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Virulence Dec 2021Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The...
Catalase is one of the most abundant enzymes on Earth. It decomposes hydrogen peroxide, thus protecting cells from dangerous reactive oxygen species. The catalase-encoding gene is conspicuously absent from the genome of most representatives of the family Trypanosomatidae. Here, we expressed this protein from the locus using a novel bicistronic expression system, which relies on the 2A peptide of . We demonstrated that catalase-expressing parasites are severely compromised in their ability to develop in insects, to be transmitted and to infect mice, and to cause clinical manifestation in their mammalian host. Taken together, our data support the hypothesis that the presence of catalase is not compatible with the dixenous life cycle of , resulting in loss of this gene from the genome during the evolution of these parasites.
Topics: Animals; Catalase; Cells, Cultured; Female; Leishmania mexicana; Life Cycle Stages; Mice; Mice, Inbred BALB C; Protozoan Proteins; Psychodidae; Teschovirus; Virulence; Virulence Factors
PubMed: 33724149
DOI: 10.1080/21505594.2021.1896830 -
Parasitology Sep 2021Telomeres are the ends of linear eukaryotic chromosomes facilitating the resolution of the ‘end replication and protection’ problems, associated with linearity. At...
Telomeres are the ends of linear eukaryotic chromosomes facilitating the resolution of the ‘end replication and protection’ problems, associated with linearity. At the nucleotide level, telomeres typically represent stretches of tandemly arranged telomeric repeats, which vary in length and sequence among different groups of organisms. Recently, a composition of the telomere-associated protein complex has been scrutinized in Trypanosoma brucei. In this work, we subjected proteins from that list to a more detailed bioinformatic analysis and delineated a core set of 20 conserved proteins putatively associated with telomeres in trypanosomatids. Out of these, two proteins (Ku70 and Ku80) are conspicuously missing in representatives of the genus Blastocrithidia, yet telomeres in these species do not appear to be affected. In this work, based on the analysis of a large set of trypanosomatids widely different in their phylogenetic position and life strategies, we demonstrated that telomeres of trypanosomatids are diverse in length, even within groups of closely related species. Our analysis showed that the expression of two proteins predicted to be associated with telomeres (those encoding telomerase and telomere-associated hypothetical protein orthologous to Tb927.6.4330) may directly affect and account for the differences in telomere length within the species of the Leishmania mexicana complex.
Topics: Leishmania mexicana; Telomere; Trypanosomatina
PubMed: 33612129
DOI: 10.1017/S0031182021000378 -
MSphere Aug 2018Glucose transporters are important for viability and infectivity of the disease-causing amastigote stages of The Δ null mutant, in which the 3 clustered glucose...
Glucose transporters are important for viability and infectivity of the disease-causing amastigote stages of The Δ null mutant, in which the 3 clustered glucose transporter genes, , , and , have been deleted, is strongly impaired in growth inside macrophages We have now demonstrated that this null mutant is also impaired in virulence in the BALB/c murine model of infection and forms lesions considerably more slowly than wild-type parasites. Previously, we established that amplification of the gene, which encodes an intraflagellar transport protein, both facilitated and accompanied the isolation of the original Δ null mutant generated in extracellular insect-stage promastigotes. We have now isolated Δ null mutants without coamplification of These amplicon-negative null mutants are further impaired in growth as promastigotes, compared to the previously described null mutants containing the amplification. In contrast, the GT3 glucose transporter plays an especially important role in promoting amastigote viability. A line that expresses only the single glucose transporter GT3 grows as well inside macrophages and induces lesions in animals as robustly as do wild-type amastigotes, but lines expressing only the GT1 or GT2 transporters replicate poorly in macrophages. Strikingly, GT3 is restricted largely to the endoplasmic reticulum in intracellular amastigotes. This observation raises the possibility that GT3 may play an important role as an intracellular glucose transporter in the infectious stage of the parasite life cycle. Glucose transport plays important roles for growth of insect-stage promastigotes and especially for viability of intramacrophage mammalian host-stage amastigotes of However, the roles of the three distinct glucose transporters, GT1, GT2, and GT3, in parasite viability inside macrophages and virulence in mice have not been fully explored. Parasite lines expressing GT1 or GT2 alone were strongly impaired in growth inside macrophages, but lines expressing GT3 alone infected macrophages and caused lesions in mice as robustly as wild-type parasites. Notably, GT3 localizes to the endoplasmic reticulum of intracellular amastigotes, suggesting a potential role for salvage of glucose from that organelle for viability of infectious amastigotes. This study establishes the unique role of GT3 for parasite survival inside host macrophages and for robust virulence in infected animals.
