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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 -
Frontiers in Cellular and Infection... 2022is the unicellular parasite transmitted by phlebotomine sand fly bite. It exists in two different forms; extracellular promastigotes, occurring in the gut of sand...
is the unicellular parasite transmitted by phlebotomine sand fly bite. It exists in two different forms; extracellular promastigotes, occurring in the gut of sand flies, and intracellular, round-shaped amastigotes residing mainly in vertebrate macrophages. As amastigotes originating from infected animals are often present in insufficient quality and quantity, two alternative types of amastigotes were introduced for laboratory experiments: axenic amastigotes and amastigotes from macrophages infected . Nevertheless, there is very little information about the degree of similarity/difference among these three types of amastigotes on proteomic level, whose comparison is crucial for assessing the suitability of using alternative types of amastigotes in experiments. In this study, amastigotes obtained from lesion of infected BALB/c mice were proteomically compared with alternatively cultivated amastigotes (axenic and macrophage-derived ones). Amastigotes of all three types were isolated, individually treated and analysed by LC-MS/MS proteomic analysis with quantification using TMT-plex isobaric labeling. Significant differences were observed in the abundance of metabolic enzymes, virulence factors and proteins involved in translation and condensation of DNA. The most pronounced differences were observed between axenic amastigotes and lesion-derived amastigotes, macrophage-derived amastigotes were mostly intermediate between axenic and lesion-derived ones.
Topics: Mice; Animals; Leishmania mexicana; Proteome; Proteomics; Chromatography, Liquid; Tandem Mass Spectrometry; Mice, Inbred BALB C
PubMed: 36439224
DOI: 10.3389/fcimb.2022.1022448 -
Journal of Immunology Research 2020Exosomes are extracellular microvesicles of endosomal origin (multivesicular bodies, MVBs) constitutively released by eukaryotic cells by fusion of MVBs to the plasma...
Exosomes are extracellular microvesicles of endosomal origin (multivesicular bodies, MVBs) constitutively released by eukaryotic cells by fusion of MVBs to the plasma membrane. The exosomes from parasites contain an array of parasite molecules such as virulence factors and survival messengers, capable of modulating the host immune response and thereby favoring the infection of the host. We here show that exosomes of amastigotes (aExo) contain the virulence proteins gp63 and PP2C. The incubation of aExo with bone marrow-derived macrophages (BMMs) infected with led to their internalization and were found to colocalize with the cellular tetraspanin CD63. Furthermore, aExo inhibited nitric oxide production of infected BMMs, permitting enhanced intracellular parasite survival. Expressions of antigen-presenting (major histocompatibility complex class I, MHC-I, and CD1d) and costimulatory (CD86 and PD-L1) molecules were modulated in a dose-dependent fashion. Whereas MHC-I, CD86 and PD-L1 expressions were diminished by exosomes, CD1d was enhanced. We conclude that aExo of are capable of decreasing microbicidal mechanisms of infected macrophages by inhibiting nitric oxide production, thereby enabling parasite survival. They also hamper the cellular immune response by diminishing MHC-I and CD86 on an important antigen-presenting cell, which potentially interferes with CD8 T cell activation. The enhanced CD1d expression in combination with reduction of PD-L1 on BMMs point to a potential shift of the activation route towards lipid presentations, yet the effectivity of this immune activation is not evident, since in the absence of costimulatory molecules, cellular anergy and tolerance would be expected.
Topics: Animals; Biomarkers; Cells, Cultured; Disease Models, Animal; Exosomes; Host-Pathogen Interactions; Leishmania mexicana; Leishmaniasis, Cutaneous; Mice
PubMed: 33344659
DOI: 10.1155/2020/8894549 -
International Journal of Molecular... Jul 2022The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by...
The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from (ΔNT1.1/1.2/2 (SUPKO)). SUPKO grew at the same rate as the parental strain and displayed no apparent deficiencies, owing to the cells' ability to synthesize pyrimidines, and the expression of the LmexNT3 purine nucleobase transporter. Nucleoside transport was barely measurable in SUPKO, but reintroduction of NT1.1, NT1.2, and NT2 restored uptake. Thus, SUPKO provides an ideal null background for the expression and characterization of single ENT transporter genes in isolation. Similarly, an LmexNT3-KO strain provides a null background for transport of purine nucleobases and was used for the functional characterization of NB2, which was determined to be adenine-specific. A 5-fluorouracil-resistant strain (Lmex5FURes) displayed null transport for uracil and 5FU, and was used to express the uracil transporter FurD.
Topics: Biological Transport; Equilibrative Nucleoside Transport Proteins; Leishmania mexicana; Membrane Transport Proteins; Nucleosides; Purines; Pyrimidines; Uracil
PubMed: 35897714
DOI: 10.3390/ijms23158139 -
International Journal For Parasitology Mar 2018Cyclopropane fatty acid synthase (CFAS) catalyzes the transfer of a methylene group from S-adenosyl methionine to an unsaturated fatty acid, generating a cyclopropane...
