-
The Journal of Biological Chemistry Nov 2014Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway...
Gluconeogenesis is an active pathway in Leishmania amastigotes and is essential for their survival within the mammalian cells. However, our knowledge about this pathway in trypanosomatids is very limited. We investigated the role of glycerol kinase (GK), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate phosphate dikinase (PPDK) in gluconeogenesis by generating the respective Leishmania mexicana Δgk, Δpepck, and Δppdk null mutants. Our results demonstrated that indeed GK, PEPCK, and PPDK are key players in the gluconeogenesis pathway in Leishmania, although stage-specific differences in their contribution to this pathway were found. GK participates in the entry of glycerol in promastigotes and amastigotes; PEPCK participates in the entry of aspartate in promastigotes, and PPDK is involved in the entry of alanine in amastigotes. Furthermore, the majority of alanine enters into the pathway via decarboxylation of pyruvate in promastigotes, whereas pathway redundancy is suggested for the entry of aspartate in amastigotes. Interestingly, we also found that l-lactate, an abundant glucogenic precursor in mammals, was used by Leishmania amastigotes to synthesize mannogen, entering the pathway through PPDK. On the basis of these new results, we propose a revision in the current model of gluconeogenesis in Leishmania, emphasizing the differences between amastigotes and promastigotes. This work underlines the importance of studying the trypanosomatid intracellular life cycle stages to gain a better understanding of the pathologies caused in humans.
Topics: Biosynthetic Pathways; Blotting, Southern; Blotting, Western; DNA, Protozoan; Gluconeogenesis; Glucose; Glycerol Kinase; Humans; Leishmania mexicana; Life Cycle Stages; Mutation; Phosphoenolpyruvate Carboxylase; Protozoan Proteins; Pyruvate, Orthophosphate Dikinase
PubMed: 25288791
DOI: 10.1074/jbc.M114.569434 -
PLoS Pathogens Jun 2020The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its...
The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.
Topics: Animals; Cell Cycle; Deubiquitinating Enzymes; Female; Gene Deletion; Leishmania mexicana; Mice; Mice, Inbred BALB C; Protozoan Proteins; Ubiquitination
PubMed: 32544189
DOI: 10.1371/journal.ppat.1008455 -
PLoS Neglected Tropical Diseases 2012While C57BL/6 mice infected in the ear with L. major mount a vigorous Th1 response and resolve their lesions, the Th1 response in C57BL/6 mice infected with L. mexicana... (Comparative Study)
Comparative Study
While C57BL/6 mice infected in the ear with L. major mount a vigorous Th1 response and resolve their lesions, the Th1 response in C57BL/6 mice infected with L. mexicana is more limited, resulting in chronic, non-healing lesions. The aim of this study was to determine if the limited immune response following infection with L. mexicana is related to a deficiency in the ability of monocyte-derived dendritic cells (mo-DCs) to prime a sufficient Th1 response. To address this issue we compared the early immune response following L. mexicana infection with that seen in L. major infected mice. Our data show that fewer monocytes are recruited to the lesions of L. mexicana infected mice as compared to mice infected with L. major. Moreover, monocytes that differentiate into mo-DCs in L. mexicana lesions produced less iNOS and migrated less efficiently to the draining lymph node as compared to those from L. major infected mice. Treatment of L. mexicana infected mice with α-IL-10R antibody resulted in increased recruitment of monocytes to the lesion along with greater production of IFN-γ and iNOS. Additionally, injection of DCs into the ear at the time of infection with L. mexicana also led to a more robust Th1 response. Taken together, these data suggest that during L. mexicana infection reduced recruitment, activation and subsequent migration of monocytes and mo-DCs to the draining lymph nodes may result in the insufficient priming of a Th1 response.
Topics: Animals; Dendritic Cells; Ear; Female; Leishmania major; Leishmania mexicana; Leishmaniasis, Cutaneous; Mice; Mice, Inbred C57BL; Monocytes; Th1 Cells
PubMed: 23094119
DOI: 10.1371/journal.pntd.0001858 -
PloS One 2015Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines...
