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PloS One 2019Macrophages can reprogram their metabolism in response to the surrounding stimuli, which affects their capacity to kill intracellular pathogens. We have investigated the...
Macrophages can reprogram their metabolism in response to the surrounding stimuli, which affects their capacity to kill intracellular pathogens. We have investigated the metabolic and immune status of human macrophages after infection with the intracellular trypanosomatid parasites Leishmania donovani, L. amazonensis and T. cruzi and their capacity to respond to a classical polarizing stimulus (LPS and IFN-γ). We found that macrophages infected with Leishmania preferentially upregulate oxidative phosphorylation, which could be contributed by both host cell and parasite, while T. cruzi infection did not significantly increase glycolysis or oxidative phosphorylation. Leishmania and T. cruzi infect macrophages without triggering a strong inflammatory cytokine response, but infection does not prevent a potent response to LPS and IFN-γ. Infection appears to prime macrophages, since the cytokine response to activation with LPS and IFN-γ is more intense in infected macrophages compared to uninfected ones. Metabolic polarization in macrophages can influence infection and immune evasion of these parasites since preventing macrophage cytokine responses would help parasites to establish a persistent infection. However, macrophages remain responsive to classical inflammatory stimuli and could still trigger inflammatory cytokine secretion by macrophages.
Topics: 3T3 Cells; Animals; Cells, Cultured; Chagas Disease; Cytokines; Healthy Volunteers; Humans; Leishmania donovani; Leishmania mexicana; Leishmaniasis; Macrophage Activation; Macrophages; Metabolome; Mice; Oxidative Phosphorylation; Primary Cell Culture; Trypanosoma cruzi; Up-Regulation
PubMed: 31841511
DOI: 10.1371/journal.pone.0225588 -
Experimental Parasitology Mar 2008Leishmania cannot synthesize purines de novo and rely on their host to furnish these compounds. To accomplish this, they possess multiple purine nucleoside and...
Leishmania cannot synthesize purines de novo and rely on their host to furnish these compounds. To accomplish this, they possess multiple purine nucleoside and nucleobase transporters. Subcellular fractionation, immunohistochemical localization with anti-adenine nucleotide translocator (ANT) antibodies and surface biotinylation show that the mitochondrial ANT is also present in the plasma membrane of both promastigotes and amastigotes. Leishmania, however, do not appear to rely on this transporter to supplement their purine or energy requirements via preformed ATP from its host. Rather, Leishmania appear to use the plasma membrane ANT as part of a chemotaxis response. ATP is a chemorepellant for Leishmania and cells treated with atractyloside, an inhibitor of ANT, no longer exhibit negative chemotaxis for this compound.
Topics: Animals; Blotting, Western; Cell Membrane; Chemotaxis; Immunohistochemistry; Leishmania mexicana; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondrial ADP, ATP Translocases
PubMed: 18031742
DOI: 10.1016/j.exppara.2007.10.010 -
PLoS Neglected Tropical Diseases Aug 2021Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by...
Micro-CT visualization of a promastigote secretory gel (PSG) and parasite plug in the digestive tract of the sand fly Lutzomyia longipalpis infected with Leishmania mexicana.
Leishmaniasis is a debilitating disease of the tropics, subtropics and southern Europe caused by Leishmania parasites that are transmitted during blood feeding by phlebotomine sand flies (Diptera: Psychodidae). Using non-invasive micro-computed tomography, we were able to visualize the impact of the laboratory model infection of Lutzomyia longipalpis with Leishmania mexicana and its response to a second blood meal. For the first time we were able to show in 3D the plug of promastigote secretory gel (PSG) and parasites in the distended midgut of whole infected sand flies and measure its volume in relation to that of the midgut. We were also able to measure the degree of opening of the stomodeal valve and demonstrate the extension of the PSG and parasites into the pharynx. Although our pilot study could only examine a few flies, it supports the hypothesis that a second, non-infected, blood meal enhances parasite transmission as we showed that the thoracic PSG-parasite plug in infected flies after a second blood meal was, on average, more than twice the volume of the plug in infected flies that did not have a second blood meal.
Topics: Animals; Female; Gastrointestinal Tract; Insect Vectors; Leishmania mexicana; Pilot Projects; Protozoan Proteins; Psychodidae; X-Ray Microtomography
PubMed: 34449767
DOI: 10.1371/journal.pntd.0009682 -
The Journal of Biological Chemistry Apr 1992Lipophosphoglycan (LPG) was isolated from the culture supernatant of Leishmania mexicana promastigotes and its structure elucidated by a combination of 1H NMR, fast atom...