Cyclopropane fatty acid synthase (CFAS) catalyzes the transfer of a methylene group from S-adenosyl methionine to an unsaturated fatty acid, generating a cyclopropane fatty acid (CFA). The gene encoding CFAS is present in many bacteria and several Leishmania spp. including Leishmania mexicana, Leishmania infantum and Leishmania braziliensis. In this study, we characterised the CFAS-null and -overexpression mutants in L. mexicana, the causative agent for cutaneous leishmaniasis in Mexico and central America. Our data indicate that L. mexicana CFAS modifies the fatty acid chain of plasmenylethanolamine (PME), the dominant class of ethanolamine glycerophospholipids in Leishmania, generating CFA-PME. While the endogenous level of CFA-PME is extremely low in wild type L. mexicana, overexpression of CFAS results in a significant increase. CFAS-null mutants (cfas) exhibit altered cell shape, increased sensitivity to acidic pH, and aberrant growth in serum-free media. In addition, the CFAS protein is preferentially expressed during the proliferative stage of L. mexicana and is required for the cell membrane targeting of lipophosphoglycan. Finally, the maturation and localization of CFAS protein are dependent upon the downstream sequence of the CFAS coding region. Without the downstream sequence, the mis-localised CFAS protein cannot fully rescue the defects of cfas. Our data suggest that CFA modification of phospholipids can significantly affect the parasite's response to certain adverse conditions. These findings are distinct from the roles of CFAS in L. infantum, highlighting the functional divergence in lipid modification among Leishmania spp.
Topics: Animals; Blotting, Southern; Blotting, Western; Cyclopropanes; Fatty Acids; Hydrogen-Ion Concentration; Leishmania mexicana; Leishmaniasis, Cutaneous; Lipids; Macrophages; Methyltransferases; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Plasmalogens; Spectrometry, Mass, Electrospray Ionization; Spectroscopy, Fourier Transform Infrared
PubMed: 29180119
DOI: 10.1016/j.ijpara.2017.09.006 -
Frontiers in Cellular and Infection... 2022and are the causative agents of cutaneous and mucocutaneous diseases. The infections' outcome depends on host-parasite interactions and Th1/Th2 response, and in...
and are the causative agents of cutaneous and mucocutaneous diseases. The infections' outcome depends on host-parasite interactions and Th1/Th2 response, and in cutaneous form, regulation of Th17 cytokines has been reported to maintain inflammation in lesions. Despite that, the Th17 regulatory scenario remains unclear. With the aim to gain a better understanding of the transcription factors (TFs) and genes involved in Th17 induction, in this study, the role of inducing factors of the Th17 pathway in -macrophage infection was addressed through computational modeling of gene regulatory networks (GRNs). The Th17 GRN modeling integrated experimentally validated data available in the literature and gene expression data from a time-series RNA-seq experiment (4, 24, 48, and 72 h post-infection). The generated model comprises a total of 10 TFs, 22 coding genes, and 16 cytokines related to the Th17 immune modulation. Addressing the Th17 induction in infected and uninfected macrophages, an increase of 2- to 3-fold in 4-24 h was observed in the former. However, there was a decrease in basal levels at 48-72 h for both groups. In order to evaluate the possible outcomes triggered by GRN component modulation in the Th17 pathway. The generated GRN models promoted an integrative and dynamic view of -macrophage interaction over time that extends beyond the analysis of single-gene expression.
Topics: Cytokines; Gene Regulatory Networks; Humans; Leishmania major; Leishmania mexicana; Leishmaniasis; Macrophages
PubMed: 35774406
DOI: 10.3389/fcimb.2022.826523 -
PLoS Neglected Tropical Diseases Sep 2022Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for...
Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.
Topics: Mice; Animals; Amphotericin B; Leishmania mexicana; Nystatin; Serum Albumin, Bovine; Sterols; Oxidative Stress; Polyenes; Transferases; Glucose; Fatty Acid Desaturases; Antiprotozoal Agents
PubMed: 36170238
DOI: 10.1371/journal.pntd.0010779 -
International Journal of Molecular... Mar 2024Twenty 2-(4-alkyloxyphenyl)-imidazolines and 2-(4-alkyloxyphenyl)-imidazoles were synthesized, with the former being synthesized in two steps by using MW and...