Chemotherapy against visceral leishmaniasis is associated with high toxicity and drug resistance. Leishmania parasites are purine auxotrophs that obtain their purines from exogenous sources. Nucleoside hydrolases release purines from nucleosides and are drug targets for anti-leishmanial drugs, absent in mammal cells. We investigated the substrate specificity of the Leishmania (L.) donovani recombinant nucleoside hydrolase NH36 and the inhibitory effect of the immucillins IA (ImmA), DIA (DADMe-ImmA), DIH (DADMe-ImmH), SMIH (SerMe-ImmH), IH (ImmH), DIG (DADMe-ImmG), SMIG (SerMe-ImmG) and SMIA (SerME-ImmA) on its enzymatic activity. The inhibitory effects of immucillins on the in vitro multiplication of L. (L.) infantum chagasi and L. (L.) amazonensis promastigotes were determined using 0.05-500 μM and, when needed, 0.01-50 nM of each drug. The inhibition on multiplication of L. (L.) infantum chagasi intracellular amastigotes in vitro was assayed using 0.5, 1, 5 and 10 μM of IA, IH and SMIH. The NH36 shows specificity for inosine, guanosine, adenosine, uridine and cytidine with preference for adenosine and inosine. IA, IH, DIH, DIG, SMIH and SMIG immucillins inhibited L. (L.) infantum chagasi and L. (L.) amazonensis promastigote growth in vitro at nanomolar to micromolar concentrations. Promastigote replication was also inhibited in a chemically defined medium without a nucleoside source. Addition of adenosine decreases the immucillin toxicity. IA and IH inhibited the NH36 enzymatic activity (Ki = 0.080 μM for IA and 0.019 μM for IH). IA, IH and SMIH at 10 μM concentration, reduced the in vitro amastigote replication inside mice macrophages by 95% with no apparent effect on macrophage viability. Transmission electron microscopy revealed global alterations and swelling of L. (L.) infantum chagasi promastigotes after treatment with IA and IH while SMIH treatment determined intense cytoplasm vacuolization, enlarged vesicles and altered kinetoplasts. Our results suggest that IA, IH and SMIH may provide new chemotherapy agents for leishmaniasis.
Topics: Adenine; Adenosine; Animals; Antiprotozoal Agents; Cell Proliferation; Enzyme Inhibitors; Female; Humans; In Vitro Techniques; Kinetics; Leishmania infantum; Leishmania mexicana; Leishmaniasis, Cutaneous; Leishmaniasis, Visceral; Mice; Mice, Inbred BALB C; Microscopy, Electron, Transmission; N-Glycosyl Hydrolases; Purine Nucleosides; Pyrimidinones; Pyrroles; Pyrrolidines
PubMed: 25909893
DOI: 10.1371/journal.pone.0124183 -
Infection and Immunity Jun 2020l-Arginine metabolism through arginase 1 (Arg-1) and inducible nitric oxide synthase (NOS2) constitutes a fundamental axis for the resolution or progression of...
Differential Regulation of l-Arginine Metabolism through Arginase 1 during Infection with Leishmania mexicana Isolates Obtained from Patients with Localized and Diffuse Cutaneous Leishmaniasis.
l-Arginine metabolism through arginase 1 (Arg-1) and inducible nitric oxide synthase (NOS2) constitutes a fundamental axis for the resolution or progression of leishmaniasis. Infection with can cause two distinct clinical manifestations: localized cutaneous leishmaniasis (LCL) and diffuse cutaneous leishmaniasis (DCL). In this work, we analyzed in an model the capacity of two isolates, one obtained from a patient with LCL and the other from a patient with DCL, to regulate the metabolism of l-arginine through Arg-1 and NOS2. Susceptible BALB/c mice were infected with isolates from both clinical manifestations, and the evolution of the infection as well as protein presence and activity of Arg-1 and NOS2 were evaluated. The lesions of mice infected with the DCL isolate were bigger, had higher parasite loads, and showed greater protein presence and enzymatic activity of Arg-1 than the lesions of mice infected with the LCL isolate. In contrast, NOS2 protein synthesis was poorly or not induced in the lesions of mice infected with the LCL or DCL isolate. The immunochemistry analysis of the lesions allowed the identification of highly parasitized macrophages positive for Arg-1, while no staining for NOS2 was found. In addition, we observed in lesions of patients with DCL macrophages with higher parasite loads and stronger Arg-1 staining than those in lesions of patients with LCL. Our results suggest that isolates obtained from patients with LCL or DCL exhibit different virulence or pathogenicity degrees and differentially regulate l-arginine metabolism through Arg-1.
Topics: Animals; Arginase; Arginine; Disease Models, Animal; Disease Susceptibility; Host-Pathogen Interactions; Humans; Leishmania mexicana; Leishmaniasis, Diffuse Cutaneous; Macrophages; Mice; Nitric Oxide Synthase Type II; Time Factors
PubMed: 32312763
DOI: 10.1128/IAI.00963-19 -
Acta Parasitologica Mar 2024Leishmaniasis is a neglected disease with high prevalence and incidence in tropical and subtropical areas. Existing drugs are limited due to cost, toxicity, declining...
INTRODUCTION
Leishmaniasis is a neglected disease with high prevalence and incidence in tropical and subtropical areas. Existing drugs are limited due to cost, toxicity, declining efficacy and unavailability in endemic places. Drug repurposing has established as an efficient way for the discovery of drugs for a variety of diseases.
PURPOSE
The objective of the present work was testing the antileishmanial activity of thioridazine, an antipsychotic agent with demonstrated effect against other intracellular pathogens.
METHODS
The cytotoxicity for mouse peritoneal macrophages as well as the activity against Leishmania amazonensis, Leishmania mexicana and Leishmania major promastigotes and intracellular amastigotes, as well as in a mouse model of cutaneous leishmaniasis, were assessed.
