Kinetic and mutational analysis of the Trypanosoma brucei NBT1 nucleobase transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS transporters.

Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.

First detection and genetic typing of Leishmania infantum MON-24 in a dog from the Moroccan Mediterranean coast: genetic diversity of MON-24.

As in the countries edging the Mediterranean basin, Leishmania infantum zymodeme MON-1 is the main causative agent of visceral leishmaniasis in Morocco, where visceral leishmaniasis is most active in the North-Eastern slopes of the Rif mountains. The dog was confirmed to be the main reservoir of L. infantum MON-1, while the reservoir of L. infantum MON-24 causative agent of both infantile visceral leishmaniasis and cutaneous leishmaniasis has not yet been identified. Here we report the first detection of this last zymodeme in a dog in Morocco. The isolated strain was first identified by the use of genotyping markers and confirmed by isoenzyme analysis. Phylogenetic analysis with the use of concatenated sequences from 26 Leishmania donovani complex strains revealed strong geographical correlation with the MON-24 strain from Morocco clustering with other East African strains whereas two other MON-24 strains clustered with L. infantum strains. Interestingly, the two distinct populations of MON-24 identified with the use of genotyping markers cannot be distinguished by multilocus enzyme electrophoresis.

Identification of the first pyrimidine nucleobase transporter in Leishmania: similarities with the Trypanosoma brucei U1 transporter and antileishmanial activity of uracil analogues.

While purine transport has been widely studied in protozoa, almost nothing is known about their capacity to salvage pyrimidines. Here, we report a Leishmania major transporter with high affinity for uracil (Km=0.32+/-0.07 microM) which we designated LmU1. This transporter displayed a high degree of specificity, as it had virtually no affinity for cytosine, thymine or purine nucleobases, nor did it transport pyrimidine nucleosides. Highest affinity was for 5-fluorouracil. The results show that the permeant binding site of LmU1 interacts strongly with the keto groups of uracil, as shown by a low affinity for 2-thio- and 4-thiouracil. LmU1 appears to further bind uracil through a weak hydrogen bond with N(1)H of the pyrimidine ring in addition to a stronger H-bond with N(3)H. Substrate binding and selectivity were strikingly similar to that of the U1 transporter in the related kinetoplastid Trypanosoma brucei. Uracil analogues likely to be transported by LmU1 were also screened for antileishmanial activity, with 5-fluorouracil displaying strong activity against promastigotes and intracellular amastigotes. Overall, the results show that, like purine nucleobase transport, pyrimidine nucleobase transport function is very similar in L. major and T. brucei insect forms.

The outcome of Leishmania major experimental infection in BALB/c mice can be modulated by exogenously delivered iron.

We previously established that Leishmania promastigotes express a transferrin receptor and that iron chelators inhibit promastigote growth in vitro. Thus, we were interested in modulating the vertebrate host iron pool and to monitor whether such changes will affect the outcome of L. major infection in BALB / c mice, inoculated in the footpad with 106 stationary phase promastigotes. Treatment of mice with desferrioxamine resulted in a slight delay of the development of cutaneous lesions. In contrast and unexpectedly, systemic iron delivery, at early time points of parasite delivery, significantly limited footpad pathology. Accordingly, parasite loads at the site of parasite delivery, the draining lymph node, liver and spleen were significantly reduced in iron-loaded mice. Importantly, the "protective" effect of iron delivery correlated with the presence, at the site of inoculation, of lower levels of IL-4 and IL-10 transcripts while both IFN-gamma and inducible nitric oxide synthase transcripts were at higher levels. The presence of more type 1 cytokine transcripts was further supported by the increased levels of IgG2a in their sera. These data strongly suggest that susceptibility to L. major as assessed in the footpad model is modifiable by interventions that alter the iron status of the host at early time points of parasite delivery.

Solution structures of the fibronectin-like Leishmania gp63 SRYD-containing sequence in the free and antibody-bound states--transferred NOE and molecular dynamics studies.