Lipophosphoglycan (LPG) was isolated from the culture supernatant of Leishmania mexicana promastigotes and its structure elucidated by a combination of 1H NMR, fast atom bombardment mass spectrometry, methylation analysis, and chemical and enzymatic modifications. It consists of the repeating phosphorylated oligosaccharides PO4-6Gal beta 1-4Man alpha 1- and PO4-6[Glc beta 1-3]Gal beta 1-4Man alpha 1-, which are linked together in linear chains by phosphodiester linkages. Each chain of repeat units is linked to a phosphosaccharide core with the structure PO4-6Gal alpha 1-6Gal alpha 1-3Galf beta 1- 3[Glc alpha 1-PO4-6]Man alpha 1-3Man alpha 1-4GlcNH2 alpha 1-6 myo-inositol, where the myo-inositol residue forms the head group of a lyso-alkylphosphatidylinositol moiety. The nonreducing terminus of the repeat chains appear to be capped with the neutral oligosaccharides Man alpha 1-2Man, Man alpha 1-2Man alpha 1-2Man, or Man alpha 1-2[Gal beta 1-4]Man. Cellular LPG, isolated from promastigotes, has a very similar structure to the culture supernatant LPG. However, it differs from culture supernatant LPG in the average number of phosphorylated oligosaccharide repeat units (20 versus 28) and in alkyl chain composition. Although culture supernatant LPG contained predominantly C24:0 alkyl chains, cellular LPG contained approximately equal amounts of C24:0 and C26:0 alkyl chains. It is suggested that culture supernatant LPG is passively shed from promastigotes and that it may contribute significantly, but not exclusively, to the "excreted factor" used for serotyping Leishmania spp. Comparison of L. mexicana LPG with the LPGs of Leishmania major and Leishmania donovani indicate that these molecules are highly conserved but that species-specific differences occur in the phosphorylated oligosaccharide repeat branches and in the relative abundance of the neutral cap structures.
Topics: Animals; Blotting, Western; Carbohydrate Sequence; Chromatography, Thin Layer; Culture Media; Electrophoresis, Polyacrylamide Gel; Glycosphingolipids; Hydrolysis; Leishmania mexicana; Magnetic Resonance Spectroscopy; Methylation; Molecular Sequence Data; Spectrometry, Mass, Fast Atom Bombardment
PubMed: 1551890
DOI: No ID Found -
MBio May 2021In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes and is catalyzed by either multisubunit cytochrome maturation system I or...
In eukaryotes, heme attachment through two thioether bonds to mitochondrial cytochromes and is catalyzed by either multisubunit cytochrome maturation system I or holocytochrome synthetase (HCCS). The former was inherited from the alphaproteobacterial progenitor of mitochondria; the latter is a eukaryotic innovation for which prokaryotic ancestry is not evident. HCCS provides one of a few exemplars of protein innovation in eukaryotes, but structure-function insight of HCCS is limited. Uniquely, euglenozoan protists, which include medically relevant kinetoplastids and parasites, attach heme to mitochondrial -type cytochromes by a single thioether linkage. Yet the mechanism is unknown, as genes encoding proteins with detectable similarity to any proteins involved in cytochrome maturation in other taxa are absent. Here, a bioinformatics search for proteins conserved in all hemoprotein-containing kinetoplastids identified kinetoplastid cytochrome synthetase (KCCS), which we reveal as essential and mitochondrial and catalyzes heme attachment to trypanosome cytochrome KCCS has no sequence identity to other proteins, apart from a slight resemblance within four short motifs suggesting relatedness to HCCS. Thus, KCCS provides a novel resource for studying eukaryotic cytochrome maturation, possibly with wider relevance, since mutations in human HCCS leads to disease. Moreover, many examples of mitochondrial biochemistry are different in euglenozoans compared to many other eukaryotes; identification of KCCS thus provides another exemplar of extreme, unusual mitochondrial biochemistry in an evolutionarily divergent group of protists. Cytochromes are essential proteins for respiratory and photosynthetic electron transfer. They are posttranslationally modified by covalent attachment of a heme cofactor. Kinetoplastids include important tropical disease-causing parasites; many aspects of their biology differ from other organisms, including their mammalian or plant hosts. Uniquely, kinetoplastids produce cytochromes with a type of heme attachment not seen elsewhere in nature and were the only cytochrome bearing taxa without evidence of protein machinery to attach heme to the apocytochrome. Using bioinformatics, biochemistry, and molecular genetics, we report how kinetoplastids make their cytochromes Unexpectedly, they use a highly diverged version of an enzyme used for heme-protein attachment in many eukaryotes. Mutations in the human enzyme lead to genetic disease. Identification of kinetoplastid cytochrome synthetase, thus, solves an evolutionary unknown, provides a possible target for antiparasite drug development, and an unanticipated resource for studying the mechanistic basis of a human genetic disease.
