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PLoS Neglected Tropical Diseases Jul 2017Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a...
BACKGROUND
Leishmania virulence factors responsible for the complicated epidemiology of the various leishmaniases remain mainly unidentified. This study is a characterization of a gene previously identified as upregulated in two of three overlapping datasets containing putative factors important for Leishmania's ability to establish mammalian intracellular infection and to colonize the gut of an insect vector.
METHODOLOGY/PRINCIPAL FINDINGS
The investigated gene encodes ATP/GTP binding motif-containing protein related to Leishmania development 1 (ALD1), a cytosolic protein that contains a cryptic ATP/GTP binding P-loop. We compared differentiation, growth rates, and infective abilities of wild-type and ALD1 null mutant cell lines of L. mexicana. Loss of ALD1 results in retarded growth kinetics but not defects in differentiation in axenic culture. Similarly, when mice and the sand fly vector were infected with the ALD1 null mutant, the primary difference in infection and colonization phenotype relative to wild type was an inability to achieve maximal host pathogenicity. While ability of the ALD1 null mutant cells to infect macrophages in vitro was not affected, replication within macrophages was clearly curtailed.
CONCLUSIONS/SIGNIFICANCE
L. mexicana ALD1, encoding a protein with no assigned functional domains or motifs, was identified utilizing multiple comparative analyses with the related and often experimentally overlooked monoxenous flagellates. We found that it plays a role in Leishmania infection and colonization in vitro and in vivo. Results suggest that ALD1 functions in L. mexicana's general metabolic network, rather than function in specific aspect of virulence as anticipated from the compared datasets. This result validates our comparative genomics approach for finding relevant factors, yet highlights the importance of quality laboratory-based analysis of genes tagged by these methods.
Topics: Animals; Female; GTP-Binding Proteins; Gene Expression Regulation, Developmental; Insect Vectors; Leishmania mexicana; Leishmaniasis, Cutaneous; Macrophages; Mice; Mice, Inbred BALB C; Protozoan Proteins; Psychodidae; Virulence
PubMed: 28742133
DOI: 10.1371/journal.pntd.0005782 -
Parasites & Vectors Nov 2017Leishmaniasis and Chagas disease are life-threatening illnesses caused by the protozoan parasites Leishmania spp. and Trypanosoma cruzi, respectively. They are known as...
BACKGROUND
Leishmaniasis and Chagas disease are life-threatening illnesses caused by the protozoan parasites Leishmania spp. and Trypanosoma cruzi, respectively. They are known as "neglected diseases" due to the lack of effective drug treatments and the scarcity of research work devoted to them. Therefore, the development of novel and effective drugs is an important and urgent need. Natural products are an important source of bioactive molecules for the development of new drugs. In this study, we evaluated the activity of enhydrin, uvedalin and polymatin B, three sesquiterpene lactones (STLs) isolated from Smallanthus sonchifolius, on Leishmania mexicana (MNYC/BZ/62/M) and Trypanosoma cruzi (Dm28c). In addition, the in vivo trypanocidal activity of enhydrin and uvedalin and the effects of these STLs on parasites' ultrastructure were evaluated.
METHODS
The inhibitory effect of the three STLs on the growth of L. mexicana amastigotes and promastigotes as well as T. cruzi epimastigotes was evaluated in vitro. The changes produced by the STLs on the ultrastructure of parasites were examined by transmission electron microscopy (TEM). Enhydrin and uvedalin were also studied in a murine model of acute T. cruzi infection (RA strain). Serum activities of the hepatic enzymes alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase were used as biochemical markers of hepatotoxicity.
RESULTS
The three compounds exhibited leishmanicidal activity on both parasite forms with IC values of 0.42-0.54 μg/ml for promastigotes and 0.85-1.64 μg/ml for intracellular amastigotes. Similar results were observed on T. cruzi epimastigotes (IC 0.35-0.60 μg/ml). The TEM evaluation showed marked ultrastructural alterations, such as an intense vacuolization and mitochondrial swelling in both L. mexicana promastigotes and T. cruzi epimastigotes exposed to the STLs. In the in vivo study, enhydrin and uvedalin displayed a significant decrease in circulating parasites (50-71%) and no signs of hepatotoxicity were detected.
CONCLUSIONS
Enhydrin, uvedalin and polymatin B possess significant leishmanicidal and trypanocidal activity on different parasite stages. These results show that these compounds may provide valuable leads for the development of new drugs against these neglected parasitic diseases.