Topics: Animals; Cell Line; Endoplasmic Reticulum; Female; Gene Knockout Techniques; Glucose Transport Proteins, Facilitative; Leishmania mexicana; Life Cycle Stages; Macrophages; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Mutation; Protozoan Proteins; Virulence
PubMed: 30068561
DOI: 10.1128/mSphere.00349-18 -
Frontiers in Cellular and Infection... 2021Protein phosphorylation and dephosphorylation are increasingly recognized as important processes for regulating multiple physiological mechanisms. Phosphorylation is...
Protein phosphorylation and dephosphorylation are increasingly recognized as important processes for regulating multiple physiological mechanisms. Phosphorylation is carried out by protein kinases and dephosphorylation by protein phosphatases. Phosphoprotein phosphatases (PPPs), one of three families of protein serine/threonine phosphatases, have great structural diversity and are involved in regulating many cell functions. PP2C, a type of PPP, is found in , a dimorphic protozoan parasite and the causal agent of leishmaniasis. The aim of this study was to clone, purify, biochemically characterize and quantify the expression of PP2C in (PP2C). Recombinant PP2C dephosphorylated a specific threonine (with optimal activity at pH 8) in the presence of the manganese divalent cation (Mn). PP2C activity was inhibited by sanguinarine (a specific inhibitor) but was unaffected by protein tyrosine phosphatase inhibitors. Western blot analysis indicated that anti-PP2C antibodies recognized a molecule of 45.2 kDa. Transmission electron microscopy with immunodetection localized PP2C in the flagellar pocket and flagellum of promastigotes but showed poor staining in amastigotes. Interestingly, PP2C belongs to the ortholog group OG6_142542, which contains only protozoa of the family Trypanosomatidae. This suggests a specific function of the enzyme in the flagellar pocket of these microorganisms.
Topics: Humans; Leishmania; Leishmania mexicana; Leishmaniasis; Phosphoprotein Phosphatases; Phosphorylation; Serine
PubMed: 33937094
DOI: 10.3389/fcimb.2021.641356 -
Molecules (Basel, Switzerland) Sep 2019Leishmanicidal drugs have many side effects, and drug resistance to all of them has been documented. Therefore, the development of new drugs and the identification of...
Leishmanicidal drugs have many side effects, and drug resistance to all of them has been documented. Therefore, the development of new drugs and the identification of novel therapeutic targets are urgently needed. trypanothione reductase (LmTR), a NADPH-dependent flavoprotein oxidoreductase important to thiol metabolism, is essential for parasite viability. Its absence in the mammalian host makes this enzyme an attractive target for the development of new anti- drugs. Herein, a tridimensional model of LmTR was constructed and the molecular docking of 20 molecules from a ZINC database was performed. Five compounds (ZINC04684558, ZINC09642432, ZINC12151998, ZINC14970552, and ZINC11841871) were selected (docking scores -10.27 kcal/mol to -5.29 kcal/mol and structurally different) and evaluated against recombinant LmTR (rLmTR) and promastigote. Additionally, molecular dynamics simulation of LmTR-selected compound complexes was achieved. The five selected compounds inhibited rLmTR activity in the range of 32.9% to 40.1%. The binding of selected compounds to LmTR involving different hydrogen bonds with distinct residues of the molecule monomers A and B is described. Compound ZINC12151998 (docking score -10.27 kcal/mol) inhibited 32.9% the enzyme activity (100 µM) and showed the highest leishmanicidal activity (IC = 58 µM) of all the selected compounds. It was more active than glucantime, and although its half-maximal cytotoxicity concentration (CC = 53 µM) was higher than that of the other four compounds, it was less cytotoxic than amphotericin B. Therefore, compound ZINC12151998 provides a promising starting point for a hit-to-lead process in our search for new anti- drugs that are more potent and less cytotoxic.