The anti-SRYD monoclonal antibody (mAbSRYD) raised against the IASRYDQL synthetic octapeptide, the 250-257 sequence of the Leishmania major surface glycoprotein gp63 recognizes both SRYD-containing peptides and the whole cognate major surface protein on intact parasites. Two SRYD-containing peptides, which antigenically and functionally mimic the RGDS sequence of fibronectin and efficiently inhibit parasite attachment to the macrophage receptors, were studied by two-dimensional transferred nuclear Overhauser effect experiments in the presence of mAbSRYD. The antibody-bound IASRYDQL octapeptide solution conformation was determined on the basis of 55 interproton-distance restraints, derived from NMR measurements. Eighteen structures which were first generated using an approach combining distance geometry and molecular dynamics, converge by energy minimization toward a folded structure with an average rmsd from the experimental data of less than 0.05 nm for the overall backbone and 0.025 nm for the SRYD motif. A distorted gamma-turn was found, stabilized by the backbone-backbone D255-NH to R253-CO hydrogen bond, while the R253 and D255 side chains are pointing in opposite directions. This latter antibody-bound structure is compared with that of the free octapeptide in dimethylsulfoxide solution, and with the crystal structure of the RYD fragment in OPG2 Fab, an antireceptor antibody that mimics the RGD cell adhesion site. On this basis, a mechanism for IASRYDQL-receptor interaction is discussed.

IL-1 beta transduces different signals than IL-1 alpha leading to class II antigen expression on beta-insulinoma RIN-5AH cells through specific receptors.

Like most cytokines, IL-1 transduces its signals for growth, differentiation and diverse cellular functions after binding to specific receptors on the cell surface. Up to now two IL-1 receptors have been reported, type I which induces signal transduction and type II which binds IL-1 but does not transduce signalling. By using the rat insulinoma RIN-5AH cell line that expresses both types of receptor mRNA, and computer-assisted binding analysis, we show that interleukin-1 beta (IL-1 beta) binds to a single class of high affinity receptors with a Kd of 155 pmol/l. The average number of receptors on adherent cell layer is calculated to be 7300 per cell. 125I-IL-1 beta binding can be competed out by unlabelled IL-1 beta. 125I-IL-1 alpha binding can be also obtained and is subject to competition by cold IL-1 alpha. Its saturation curve, however, varies within experiments due to differential receptor up-regulation. These results have also been confirmed by FACS analysis using specific antibodies to type I and II IL-1 receptors, where type I receptor antibody binds strongly to RIN-5AH cells, and type II receptor antibody shows weak staining, also due to inadequate receptor up-regulation. In order to determine whether functional signal transduction occurs via the receptors detected, it is shown that IL-1 beta is able to induce MHC class II antigen expression on the surface of the RIN cells, whereas IL-1 alpha is unable to do so, indicating different signal reception by the cells. IL-1 beta-induced class II upregulation shows moderate signs of p21ras or/and PKC dependency, whereas IL-1 alpha strongly activates both pathways that probably regulate different functions. Finally, both IL-1 alpha and beta induce nitric oxide (NO) production in a time-dependent fashion which appears to be unrelated to the signals and pathways described, but may be involved in the onset of autoimmune type 1 diabetes.

Use of sequential oligopeptide carriers (SOCn) in the design of potent Leishmania gp63 immunogenic peptides.

The antigenic sequence Ac-IASRYDQL (gp63-SRYD) of the major surface glycoprotein of Leishmania, gp63, was covalently attached to the Lys-N epsilon H2 groups of a new sequential oligopeptide carrier (SOCn), namely, (Lys-Aib-Gly)n (n = 5.6), in order to obtain potent immunogens and site-specific antibodies. It was shown, using 1H-NMR spectroscopy, that the gp63-SRYD octapeptides bound to the SOCn retain their original structural profile outlined by an ionic interaction between R and D side chains and a type 1 beta-turn involving the QNH-->RCO hydrogen bonding. Also, the gp63-SRYD octapeptides linked to the carrier do not experience conformational restrictions, probably because of the favorable conformation of the SOCn. Immunizations of outbred rabbits with the peptide carriers designed resulted in high-titered antibody response to the gp63-SRYD octapeptide and the gp63 cognate protein. Thus, this chemically defined model may be used for incorporating "protective" Leishmania epitopes and ultimately for the design of a multivalent synthetic vaccine against leishmaniosis.