Topics: Animals; Asteraceae; Chagas Disease; Disease Models, Animal; Lactones; Leishmania mexicana; Leishmaniasis; Liver; Mice; Microscopy, Electron, Transmission; Plant Extracts; Sesquiterpenes; Sesquiterpenes, Germacrane; Trypanosoma cruzi
PubMed: 29132413
DOI: 10.1186/s13071-017-2509-6 -
Molecular and Biochemical Parasitology Jun 2024In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to...
In eukaryotic cells, molecular fate and cellular responses are shaped by multicomponent enzyme systems which reversibly attach ubiquitin and ubiquitin-like modifiers to target proteins. The extent of the ubiquitin proteasome system in Leishmania mexicana and its importance for parasite survival has recently been established through deletion mutagenesis and life-cycle phenotyping studies. The ubiquitin conjugating E2 enzyme UBC2, and the E2 enzyme variant UEV1, with which it forms a stable complex in vitro, were shown to be essential for the differentiation of promastigote parasites to the infectious amastigote form. To investigate further, we used immunoprecipitation of Myc-UBC2 or Myc-UEV1 to identify interacting proteins in L. mexicana promastigotes. The interactome of UBC2 comprises multiple ubiquitin-proteasome components including UEV1 and four RING E3 ligases, as well as potential substrates predicted to have roles in carbohydrate metabolism and intracellular trafficking. The smaller UEV1 interactome comprises six proteins, including UBC2 and shared components of the UBC2 interactome consistent with the presence of intracellular UBC2-UEV1 complexes. Recombinant RING1, RING2 and RING4 E3 ligases were shown to support ubiquitin transfer reactions involving the E1, UBA1a, and UBC2 to available substrate proteins or to unanchored ubiquitin chains. These studies define additional components of a UBC2-dependent ubiquitination pathway shown previously to be essential for promastigote to amastigote differentiation.
Topics: Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Protozoan Proteins; Leishmania mexicana; Protein Binding; Protein Interaction Mapping; Immunoprecipitation
PubMed: 38556171
DOI: 10.1016/j.molbiopara.2024.111619 -
Molecular and Cellular Biology Dec 2001Leishmania parasites synthesize an abundance of mannose (Man)-containing glycoconjugates thought to be essential for virulence to the mammalian host and for viability....
Leishmania parasites synthesize an abundance of mannose (Man)-containing glycoconjugates thought to be essential for virulence to the mammalian host and for viability. These glycoconjugates include lipophosphoglycan (LPG), proteophosphoglycans (PPGs), glycosylphosphatidylinositol (GPI)-anchored proteins, glycoinositolphospholipids (GIPLs), and N-glycans. A prerequisite for their biosynthesis is an ample supply of the Man donors GDP-Man and dolicholphosphate-Man. We have cloned from Leishmania mexicana the gene encoding the enzyme phosphomannomutase (PMM) and the previously described dolicholphosphate-Man synthase gene (DPMS) that are involved in Man activation. Surprisingly, gene deletion experiments resulted in viable parasite lines lacking the respective open reading frames (DeltaPMM and DeltaDPMS), a result against expectation and in contrast to the lethal phenotype observed in gene deletion experiments with fungi. L. mexicana DeltaDPMS exhibits a selective defect in LPG, protein GPI anchor, and GIPL biosynthesis, but despite the absence of these structures, which have been implicated in parasite virulence and viability, the mutant remains infectious to macrophages and mice. By contrast, L. mexicana DeltaPMM are largely devoid of all known Man-containing glycoconjugates and are unable to establish an infection in mouse macrophages or the living animal. Our results define Man activation leading to GDP-Man as a virulence pathway in Leishmania.
Topics: Amino Acid Sequence; Animals; Carbohydrate Sequence; Cloning, Molecular; Dolichol Monophosphate Mannose; Down-Regulation; Flow Cytometry; Gene Deletion; Gene Targeting; Glycoconjugates; Glycosylation; Guanosine Diphosphate Mannose; Leishmania mexicana; Macrophages; Mannosyltransferases; Mice; Microscopy, Fluorescence; Molecular Sequence Data; Mutation; Phenotype; Phosphotransferases (Phosphomutases); Sequence Homology, Amino Acid; Virulence
PubMed: 11689705
DOI: 10.1128/MCB.21.23.8168-8183.2001 -
PloS One May 2011Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS)...