Topics: Computational Biology; Cytochromes c; Eukaryota; Leishmania mexicana; Lyases; Trypanosoma brucei brucei
PubMed: 33947751
DOI: 10.1128/mBio.00166-21 -
Journal of Cell Science Aug 2019Analysis of flagellum and cilium beating in three dimensions (3D) is important for understanding cell motility, and using fluorescence microscopy to do so would be...
Analysis of flagellum and cilium beating in three dimensions (3D) is important for understanding cell motility, and using fluorescence microscopy to do so would be extremely powerful. Here, high-speed multifocal plane fluorescence microscopy, where the light path is split to visualise multiple focal planes simultaneously, was used to reconstruct and movement in 3D. These species are uniflagellate unicellular parasites for which motility is vital. It was possible to use either a fluorescent stain or a genetically-encoded fluorescent protein to visualise flagellum and cell movement at 200 Hz frame rates. This addressed two open questions regarding and flagellum beating, which contributes to their swimming behaviours: 1) how planar is the flagellum beat, and 2) what is the nature of flagellum beating during 'tumbling'? We showed that has notable deviations from a planar flagellum beat, and that during tumbling the flagellum bends the cell and beats only in the distal portion to achieve cell reorientation. This demonstrates high-speed multifocal plane fluorescence microscopy as a powerful tool for the analysis of beating flagella.
Topics: Flagella; Leishmania mexicana; Microscopy, Fluorescence; Microscopy, Video; Motion; Trypanosoma brucei brucei
PubMed: 31371486
DOI: 10.1242/jcs.231795 -
Frontiers in Cellular and Infection... 2023Farnesol, derived from farnesyl pyrophosphate in the sterols biosynthetic pathway, is a molecule with three unsaturations and four possible isomers. predominantly...
INTRODUCTION
Farnesol, derived from farnesyl pyrophosphate in the sterols biosynthetic pathway, is a molecule with three unsaturations and four possible isomers. predominantly secretes the , -farnesol (, -FOH) isomer, known for its role in regulating the virulence of various fungi species and modulating morphological transition processes. Notably, the evolutionary divergence in sterol biosynthesis between fungi, including , and trypanosomatids resulted in the synthesis of sterols with the ergostane skeleton, distinct from cholesterol. This study aims to assess the impact of exogenously added , -farnesol on the proliferative ability of and to identify its presence in the lipid secretome of the parasite.
METHODS
The study involved the addition of exogenous , -farnesol to evaluate its interference with the proliferation of promastigotes. Proliferation, cell cycle, DNA fragmentation, and mitochondrial functionality were assessed as indicators of the effects of , -farnesol. Additionally, lipid secretome analysis was conducted, focusing on the detection of , -farnesol and related products derived from the precursor, farnesyl pyrophosphate. analysis was employed to identify the sequence for the farnesene synthase gene responsible for producing these isoprenoids in the genome.
RESULTS
Exogenously added , -farnesol was found to interfere with the proliferation of promastigotes, inhibiting the cell cycle without causing DNA fragmentation or loss of mitochondrial functionality. Despite the absence of , -farnesol in the culture supernatant, other products derived from farnesyl pyrophosphate, specifically α-farnesene and β-farnesene, were detected starting on the fourth day of culture, continuing to increase until the tenth day. Furthermore, the identification of the farnesene synthase gene in the genome through in silico analysis provided insights into the enzymatic basis of isoprenoid production.
DISCUSSION
The findings collectively offer the first insights into the mechanism of action of farnesol on . While , -farnesol was not detected in the lipid secretome, the presence of α-farnesene and β-farnesene suggests alternative pathways or modifications in the isoprenoid metabolism of the parasite. The inhibitory effects on proliferation and cell cycle without inducing DNA fragmentation or mitochondrial dysfunction raise questions about the specific targets and pathways affected by exogenous , -farnesol. The identification of the farnesene synthase gene provides a molecular basis for understanding the synthesis of related isoprenoids in . Further exploration of these mechanisms may contribute to the development of novel therapeutic strategies against infections.
Topics: Farnesol; Leishmania mexicana; Leishmania; Sterols; Candida albicans
PubMed: 38035328
DOI: 10.3389/fcimb.2023.1221246 -
Folia Parasitologica May 2016In our previous work we established a T7 polymerase-driven Tetracycline-inducible protein expression system in Leishmania mexicana (Biagi, 1953). We used this system to...
In our previous work we established a T7 polymerase-driven Tetracycline-inducible protein expression system in Leishmania mexicana (Biagi, 1953). We used this system to analyse gene expression profiles during development of L. mexicana in procyclic and metacyclic promastigotes and amastigotes. The transcription of the gene of interest and the T7 polymerase genes was significantly reduced upon cell differentiation. This regulation is not locus-specific. It depends on untranslated regions flanking open reading frames of the genes analysed. In this paper, we report that the previously established conventional inducible protein expression system may not be suitable for studies on differentiation of species of Leishmania Ross, 1903 and protein expression systems might have certain limitations.