Antigenicity and conformational analysis of the Zn(2+)-binding sites of two Zn(2+)-metalloproteases: Leishmania gp63 and mammalian endopeptidase-24.11.

The antigenic properties of the Zn(2+)-binding region of two Zn(2+)-metalloproteases, Leishmania surface protease gp63 and mammalian endopeptidase-24.11 (E-24.11), possessing in their active site the characteristic amino acid sequence HEXXH, were investigated by using oligoclonal antibodies raised against two synthetic peptides, V1VTHEMAHALG11 (pepgp63) and V1IGHEITHGFD11 (pepE-24.11), containing the respective Zn(2+)-binding sites of the cognate protein. The affinity-purified antibodies, tested on synthetic peptides modelled from the active sites of ten different Zn(2+)-metalloproteases, showed high selectivity for their respective peptides. However, cross-reactivity was revealed when the antibodies were tested against the gp63 and E-24.11 molecules. A panel of synthetic peptide analogues and peptides of various size was synthesized and used for the fine antigenic characterization of pepgp63 and pepE-24.11. The shortest peptides capable of significant antibody binding were the pentapeptides V1VTHE5 and E5ITHG9 for pepgp63 and pepE-24.11 respectively. His4 and Glu5 were found to be indispensable for anti-pepgp63 binding to pepgp63, whereas in the case of pepE-24.11, Glu5 and His8 were found to be critical. The conformational characteristics of the two peptides correlate well with the observed differences in their antigenicity. 1H-NMR studies showed that pepgp63 adopts a folded structure whereas pepE-24.11 takes up a rather flexible conformation. Moreover, the antigenically critical His4 of pepgp63 contributes to the structural stabilization of the peptide. Similarly, the antigenically critical His8 of pepE-24.11 is involved in partial structural stabilization of its C-terminal region. The generated antibodies may be useful tools for identifying and classifying proteins possessing similar Zn(2+)-binding motifs and/or environments.

Effect of iron chelation on the in-vitro growth of Leishmania promastigotes.

The development of vaccines and drugs to control leishmaniasis is urgently needed. The presence of a leishmania transferrin receptor on the parasite suggests that an adequate supply of iron is needed for the life cycle of leishmania. We have investigated the effect of iron deprivation on the growth of leishmania promastigotes in vitro using an iron chelation approach. All chelators tested reduced the rate of promastigote multiplication in a dose-dependent fashion, whereas referrated ones did not. The hydroxypyridin-4-one chelators CP94 and L1 were found to be more efficient than desferrioxamine. We suggest that iron depletion may be an effective mechanism against leishmania infection.

Expression of the major surface glycoprotein of Leishmania, gp63, in wild-type and sinefungin-resistant promastigotes.