Protozoan parasites of genus Leishmania are the causative agents of leishmaniasis. These digenetic microorganisms undergo a marked environmental temperature shift (TS) during transmission from the sandfly vector (ambient temperature, 25-26°C) to the mammalian host (37°C). We have observed that this TS induces a rapid and dramatic increase in protein release from Leishmania mexicana (cutaneous leishmaniasis) within 4 h. Proteomic identification of the TS-induced secreted proteins revealed 72 proteins, the majority of which lack a signal peptide and are thus thought to be secreted via nonconventional mechanisms. Interestingly, this protein release is accompanied by alterations in parasite morphology including an augmentation in the budding of exovesicles from its surface. Here we show that the exoproteome of L. mexicana upon TS induces cleavage and activation of the host protein tyrosine phosphatases, specifically SHP-1 and PTP1-B, in a murine bone-marrow-derived macrophage cell line. Furthermore, translocation of prominent inflammatory transcription factors, namely NF-κB and AP-1 is altered. The exoproteome also caused inhibition of nitric oxide production, a crucial leishmanicidal function of the macrophage. Overall, our results provide strong evidence that within early moments of interaction with the mammalian host, L. mexicana rapidly releases proteins and exovesicles that modulate signalling and function of the macrophage. These modulations can result in attenuation of the inflammatory response and deactivation of the macrophage aiding the parasite in the establishment of infection.
Topics: Animals; Chromatography, Liquid; Environment; Inflammation; Leishmania mexicana; Macrophages; Mice; Models, Biological; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Proteome; Proteomics; Signal Transduction; Tandem Mass Spectrometry; Temperature
PubMed: 21559274
DOI: 10.1371/journal.pone.0018724 -
The Journal of Biological Chemistry Feb 2001The human pathogen Leishmania synthesizes phosphoglycans (PGs) formed by variably modified phosphodisaccharide [6-Galbeta1-4Manalpha1-PO(4)] repeats and...
The human pathogen Leishmania synthesizes phosphoglycans (PGs) formed by variably modified phosphodisaccharide [6-Galbeta1-4Manalpha1-PO(4)] repeats and mannooligosaccharide phosphate [(Manalpha1-2)(0-5)Manalpha1-PO(4)] caps that occur lipid-bound on lipophosphoglycan, protein-bound on proteophosphoglycans, and as an unlinked form. PG repeat synthesis has been described as essential for survival and development of Leishmania throughout their life cycle, including for virulence to the mammalian host. In this study, this proposal was investigated in Leishmania mexicana using a spontaneous mutant that was fortuitously isolated from an infected mouse, and by generating a lmexlpg2 gene deletion mutant (Deltalmexlpg2), that lacks a Golgi GDP-Man transporter. The spontaneous mutant lacks PG repeats but synthesizes normal levels of mannooligosaccharide phosphate caps, whereas the Deltalmexlpg2 mutant is deficient in PG repeat synthesis and down-regulates cap expression. In contrast to expectations, both L. mexicana mutants not only retain their ability to bind to macrophages, but are also indistinguishable from wild type parasites with respect to colonization of and multiplication within host cells. Moreover, in mouse infection studies, the spontaneous L. mexicana repeat-deficient mutant and the Deltalmexlpg2 mutant showed no significant difference to a wild type strain with respect to the severity of disease caused by these parasites. Therefore, at least in Leishmania mexicana, PG repeat synthesis is not an absolute requirement for virulence.
Topics: Animals; Carbohydrate Sequence; Carrier Proteins; Cells, Cultured; Gene Targeting; Genes, Protozoan; Glycosphingolipids; Glycosylphosphatidylinositols; Leishmania mexicana; Leishmaniasis; Macrophages; Mannosephosphates; Membrane Proteins; Membrane Transport Proteins; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Polysaccharides; Protozoan Proteins
PubMed: 11071892
DOI: 10.1074/jbc.M008030200 -
European Review For Medical and... Oct 2012Previous studies have shown that CRK3 protein kinase of Leishmania mexicana is a potential drug target. Therefore, the aim of this study was to provide an active protein...
OBJECTIVES AND METHODS
Previous studies have shown that CRK3 protein kinase of Leishmania mexicana is a potential drug target. Therefore, the aim of this study was to provide an active protein kinase for chemical inhibitors testing. A system was developed to express and affinity-purify recombinant L. mexicana CRK3 protein from Escherichia coli.
RESULTS
Biochemical analysis has confirmed the expression of the pure kinase. The bacterial-expressed kinase was found to be inactive as a monomer. The mutated CRK3-E178 protein kinase was also found to be inactive.
