagellum and flagellar pocket are distinctive organelles present among all of the trypanosomatid protozoa. Currently, recognized functions for these organelles include generation of motility for the flagellum and dedicated secretory and endocytic activities for the flagellar pocket. The flagellar and flagellar pocket membranes have long been recognized as morphologically separate domains that are component parts of the plasma membrane that surrounds the entire cell. The structural and functional specialization of these two membranes has now been underscored by the identification of multiple proteins that are targeted selectively to each of these domains, and non-membrane proteins have also been identified that are targeted to the internal lumina of these organelles. Investigations on the functions of these organelle-specific proteins should continue to shed light on the unique biological activities of the flagellum and flagellar pocket. In addition, work has begun on identifying signals or modifications of these proteins that direct their targeting to the correct subcellular location. Future endeavors should further refine our knowledge of targeting signals and begin to dissect the molecular machinery involved in transporting and retaining each polypeptide at its designated cellular address.

Substrate depletion upregulates uptake of myo-inositol, glucose and adenosine in Leishmania.

Leishmania flagellates undergo a digenetic life cycle in the gut of the sandfly insect vector and in macrophage phagolysosomes of the mammalian host. This involves vast changes of the environment to which the parasite has to adapt, including temperature, pH and concentration of nutrients between different types of meals of the insect vector or within the enclosed intracellular environment of the phagolysosome. The regulation of transporters for important organic substrates in Leishmania donovani, Leishmania mexicana and Leishmania enriettii has been investigated. A pronounced upregulation of inositol (25-fold), adenosine (11-fold) or glucose (5-fold) uptake activities was found when cells were depleted of the respective substrates during culture. Inositol-depleted cells showed a half-maximal uptake rate at nanomolar inositol concentration. Depletion of inositol only affected inositol uptake but did not affect uptake of glucose analog or proline in control experiments, indicating the specificity of the mechanism(s) underlying transport regulation. Adenosine-depleted cells showed an approximately 10-fold increase in both adenosine and uridine uptake, both mediated by the L. donovani nucleoside transporter 1 (LdNT1), but no change in guanosine uptake, which is mediated by the L. donovani nucleoside transporter 2 (LdNT2). These results suggest that extracellular adenosine concentration specifically regulates LdNT1 transport activity and does not affect LdNT2. The data imply that upregulation of transport activities by substrate depletion is a general phenomenon in protozoan flagellates, which is in remarkable contrast to bacteria where upregulation typically follows an increase of extracellular organic substrate. Hence, the parasites can maximize the uptake of important nutrients from the host even under limiting conditions, whereas bacteria often have dormant stages (spores) to overcome unfavorable environmental conditions or are heterotrophic for organic substrates.

Transcriptional mapping of Leishmania enrietti tubulin mRNAs.

We have mapped the mRNAs for alpha- and beta-tubulins of Leishmania enriettii promastigotes and amastigotes and have demonstrated that each RNA contains a 35 nucleotide sequence on its 5' end which is not encoded contiguously with the rest of the message. Sequencing of the 5' end of the alpha- and beta-tubulin mRNAs revealed that this 35 nucleotide leader sequence is identical in both messages and that it is homologous to the spliced leader found on the 5' end of mRNAs in the related parasite Trypanosoma brucei. Additionally, we have sequenced regions of the alpha- and beta-tubulin genomic clones upstream from the mRNA encoding regions and have shown that the leader sequence is not encoded within these regions of DNA. Hence, Leishmania tubulin mRNAs may be synthesized by a discontinuous transcription process that links together the transcription products of two separate gene families, as suggested previously by others for the assembly of T. brucei mRNAs. Homologies between the Leishmania alpha- and beta-tubulin genes themselves and between these genes and the T. brucei alpha- and beta-tubulin genes have revealed sequences which may be important in synthesis or processing of Leishmania tubulin mRNAs.

Molecular characterization of two genes encoding members of the glucose transporter superfamily in the parasitic protozoan Leishmania donovani.

The polymerase chain reaction was used to clone two genes from Leishmania donovani, each of which encodes a member of a superfamily of membrane transporters which include the mammalian facilitative glucose transporters. One of these transporters, designated D2, is similar in sequence and overall structural features to a previously cloned Leishmania transporter Pro-1. Both D2 and Pro-1 are developmentally regulated genes which are expressed primarily in the insect stage of the parasite life cycle. In contrast, the second novel transporter, D1, is structurally quite different from either D2 or Pro-1, and its expression is not regulated during the parasite life cycle. All three genes are located on different chromosomes in L. donovani.

Molecular genetics of nucleoside transporters in Leishmania and African trypanosomes.

Nucleoside transporters play central roles in the biochemistry of parasitic protozoa such as Leishmania and African trypanosomes, because these parasites cannot synthesize purines de novo and are absolutely reliant upon purine salvage from their hosts. Furthermore, nucleoside transporters are important to the pharmacology of these significant human pathogens, because they mediate the uptake of purine analogs, as well as some non-purine drugs, that are selectively cytotoxic to the parasites. Recent advances in molecular biology and genomics have allowed the cloning and functional expression of several nucleoside transporter genes from Leishmania donovani and Trypanosoma brucei, providing molecular reagents for a detailed functional examination of these permeases and their role in the delivery of nutrients and drugs to the parasites. Furthermore, the molecular basis of drug-resistant mutants that are deficient in nucleoside transport functions can now be fathomed.

18S rRNA sequences of Leishmania enriettii promastigote and amastigote.