Twenty 2-(4-alkyloxyphenyl)-imidazolines and 2-(4-alkyloxyphenyl)-imidazoles were synthesized, with the former being synthesized in two steps by using MW and ultrasonication energy, resulting in good to excellent yields. Imidazoles were obtained in moderate yields by oxidizing imidazolines with MnO and MW energy. In response to the urgent need to treat neglected tropical diseases, a set of 2-(4-alkyloxyphenyl)- imidazolines and imidazoles was tested in vitro on and . The leishmanicidal activity of ten compounds was evaluated, showing an IC < 10 µg/mL. Among these compounds, - were the most active, with IC values < 1 µg/mL (similar to the reference drugs). In the evaluation on epimastigotes of , only and reached an IC < 1 µg/mL, showing better inhibition than both reference drugs. However, compounds , , and also demonstrated attractive trypanocidal activities, with IC values < 10 µg/mL, similar to the values for benznidazole and nifurtimox.
Topics: Humans; Imidazoles; Imidazolines; Trypanosoma cruzi; Leishmania mexicana; Manganese Compounds; Oxides; Antiprotozoal Agents; Chagas Disease
PubMed: 38612484
DOI: 10.3390/ijms25073673 -
The Biochemical Journal Apr 2002Free glycosylphosphatidylinositols (GPIs) are an important class of membrane lipids in many pathogenic protozoa. In this study, we have investigated the subcellular...
Free glycosylphosphatidylinositols (GPIs) are an important class of membrane lipids in many pathogenic protozoa. In this study, we have investigated the subcellular distribution and intracellular trafficking of an abundant class of free GPIs [termed glycosylinositolphospholipids (GIPLs)] in Leishmania mexicana promastigotes. The intracellular transport of the GIPLs and the major GPI-anchored glycoprotein gp63 was measured by following the incorporation of these molecules into sphingolipid-rich, detergent-resistant membranes (DRMs) in the plasma membrane. In metabolic-labelling experiments, mature GIPLs and gp63 were transported to DRMs in the plasma membrane with a t(1/2) of 70 and 40 min, respectively. Probably, GIPL transport to the DRMs involves a vesicular mechanism, as transport of both the GIPLs and gp63 was inhibited similarly at 10 degrees C. All GIPL intermediates were quantitatively recovered in Triton X-100-soluble membranes and were largely orientated on the cytoplasmic face of the endoplasmic reticulum, as shown by their sensitivity to exogenous phosphatidylinositol-specific phospho-lipase C. On the contrary, a significant proportion of the mature GIPLs ( approximately 50% of iM4) were accessible to membrane-impermeable probes on the surface of live promastigotes. These results suggest that the GIPLs are flipped across intracellular or plasma membranes during surface transport and that a significant fraction may populate the cytoplasmic leaflet of the plasma membrane. Finally, treatment of L. mexicana promastigotes with myriocin, an inhibitor of sphingolipid biosynthesis, demonstrated that ongoing sphingolipid biosynthesis is not required for the plasma-membrane transport of either gp63 or the GIPLs and that DRMs persist even when cellular levels of the major sphingolipid are depleted by 70%.
Topics: Animals; Biological Transport, Active; Cell Membrane; Endoplasmic Reticulum; Glycosylphosphatidylinositols; Intracellular Fluid; Kinetics; Leishmania mexicana; Metalloendopeptidases; Protozoan Proteins; Sphingolipids; Temperature
PubMed: 11931667
DOI: 10.1042/0264-6021:3630365 -
Revista Do Instituto de Medicina... 2022Localized cutaneous leishmaniasis (LCL) is an endemic disease in several Mexican States with the main endemic areas located in the South-Southeast region of the country,...
Localized cutaneous leishmaniasis (LCL) is an endemic disease in several Mexican States with the main endemic areas located in the South-Southeast region of the country, where 90% of Leishmania (Leishmania) mexicana cases are registered. The Southeast region is located in the Yucatan Peninsula, including Campeche, Quintana Roo and Yucatan States. Campeche and Quintana Roo register more than 60% of the cases in the country each year, while in Yucatan the reports are of imported cases due to residents traveling to endemic areas. However, since 2015, autochthonous cases have been diagnosed by health authorities in municipalities with no previous transmission records. We aimed to identify Leishmania parasite species involved in autochthonous cases by means of the PCR technique. The present study included 13 autochthonous cases of LCL with clinical and parasitological diagnoses during 2018 and 2019 by health authorities, without specific identification of the causal agent. Tissue samples were taken by scraping the margins of active lesions and then they were spotted onto an FTATM Elute Microcard. Next, DNA was eluted and used for PCR amplification of specific Leishmania genus and L. (L.) mexicana species-specific fragments. Molecular analysis showed evidence that L. (L.) mexicana was the causal agent of LCL in 12 of the 13 patients; in one patient, PCR was not performed due to the patient's refusal to participate in the study. Identifying Leishmania species that cause LCL is necessary to define efficient treatment schemes and control strategies for the disease in vulnerable and susceptible areas of the Yucatan State's municipalities.
Topics: Endemic Diseases; Humans; Leishmania; Leishmania mexicana; Leishmaniasis, Cutaneous; Mexico
PubMed: 35648988
DOI: 10.1590/S1678-9946202264035