CONCLUSION
This study suggests that cyclin binding and phosphorylation status are both important for reconstituting protein kinase activity. Work presented by this paper has confirmed the usefulness of the prokaryotic system for production of pure homogenous recombinant protein kinase of Leishmania parasite, though this system is unable to produce active CRK3 protein kinase
Topics: Escherichia coli; Immunoblotting; Leishmania mexicana; Phosphorylation; Protein Kinases; Proto-Oncogene Proteins c-crk; Recombinant Proteins
PubMed: 23104649
DOI: No ID Found -
International Journal of Molecular... Dec 2021Leishmaniasis is a disease caused by parasites of the genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people...
Leishmaniasis is a disease caused by parasites of the genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people are at risk. The use of the actual treatments is limited due to toxicity concerns and the apparition of resistance strains. Therefore, there is an urgent necessity to find new drugs for the treatment of this disease. In this context, enzymes from the polyamine biosynthesis pathway, such as arginase, have been considered a good target. In the present work, a chemical library of benzimidazole derivatives was studied performing computational, enzyme kinetics, biological activity, and cytotoxic effect characterization, as well as in silico ADME-Tox predictions, to find new inhibitors for arginase from (LmARG). The results show that the two most potent inhibitors (compounds and ) have an I values of 52 μM and 82 μM, respectively. Moreover, assays with human arginase 1 (HsARG) show that both compounds are selective for LmARG. According to molecular dynamics simulation studies these inhibitors interact with important residues for enzyme catalysis. Biological activity assays demonstrate that both compounds have activity against promastigote and amastigote, and low cytotoxic effect in murine macrophages. Finally, in silico prediction of their ADME-Tox properties suggest that these inhibitors support the characteristics to be considered drug candidates. Altogether, the results reported in our study suggest that the benzimidazole derivatives are an excellent starting point for design new drugs against leishmanisis.
Topics: Animals; Antiprotozoal Agents; Arginase; Benzimidazoles; Cell Line; Drug Discovery; Humans; Leishmania mexicana; Leishmaniasis, Cutaneous; Mice; Models, Molecular; Protozoan Proteins
PubMed: 34948408
DOI: 10.3390/ijms222413613 -
Molecular & Cellular Proteomics : MCP Jul 2019parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require...
parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few -regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the -regulatory mRNA:Protein (mRNP) complexes that drive parasite lifecycle progression.
Topics: Animals; Gene Ontology; Leishmania mexicana; Life Cycle Stages; Mice, Inbred BALB C; Parasites; Principal Component Analysis; Proteome; Proteomics; Protozoan Proteins; RNA, Messenger; RNA-Binding Proteins; Reproducibility of Results; Transcriptome
PubMed: 30948621
DOI: 10.1074/mcp.RA118.001307 -
Antimicrobial Agents and Chemotherapy Jan 2011Paromomycin, an aminoglycoside antibiotic having low mammalian cell toxicity, is one of the drugs currently used in the chemotherapy of cutaneous and visceral...
Paromomycin, an aminoglycoside antibiotic having low mammalian cell toxicity, is one of the drugs currently used in the chemotherapy of cutaneous and visceral leishmaniasis. In order to understand the mode of action of this antibiotic at the molecular level, we have investigated the effects of paromomycin on protein synthesis in Leishmania and its mammalian hosts. We were able to demonstrate that in vivo protein synthesis in the promastigote stage of the parasite and its proliferation rate are markedly inhibited by paromomycin while being only slightly affected by other aminoglycoside antibiotics, such as streptomycin and neomycin B. Furthermore, both in vitro polypeptide synthesis induced by poly(U) as mRNA and accuracy of translation are significantly decreased by paromomycin in cell-free systems containing ribosomal particles of Leishmania promastigotes. Conversely, when ribosomes from mammalian cells are used instead of the protozoan particles, polyphenylalanine synthesis is only barely reduced by the antibiotic and the translation misreading remains almost unaltered. Surface plasmon resonance analysis of the interaction between paromomycin and protozoan or mammalian cell ribosomal RNAs shows a strong binding of antibiotic to the parasite ribosomal decoding site and practically no interaction with the mammalian cell counterpart. Our results indicating differential effects of paromomycin on the translation processes of the Leishmania parasite and its mammalian hosts can explain the therapeutic efficiency of this antibiotic as an antileishmaniasis agent.
Topics: Animals; Anti-Bacterial Agents; Cells, Cultured; Crithidia fasciculata; Leishmania mexicana; Paromomycin; Peptides; Protein Biosynthesis; Rats; Ribosomes; Surface Plasmon Resonance
PubMed: 20956601
DOI: 10.1128/AAC.00506-10