In this study, we have surveyed gp63 expression in sinefungin-(SF)-resistant and wild-type Leishmania promastigotes. Documentation of gp63 expression in Leishmania promastigotes was carried out by Western blotting, purification of the protein and assessment of gp63 protease activity. We demonstrated a 3-4-fold and 1.5-2-fold increase of gp63 protein in SF-resistant Leishmania donovani and Leishmania tropica promastigotes compared to wild-type, respectively. Northern blot analysis showed that the increase in the amount of gp63 protein in SF-resistant compared to wild-type parasites was concomitant with an increase in gp63 mRNA. No extrachromosomal DNA was identified by alkaline lysis of isolated DNA samples and Southern blot analysis. Treatment of SF-resistant and wild-type L. donovani promastigotes with cycloheximide resulted in an increase of the steady state levels of gp63 mRNA in the SF-resistant parasites to approximately fivefold that of the wild type. After treating parasites with actinomycin D, estimated gp63 mRNA t1/2 in the wild type was 40 min and increased to 83 min in SF-resistant promastigotes. Therefore, the overexpression of gp63 may be mediated, at least in part, by post-transcriptional stabilization of a gp63 transcript by a protein factor. Down regulation of the latter factor may account for the observed increase in gp63 expression in SF-resistant promastigotes. Attempts to correlate gp63 expression with promastigote virulence suggested that the observed increase in gp63 expression did not result in a significant change in the virulence of SF-resistant compared to wild-type L. donovani promastigotes.

The Ser-Arg-Tyr-Asp region of the major surface glycoprotein of Leishmania mimics the Arg-Gly-Asp-Ser cell attachment region of fibronectin.

The major surface glycoprotein of Leishmania, gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. Binding of gp63 to macrophage receptors is inhibited by Arg-Gly-Asp-Ser (RGDS)-containing synthetic peptides of fibronectin and by antibodies to these peptides. However, gp63 lacks an RGDS tetrapeptide. We sought to identify the region of gp63 that antigenically and functionally mimics the RGDS-containing region of fibronectin. We thus synthesized on polyethylene rods overlapping tetracosapeptides covering the whole sequence of Leishmania major gp63. gp63 affinity-purified antibodies raised against fibronectin and against the RGDS-containing fibronectin decapeptide RGDSPASSKP bound specifically to gp63 residues 241-264. Subsequently, by the use of smaller peptides, the gp63 tetrapeptide 252-255 (SRYD) was identified as the minimum antibody binding segment. Single residue substitution peptide analogues showed that indeed Tyr and Gly can be alternatively substituted in the SRYD- and RGDS-containing peptides of gp63 and fibronectin, respectively, without major effects on their antibody binding capacity. Subsequently, we investigated the effect of an SRYD peptide on promastigote-macrophage interaction in vitro; treatment of macrophages with an SRYD-containing gp63 octapeptide efficiently inhibited parasite attachment to macrophage receptors. Thus, the conserved among species sequence SRYD of gp63, with significant hydrophilicity, flexibility, and beta-turn propensity features, mimics antigenically and functionally the RGDS sequence of fibronectin. We suggest that this segment constitutes the putative gp63 adhesion site.

Identification and isolation of the Leishmania transferrin receptor.

In a previous report, we have presented several lines of evidence, derived from widely different methodologies, suggesting that Leishmania has specific receptors for transferrin with a Kd similar to the mammalian transferrin receptor. This paper describes the identification, purification, and biochemical characterization of Leishmania transferrin receptor. The Leishmania transferrin receptor, detected on intact parasites by immunoperoxidase staining, was first identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blot analysis, using 125I-transferrin, as a 70-kDa protein. It has been isolated initially from Leishmania infantum promastigotes using affinity chromatography on a transferrin-Sepharose column and, subsequently, from Leishmania major promastigotes. The use of polyclonal antisera to the purified 70-kDa Leishmania transferrin receptor and to the purified rat transferrin receptor showed that the two receptors are antigenically distinct. The 70-kDa Leishmania transferrin receptor was subsequently characterized as an integral membrane glycoprotein. The monomeric state of the Leishmania transferrin receptor was demonstrated by gel filtration of purified receptor complexed with 125I-transferrin. Thus, the Leishmania transferrin receptor, unlike the mammalian receptor, is not a disulfide-linked dimer but a single 70-kDa polypeptide.

Substrate-dependent pH optima of gp63 purified from seven strains of Leishmania.