Topics: Amino Acid Sequence; Binding Sites; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydrogen Bonding; Leishmania mexicana; Molecular Conformation; Molecular Docking Simulation; Molecular Dynamics Simulation; Molecular Structure; NADH, NADPH Oxidoreductases; Pharmacokinetics; Recombinant Proteins; Structure-Activity Relationship; Trypanocidal Agents
PubMed: 31487860
DOI: 10.3390/molecules24183216 -
Antimicrobial Agents and Chemotherapy Feb 2020There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million...
There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. We investigated the activity of chitosan and several of its derivatives and showed that the pH of the culture medium plays a critical role in antileishmanial activity of chitosan against both extracellular promastigotes and intracellular amastigotes of and Chitosan and its derivatives were approximately 7 to 20 times more active at pH 6.5 than at pH 7.5, with high-molecular-weight chitosan being the most potent. High-molecular-weight chitosan stimulated the production of nitric oxide and reactive oxygen species by uninfected and -infected macrophages in a time- and dose-dependent manner at pH 6.5. Despite the activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, we showed that the antileishmanial activity of chitosan was not mediated by these metabolites. Finally, we showed that rhodamine-labeled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of -infected macrophages.
Topics: Amphotericin B; Animals; Antiprotozoal Agents; Chitosan; Culture Media; Dose-Response Relationship, Drug; Female; Humans; Hydrogen-Ion Concentration; Leishmania major; Leishmania mexicana; Life Cycle Stages; Macrophages; Macrophages, Peritoneal; Mice; Mice, Inbred BALB C; Molecular Weight; Nitric Oxide; Parasitic Sensitivity Tests; Pinocytosis; Primary Cell Culture; Reactive Oxygen Species; THP-1 Cells; Tumor Necrosis Factor-alpha
PubMed: 31871082
DOI: 10.1128/AAC.01772-19 -
Molecular and Biochemical Parasitology Jun 2019The kinetoplastids Trypanosoma brucei and Leishmania mexicana are eukaryotes with a highly structured cellular organisation that is reproduced with great fidelity in...
The kinetoplastids Trypanosoma brucei and Leishmania mexicana are eukaryotes with a highly structured cellular organisation that is reproduced with great fidelity in each generation. The pattern of signal from a fluorescently tagged protein can define the specific structure/organelle that this protein localises to, and can be extremely informative in phenotype analysis in experimental perturbations, life cycle tracking, post-genomic assays and functional analysis of organelles. Using the vast coverage of protein subcellular localisations provided by the TrypTag project, an ongoing project to determine the localisation of every protein encoded in the T. brucei genome, we have generated an inventory of reliable reference organelle markers for both parasites that combines epifluorescence images with a detailed description of the key features of each localisation. We believe this will be a useful comparative resource that will enable researchers to quickly and accurately pinpoint the localisation of their proteins of interest and will provide cellular markers for many types of cell biology studies. We see this as another important step in the post-genomic era analyses of these parasites, in which ever expanding datasets generate numerous candidates to analyse. Adoption of these reference proteins by the community is likely to enhance research studies and enable better comparison of data.
Topics: Leishmania mexicana; Microscopy, Fluorescence; Organelles; Protein Transport; Protozoan Proteins; Recombinant Fusion Proteins; Staining and Labeling; Trypanosoma brucei brucei
PubMed: 30550896
DOI: 10.1016/j.molbiopara.2018.12.003 -
Molecules (Basel, Switzerland) Jun 2021Ethanolic extracts of samples of temperate zone propolis, four from the UK and one from Poland, were tested against three strains and displayed EC values < 20 µg/mL....
Ethanolic extracts of samples of temperate zone propolis, four from the UK and one from Poland, were tested against three strains and displayed EC values < 20 µg/mL. The extracts were fractionated, from which 12 compounds and one two-component mixture were isolated, and characterized by NMR and high-resolution mass spectrometry, as 3-acetoxypinobanksin, tectochrysin, kaempferol, pinocembrin, 4'-methoxykaempferol, galangin, chrysin, apigenin, pinostrobin, cinnamic acid, coumaric acid, cinnamyl ester/coumaric acid benzyl ester (mixture), 4',7-dimethoxykaempferol, and naringenin 4',7-dimethyl ether. The isolated compounds were tested against drug-sensitive and drug-resistant strains of and , with the highest activities ≤ 15 µM. The most active compounds against were naringenin 4',7 dimethyl ether and 4'methoxy kaempferol with activity of 15-20 µM against the three strains. The most active compounds against were 4',7-dimethoxykaempferol and the coumaric acid ester mixture, with EC values of 12.9 ± 3.7 µM and 13.1 ± 1.0 µM. No loss of activity was found with the diamidine- and arsenical-resistant or phenanthridine-resistant strains, or the miltefosine-resistant strain; no clear structure activity relationship was observed for the isolated compounds. Temperate propolis yields multiple compounds with anti-kinetoplastid activity.
