The Akt-like kinase of Leishmania panamensis: As a new molecular target for drug discovery.

The Akt-like kinase of Leishmania spp. is a cytoplasmic orthologous protein of the serine/threonine kinase B-PKB/human-Akt group, which is involved in the cellular survival of these parasites. By the application of a computational strategy we obtained two specific inhibitors of the Akt-like protein of L. panamensis (UBMC1 and UBMC4), which are predicted to bind specifically to the pleckstrin domain (PH) of the enzyme. We show that the Akt-like of Leishmania panamensis is phospho-activated in parasites under nutritional and thermic stress, this phosphorylation is blocked by the UBMC1 and UMBC2 and such inhibition leads to cell death. Amongst the effects caused by the inhibitors on the parasites we found high percentage of hypodiploidy and loss of mitochondrial membrane potential. Ultrastructural studies showed highly vacuolated cytoplasm, as well as shortening of the flagellum, loss of nuclear membrane integrity and DNA fragmentation. Altogether the presented results suggest that the cell death caused by UMBC1 and UMBC4 may be associated to an apoptosis-like process. The compounds present an inhibitory concentration (IC50) over intracellular amastigotes of L. panamensis of 9.2±0.8µM for UBMC1 and 4.6±1.9µM for UBMC4. The cytotoxic activity for UBMC1 and UBMC4 in human macrophages derived from monocytes (huMDM) was 29±1.2µM and >40µM respectively. Our findings strongly support that the presented compounds can be plausible candidates as a new therapeutic alternative for the inhibition of specific kinases of the parasite.

Comparative protein profiling identifies elongation factor-1beta and tryparedoxin peroxidase as factors associated with metastasis in Leishmania guyanensis.

Parasites of the Leishmania Viannia subgenus are major causative agents of mucocutaneous leishmaniasis (MCL), a disease characterised by parasite dissemination (metastasis) from the original cutaneous lesion to form debilitating secondary lesions in the nasopharyngeal mucosa. We employed a protein profiling approach to identify potential metastasis factors in laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) through infrequently metastatic (M+/M-) to non-metastatic (M-). Comparison of the soluble proteomes of promastigotes by two-dimensional electrophoresis revealed two abundant protein spots specifically associated with M+ and M+/M- clones (Met2 and Met3) and two others exclusively expressed in M- parasites (Met1 and Met4). The association between clinical disease phenotype and differential expression of Met1-Met4 was less clear in L. Viannia strains from mucosal (M+) or cutaneous (M-) lesions of patients. Identification of Met1-Met4 by biological mass spectrometry (LC-ES-MS/MS) and bioinformatics revealed that M+ and M- clones express distinct acidic and neutral isoforms of both elongation factor-1 subunit beta (EF-1beta) and cytosolic tryparedoxin peroxidase (TXNPx). This interchange of isoforms may relate to the mechanisms by which the activities of EF-1beta and TXNPx are modulated, and/or differential post-translational modification of the gene product(s). The multiple metabolic functions of EF-1 and TXNPx support the plausibility of their participation in parasite survival and persistence and thereby, metastatic disease. Both polypeptides are active in resistance to chemical and oxidant stress, providing a basis for further elucidation of the importance of antioxidant defence in the pathogenesis underlying MCL.

The glycoinositolphospholipids from Leishmania panamensis contain unusual glycan and lipid moieties.

The cell surface of Leishmania parasites is coated by glycosylphosphatidylinositol (GPI)-anchored macromolecules (glycoproteins and a lipophosphoglycan) and a polymorphic family of free GPI glycolipids or glycoinositolphospholipids (GIPLs). Here we show that GIPLs with unusual glycan and lipid moieties are likely to be major cell surface components of L. panamensis (subgenus Viannia) promastigotes. These glycolipids were purified by high performance thin layer chromatography and their structures determined by gas-liquid chromatography-mass spectrometry, fast-atom bombardment mass spectrometry, methylation analysis and chemical and enzymatic sequencing of the glycan headgroups. The major GIPLs contained two glycan core sequences, Manalpha1-3Manalpha1-4GlcN-phosphatidylinositol (type-2 series) or Manalpha1-3[Manalpha1-2Manalpha1-6]Manalpha1- 4GlcN-phosphatidylinosit ol (hybrid series), which were elaborated with Galalpha1-2Galbeta1- or Galalpha1-2/3Galalpha1-2Galbeta1- extensions that were attached to the 3-position of the alpha1-3 linked mannose. The phosphatidylinositol moiety contained exclusively diacylglycerol with palmitoyl, stearoyl and heptadecanoyl chains. Non-galactosylated GIPL species with the same core structures were also found. The galactose extensions and the presence of diacylglycerol in the lipid moieties are novel features for the GIPLs of Leishmania spp. The implications of these structures for the biosynthesis of leishmanial GIPLs and their putative function in the mammalian host are discussed.