Topics: DNA-Directed DNA Polymerase; Gene Expression Regulation; Leishmania mexicana; Life Cycle Stages
PubMed: 27311571
DOI: 10.14411/fp.2016.016 -
International Journal For Parasitology.... Aug 2017Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon...
Leishmania pyrimidine salvage is replete with opportunities for therapeutic intervention with enzyme inhibitors or antimetabolites. Their uptake into cells depends upon specific transporters; therefore it is essential to establish whether various Leishmania species possess similar pyrimidine transporters capable of drug uptake. Here, we report a comprehensive characterization of pyrimidine transport in L. major and L. mexicana. In both species, two transporters for uridine/adenosine were detected, one of which also transported uracil and the antimetabolites 5-fluoruracil (5-FU) and 5F,2'deoxyuridine (5F,2'dUrd), and was designated uridine-uracil transporter 1 (UUT1); the other transporter mediated uptake of adenosine, uridine, 5F,2'dUrd and thymidine and was designated Nucleoside Transporter 1 (NT1). To verify the reported L. donovani model of two NT1-like genes encoding uridine/adenosine transporters, and an NT2 gene encoding an inosine transporter, we cloned the corresponding L. major and L. mexicana genes, expressing each in T. brucei. Consistent with the L. donovani reports, the NT1-like genes of either species mediated the adenosine-sensitive uptake of [H]-uridine but not of [H]-inosine. Conversely, the NT2-like genes mediated uptake of [H]-inosine but not [H]-uridine. Among pyrimidine antimetabolites tested, 5-FU and 5F,2'dUrd were the most effective antileishmanials; resistance to both analogs was induced in L. major and L. mexicana. In each case it was found that the resistant cells had lost the transport capacity for the inducing drug. Metabolomics analysis found that the mechanism of action of 5-FU and 5F-2'dUrd was similar in both Leishmania species, with major changes in deoxynucleotide metabolism. We conclude that the pyrimidine salvage system is highly conserved in Leishmania species - essential information for the development of pyrimidine-based chemotherapy.
Topics: Biological Transport; Cloning, Molecular; Drug Resistance; Leishmania major; Leishmania mexicana; Membrane Transport Proteins; Pyrimidines; Selection, Genetic; Substrate Specificity; Trypanosoma brucei brucei
PubMed: 28453984
DOI: 10.1016/j.ijpddr.2017.04.003 -
PLoS Neglected Tropical Diseases Jul 2018The protozoan parasite Leishmania causes leishmaniasis; a spectrum of diseases of which there are an estimated 1 million new cases each year. Current treatments are...
The protozoan parasite Leishmania causes leishmaniasis; a spectrum of diseases of which there are an estimated 1 million new cases each year. Current treatments are toxic, expensive, difficult to administer, and resistance to them is emerging. New therapeutics are urgently needed, however, screening the infective amastigote form of the parasite is challenging. Only certain species can be differentiated into axenic amastigotes, and compound activity against these does not always correlate with efficacy against the parasite in its intracellular niche. Methods used to assess compound efficacy on intracellular amastigotes often rely on microscopy-based assays. These are laborious, require specialist equipment and can only determine parasite burden, not parasite viability. We have addressed this clear need in the anti-leishmanial drug discovery process by producing a transgenic L. mexicana cell line that expresses the luciferase NanoLuc-PEST. We tested the sensitivity and versatility of this transgenic strain, in comparison with strains expressing NanoLuc and the red-shifted firefly luciferase. We then compared the NanoLuc-PEST luciferase to the current methods in both axenic and intramacrophage amastigotes following treatment with a supralethal dose of Amphotericin B. NanoLuc-PEST was a more dynamic indicator of cell viability due to its high turnover rate and high signal:background ratio. This, coupled with its sensitivity in the intramacrophage assay, led us to validate the NanoLuc-PEST expressing cell line using the MMV Pathogen Box in a two-step process: i) identify hits against axenic amastigotes, ii) screen these hits using our bioluminescence-based intramacrophage assay. The data obtained from this highlights the potential of compounds active against M. tuberculosis to be re-purposed for use against Leishmania. Our transgenic L. mexicana cell line is therefore a highly sensitive and dynamic system suitable for Leishmania drug discovery in axenic and intramacrophage amastigote models.
Topics: Antiprotozoal Agents; Cell Line; Drug Discovery; Drug Evaluation, Preclinical; Humans; Leishmania mexicana; Leishmaniasis; Luciferases; Macrophages; Parasitic Sensitivity Tests
PubMed: 30001317
DOI: 10.1371/journal.pntd.0006639