Topics: Cinnamates; Flavanones; Flavonoids; Kaempferols; Leishmania mexicana; Magnetic Resonance Spectroscopy; Mass Spectrometry; Poland; Propolis; Trypanocidal Agents; Trypanosoma brucei brucei; United Kingdom
PubMed: 34206940
DOI: 10.3390/molecules26133912 -
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 -
Microbiology Spectrum Feb 2022Proteomic profiling of RNA-binding proteins in is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs,...
Proteomic profiling of RNA-binding proteins in is currently limited to polyadenylated mRNA-binding proteins, leaving proteins that interact with nonadenylated RNAs, including noncoding RNAs and pre-mRNAs, unidentified. Using a combination of unbiased orthogonal organic phase separation methodology and tandem mass tag-labeling-based high resolution quantitative proteomic mass spectrometry, we robustly identified 2,417 RNA-binding proteins, including 1289 putative novel non-poly(A)-RNA-binding proteins across the two main life cycle stages. Eight out of 20 deubiquitinases, including the recently characterized L. mexicana DUB2 with an elaborate RNA-binding protein interactome were exclusively identified in the non-poly(A)-RNA-interactome. Additionally, an increased representation of WD40 repeat domains were observed in the non-poly(A)-RNA-interactome, thus uncovering potential involvement of this protein domain in RNA-protein interactions in . We also characterize the protein-bound RNAs using RNA-sequencing and show that in addition to protein coding transcripts ncRNAs are also enriched in the protein-RNA interactome. Differential gene expression analysis revealed enrichment of 142 out of 195 total L. mexicana protein kinase genes in the protein-RNA-interactome, suggesting important role of protein-RNA interactions in the regulation of the protein kinome. Additionally, we characterize the quantitative changes in RNA-protein interactions in hundreds of proteins following inhibition of heat shock protein 90 (Hsp90). Our results show that the Hsp90 inhibition in causes widespread disruption of RNA-protein interactions in ribosomal proteins, proteasomal proteins and translation factors in both life cycle stages, suggesting downstream effect of the inhibition on protein synthesis and degradation pathways in . This study defines the comprehensive RNA interactome of and provides in-depth insight into the widespread involvement of RNA-protein interactions in biology. Advances in proteomics and mass spectrometry have revealed the mRNA-binding proteins in many eukaryotic organisms, including the protozoan parasites spp., the causative agents of leishmaniasis, a major infectious disease in over 90 tropical and subtropical countries. However, in addition to mRNAs, which constitute only 2 to 5% of the total transcripts, many types of non-coding RNAs participate in crucial biological processes. In , RNA-binding proteins serve as primary gene regulators. Therefore, transcriptome-wide identification of RNA-binding proteins is necessary for deciphering the distinctive posttranscriptional mechanisms of gene regulation in . Using a combination of highly efficient orthogonal organic phase separation method and tandem mass tag-labeling-based quantitative proteomic mass spectrometry, we provide unprecedented comprehensive molecular definition of the total RNA interactome across the two main life cycle stages. In addition, we characterize for the first time the quantitative changes in RNA-protein interactions in following inhibition of heat shock protein 90, shedding light into hitherto unknown large-scale downstream molecular effect of the protein inhibition in the parasite. This work provides insight into the importance of total RNA-protein interactions in , thus significantly expanding our knowledge of the emergence of RNA-protein interactions in biology.
Topics: Leishmania mexicana; Mass Spectrometry; Open Reading Frames; Protein Binding; Proteomics; Protozoan Proteins; RNA, Protozoan; RNA, Untranslated; RNA-Binding Proteins; Ribosomal Proteins; Transcriptome
PubMed: 35138191
DOI: 10.1128/spectrum.02422-21