The major surface glycoprotein of Leishmania, gp63, is a membrane-bound metalloprotease. Contradictory data supporting a neutral or acidic nature of this enzyme have been presented. Seven strains of Old and New World Leishmania, including Leishmania donovani complex (Leishmania infantum and L. donovani), Leishmania major, Leishmania tropica and Leishmania mexicana amazonensis were used for the purification and comparative study of gp63. The protein was extracted from promastigotes by phase separation in Triton X-114 and purified by anion exchange chromatography. In agreement with previous reports, all purified gp63 were found to be structurally and immunologically related. Both membrane-bound gp63, on the surface of promastigotes, and the purified proteases had optimal activity at neutral to alkaline pH on azocasein, whereas their activity was optimal at acidic to neutral pH against 125I-insulin B-chain. The IC50 concentrations of 1,10-phenathroline against the two substrates, at the optimal pH, were comparable, suggesting that both activities measured were associated with gp63 rather than another contaminating enzyme. This was further supported by the comparable enrichment values, estimated from the specific activity of the enzyme during purification, using both assays. These results explain the earlier apparent discrepancies and suggest that the optimum pH of gp63 is substrate-dependent and not related to species differences or to the different purification procedures applied.

Evidence of transferrin binding sites on the surface of Leishmania promastigotes.

A glycoprotein of 78,000 molecular mass (78 kDa), associated with the membrane of Leishmania infantum promastigotes, was identified and immunopurified by monoclonal antibody (mAb) LD9 produced against isolated membrane preparations. mAb LD9 was subsequently found to bind to human transferrin, also of 78 kDa. Binding of LD9 to transferrin was completely abolished when the mAb was preabsorbed by Leishmania membranes, thereby indicating that the 78-kDa Leishmania membrane-associated glycoprotein and transferrin have common antigenic epitope(s). The 78-kDa Leishmania membrane-associated protein was released in soluble nonaggregated form by mild treatment with acetic acid saline. Anti-transferrin polyclonal antibodies, recognized both the membrane-associated and the soluble form of the 78-kDa glycoprotein. The 78-kDa soluble form was characterized further as an iron-containing protein. The above data combined with iron uptake by promastigotes as demonstrated by the Prussian blue reaction indicate that the 78-kDa Leishmania membrane-associated glycoprotein is transferrin. The binding of 125I-human transferrin to Leishmania-purified membrane preparations was then investigated. The results indicate the presence of a high affinity saturable binding site (Kd = 2.2 10(-8) M) that is specific for transferrin. We suggest that the 78-kDa glycoprotein recognized by mAb LD9 is transferrin that binds to the surface of Leishmania promastigotes via a transferrin receptor.

Identification of monomeric and oligomeric forms of a major Leishmania infantum antigen by using monoclonal antibodies.

Ten monoclonal antibodies (MAbs) produced against isolated Leishmania infantum membranes were used as probes of L. infantum membrane antigens. Western blots of L. infantum membranes, sodium dodecyl sulfate solubilized and heated at 100 degrees C before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that all 10 MAbs recognized a band at 58 kilodaltons (kDa). However, when solubilized membranes were not heated, 2 of the 10 MAbs recognized, in addition to the 58-kDa band, bands of higher molecular weight. Limited digestion of heated or nonheated membranes showed that both groups of MAbs (i.e., not capable or capable of binding to the high-molecular-weight bands) recognized the same proteolytic digests. Hydrophilic forms of the above proteins, possessing proteolytic activity, were detected and isolated by gel filtration. Protein staining of the isolated monomer analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing and heating conditions, revealed incomplete reduction of the 58-kDa protein. The reduced form of the 58-kDa protein migrated at 63 to 65 kDa and was not recognized by the MAbs. These results suggest the existence of a monomeric and an oligomeric form of the 58-kDa antigen. The observed inhibition of Leishmania promastigote-macrophage binding caused by MAbs representative of the two groups (capable of oligomeric and/or monomeric antigen recognition) suggest that the 58-kDa monomer and oligomer play an important role in promastigote-macrophage interaction. We suggest that the 58-kDa L. infantum antigen is the major surface Leishmania antigen (p63) identified by others.