Discovery of factors linked to antimony resistance in Leishmania panamensis through differential proteome analysis.

The rate of treatment failure to antileishmanial chemotherapy in Latin America is up to 64%. Parasite drug resistance contributes to an unknown proportion of treatment failures. Identification of clinically relevant molecular mechanisms responsible for parasite drug resistance is critical to the conservation of available drugs and to the discovery of novel targets to reverse the resistant phenotype. We conducted comparative proteomic-based analysis of Leishmania (Viannia) panamensis lines selected in vitro for resistance to trivalent antimony (Sb(III)) to identify factors associated with antimony resistance. Using 2-dimensional gel electrophoresis, two distinct sub-proteomes (soluble in NP-40/urea and Triton X-114, respectively) of promastigotes of WT and Sb(III)-resistant lines were generated. Overall, 9 differentially expressed putative Sb-resistance factors were detected and identified by mass spectrometry. These constituted two major groups: (a) proteins involved in general stress responses and (b) proteins with highly specific metabolic and transport functions, potentially directly contributing to the Sb-resistance mechanism. Notably, the sulfur amino acid-metabolizing enzymes S-adenosylmethionine synthetase (SAMS) and S-adenosylhomocysteine hydrolase (SAHH) were over-expressed in Sb(III)-resistant lines and Sb(III)-resistant clinical isolates. These enzymes play a central role in the upstream synthesis of precursors of trypanothione, a key molecule involved in Sb-resistance in Leishmania parasites, and suggest involvement of epigenetic regulation in response to drug exposure. These data re-enforce the importance of thiol metabolism in Leishmania Sb resistance, reveal previously unrecognized steps in the mechanism(s) of Sb tolerance, and suggest a cross-talk between drug resistance, metabolism and virulence.

Molecular and antigenic characterization of the Leishmania (Viannia) panamensis kinetoplastid membrane protein-11.

The kinetoplastid membrane protein 11 (KMP-11) has been recently described in Leishmania (Leishmania) donovani as a major component of the promastigote membrane. Two oligonucleotide primers were synthesized to PCR-amplify the entire encoding region of New World Leishmania species. The Leishmania (Viannia) panamensis amplification product was clone, sequenced and the putative amino acid sequence determined. A remarkably high degree of sequence homology was observed with the corresponding molecule of L. (L) donovani and L. (L) infantum (97% and 96%, respectively). Southern blot analysis showed that the KMP-11 locus is conformed by three copies of the gene. the L. (V) panamensis ORF was subsequently clone in a high expression vector and the recombinant protein was induced and purified from Escherichia coli cultures. Immunoblot analysis showed that 80%, 77% and 100% sera from cutaneous, mucocutaneous and visceral leishmaniasis patients, respectively, recognized the recombinant KMP-11 protein. In a similar assay, 86% of asymptomatic Leishmania-infected individuals showed IgG antibodies against the rKMP-11. We proposed that KMP-11 could be used as a serologic marker for infection and disease caused by Leishmania in America.

Glucantime susceptibility of Leishmania promastigotes under variable growth conditions.

Growth inhibition of Leishmania promastigotes by glucantime was compared in three different media. Glucantime inhibited the growth of Leishmania cultured in complex medium but did not affect parasite growth when added to cells cultured in defined or semi-defined media. Supplementation of the complex medium with biopterin partially reversed the glucantime effect in sensitive strains, although the addition of folic acid or oleic acid did not alter the activity of glucantime. Differences in fatty acid composition were observed between strains showing different degrees of glucantime susceptibility.

Leishmania: fine mapping of the Leishmanolysin molecule's conserved core domains involved in binding and internalization.

The Leishmanolysin molecule's role in the uptake of Leishmania parasites by the human U937 pro-myelocytic cell line was studied, using synthetic peptides representing the complete Leishmania (Viannia) guyanensis Leishmanolysin protein amino acid sequence. The particular peptides present in two protein's core domains efficiently impaired the internalization of promastigotes from four different Leishmania species and modified the kinetics of the binding of heterologous recombinant Leishmanolysin protein. The functional domains which exhibited this property represent a highly conserved portion of the sequence among different Leishmania species. The peptides' inhibitory activity correlated with their ability to bind molecules present on the surface of the human cell line. One of the two functional core domains identified involves the previously described adhesive sequence (SRYD) and the putative zinc-binding motif (HExxH). The second functional core domain includes a third histidine residue coordinated with zinc which determines the molecule's structural features. These findings indicate that the molecular interactions between Leishmanolysin's conserved domains and the macrophage surface molecules efficiently contribute to the parasite's internalization. Induction of neutralizing immune responses, which impair the early parasite-host interaction described here, may be an important alternative in designing synthetic subunit human leishmaniasis vaccines.