Dideoxy sequencing with reverse transcriptase and universal primers was used to obtain partial sequences of the 18S rRNAs from the promastigote and amastigote life-cycle stages of L. enriettii. Approximately 1400 nucleotides of sequence from the two stages were compared. Unlike Plasmodium berghei, in which 18S rRNAs from the mosquito stage and the mammalian stage of the life cycle are only 96.5% similar, the amastigote and promastigote rRNAs of L. enriettii are identical. In addition, a comparison of 1425 bases of the L. enriettii promastigote sequence with the published sequence of L. donovani revealed only four differences; the two sequences are 99.8% similar. A likely explanation for this high similarity, considering the 97% similarity between L. donovani and the related genus Crithidia fasciculata, is that the two species are closely related and of comparatively recent origin. The low diversity between the 18S rRNA sequences of Leishmania species is similar to that reported for 13 Tetrahymena species, where similarities ranged from 98.1 to 99.9%, but different from the pattern reported in the genus Naegleria, where divergence was greater.

Transporters for drug delivery and as drug targets in parasitic protozoa.

Parasitic protozoa cause devastating diseases across large regions of the globe, but a lack of economic incentives has resulted in the limited development of drugs against these "neglected diseases." Transporters expressed in the plasma membranes of these parasites offer potential for the development of new drugs. These permeases could be employed in two distinct strategies for drug development: (i) targeting selective delivery of drugs to the parasite and (ii) developing drugs that inhibit essential parasite permeases.

Control of tubulin gene expression in the parasitic protozoan Leishmania enriettii.

The life cycle of parasitic protozoa of the genus Leishmania consists of two morphologically distinct forms: (1) amastigotes, the form of the parasite that resides inside macrophages of the mammalian host, which are non-motile and possess only a residual flagellum; and (2) promastigotes, the extracellular forms that live in the gut of the sandfly vector, which are highly motile and possess a single prominent flagellum. During the developmental transformation of amastigotes to promastigotes, the biosynthesis of alpha- and beta-tubulin proteins is dramatically increased, presumably to provide sufficient tubulin for synthesis and maintenance of the flagellum. We show here that the level of alpha- and beta-tubulin mRNA in Leishmania enriettii promastigotes is significantly greater than that in amastigotes, as determined by both Northern blot analysis and by in vitro translation of cellular RNA. These results show that the regulated expression of the tubulin genes is controlled at the level of mRNA accumulation in L. enriettii , by contrast with Leishmania mexicana, in which the control of gene expression has been reported to be at the level of translation.

Differential regulation of multiple glucose transporter genes in Leishmania mexicana.

We have studied the structure and expression of glucose transporter genes in the parasitic protozoan Leishmania mexicana. Three distinct glucose transporter isoforms, LmGT1, LmGT2, and LmGT3, are encoded by single copy genes that are clustered together at a single locus. Quantitation of Northern blots reveals that LmGT2 mRNA is present at approximately 15-fold higher level in promastigotes, the insect stage of the parasite life cycle, compared with amastigotes, the intracellular stage of the life cycle that lives within the mammalian host. In contrast, LmGT1 and LmGT3 mRNAs are expressed at similar levels in both life cycle stages. Transcription of the LmGT genes in promastigotes and axenically cultured amastigotes occurs at similar levels, as measured by nuclear run-on transcription. Consequently, the approximately 15-fold up-regulation of LmGT2 mRNA levels in promastigotes compared with amastigotes must be controlled at the post-transcriptional level. Measurement of LmGT2 RNA decay in promastigotes and axenic amastigotes treated with actinomycin D suggests that differential mRNA stability may play a role in regulating glucose transporter mRNA levels in the two life cycle stages.

Four conserved cytoplasmic sequence motifs are important for transport function of the Leishmania inositol/H(+) symporter.

The protozoan Leishmania donovani has a myo-inositol/proton symporter (MIT) that is a member of a large sugar transporter superfamily. Active transport by MIT is driven by the proton electrochemical gradient across the parasite membrane, and MIT is a prototype for understanding the function of an active transporter in lower eukaryotes. MIT contains two duplicated 6- or 7-amino acid motifs within cytoplasmic loops, which are highly conserved among 50 members of the sugar transporter superfamily and are designated A(1), A(2) ((V)(D/E)(R/K)PhiGR(R/K)), and B(1) (PESPRPhiL), B(2) (VPETKG). In particular, the three acidic residues within these motifs, Glu(187)(B(1)), Asp(300)(A(2)), and Glu(429)(B(2)) in MIT, are highly conserved with 96, 78, and 96% amino acid identity within the analyzed members of this transporter superfamily ranging from bacteria, archaea, and fungi to plants and the animal kingdom. We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopus oocytes and overexpression in Leishmania transfectants to investigate the significance of these three acidic residues in the B(1), A(2), and B(2) motifs. Alteration to the uncharged amides greatly reduced MIT transport function to 23% (E187Q), 1.4% (D300N), and 3% (E429Q) of wild-type activity, respectively, by affecting V(max) but not substrate affinity. Conservative mutations that retained the charge revealed a less pronounced effect on inositol transport with 39% (E187D), 16% (D300E) and 20% (E429D) remaining transport activity. Immunofluorescence microscopy of oocyte cryosections confirmed that MIT mutants were expressed on the oocyte surface in similar quantity to MIT wild type. The proton uncouplers carbonylcyanide-4-(trifluoromethoxy) phenylhydrazone and dinitrophenol inhibited inositol transport by 50-70% in the wild type as well as in E187Q, D300N, and E429Q, despite their reduced transport activities, suggesting that transport in these mutants is still proton-coupled. Furthermore, temperature-dependent uptake studies showed an increased Arrhenius activation energy for the B(1)-E187Q and the B(2)-E429Q mutants, which supports the idea of an impaired transporter cycle in these mutants. We conclude that the conserved acidic residues Glu(187), Asp(300), and Glu(429) are critical for transport function of MIT.