RESULTS
Thioridazine inhibited the in vitro proliferation of promastigotes (50% inhibitory concentration-IC-values in the range of 0.73 µM to 3.8 µM against L. amazonensis, L. mexicana and L. major) and intracellular amastigotes (IC values of 1.27 µM to 4.4 µM for the same species). In contrast, in mouse peritoneal macrophages, the 50% cytotoxic concentration was 24.0 ± 1.89 µM. Thioridazine inhibited the growth of cutaneous lesions and reduced the number of parasites in the infected tissue of mice. The dose of thioridazine that inhibited lesion development by 50% compared to controls was 23.3 ± 3.1 mg/kg and in terms of parasite load, it was 11.1 ± 0.97 mg/kg.
CONCLUSIONS
Thioridazine was effective against the promastigote and intracellular amastigote stages of three Leishmania species and in a mouse model of cutaneous leishmaniasis, supporting the potential repurposing of this drug as an antileishmanial agent.
Topics: Animals; Thioridazine; Mice; Antiprotozoal Agents; Macrophages, Peritoneal; Leishmaniasis, Cutaneous; Leishmania; Mice, Inbred BALB C; Female; Inhibitory Concentration 50; Disease Models, Animal; Leishmania mexicana; Drug Repositioning; Leishmania major
PubMed: 38070122
DOI: 10.1007/s11686-023-00746-2 -
PloS One 2018Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these...
Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these parasites based on differential mRNA expression data. Due to unusual gene expression mechanism, functions of such genes must be further validated experimentally. Here, we investigated a role of one of the putative virulence factors, LmxM.22.0010-encoded BTN1 (a protein involved in Batten disease in humans), in L. mexicana infectivity. Due to the incredible plasticity of the L. mexicana genome, we failed to obtain a complete knock-out of LmxM.22.0010 using conventional recombination-based approach even after ablating four alleles of this gene. To overcome this, we established a modified CRISPR-Cas9 system with genomic expression of Cas9 nuclease and gRNA. Application of this system allowed us to establish a complete BTN1 KO strain of L. mexicana. The mutant strain did not show any difference in growth kinetics and differentiation in vitro, as well as in the infectivity for insect vectors and mice hosts. Based on the whole-transcriptome profiling, LmxM.22.0010-encoded BTN1 was considered a putative factor of virulence in Leishmania. Our study suggests that ablation of LmxM.22.0010 does not influence L. mexicana infectivity and further illustrates importance of experimental validation of in silico-predicted virulence factors. Here we also describe the whole genome sequencing of the widely used model isolate L. mexicana M379 and report a modified CRISPR/Cas9 system suitable for complete KO of multi-copy genes in organisms with flexible genomes.
Topics: Animals; CRISPR-Cas Systems; Computer Simulation; Female; Gene Expression Profiling; Gene Knockout Techniques; Genes, Protozoan; Humans; Insect Vectors; Leishmania mexicana; Leishmaniasis, Cutaneous; Mice; Mice, Inbred BALB C; Psychodidae; Virulence
PubMed: 29438445
DOI: 10.1371/journal.pone.0192723 -
Journal of Proteomics Feb 2017Leishmania parasites multiply and develop in the gut of a sand fly vector in order to be transmitted to a vertebrate host. During this process they encounter and exploit...
UNLABELLED
Leishmania parasites multiply and develop in the gut of a sand fly vector in order to be transmitted to a vertebrate host. During this process they encounter and exploit various nutrients, including sugars, and amino and fatty acids. We have previously generated a mutant Leishmania line that is deficient in glucose transport and which displays some biologically important phenotypic changes such as reduced growth in axenic culture, reduced biosynthesis of hexose-containing virulence factors, increased sensitivity to oxidative stress, and dramatically reduced parasite burden in both insect vector and macrophage host cells. Here we report the generation and integration of proteomic and metabolomic approaches to identify molecular changes that may explain these phenotypes. Our data suggest changes in pathways of glycoconjugate production and redox homeostasis, which likely represent adaptations to the loss of sugar uptake capacity and explain the reduced virulence of this mutant in sand flies and mammals. Our data contribute to understanding the mechanisms of metabolic adaptation in Leishmania and illustrate the power of integrated proteomic and metabolomic approaches to relate biochemistry to phenotype.
BIOLOGICAL SIGNIFICANCE
This paper reports the application of comparative proteomic and metabolomic approaches to reveal the molecular basis for important phenotypic changes Leishmania parasites that are deficient in glucose uptake. Leishmania cause a very significant disease burden across the world and there are few effective drugs available for control. This work shows that proteomics and metabolomics can produce complementary data that advance understanding of parasite metabolism and highlight potential new targets for chemotherapy.
Topics: Adaptation, Physiological; Leishmania mexicana; Metabolome; Metabolomics; Proteome; Proteomics; Protozoan Proteins
PubMed: 28040509
DOI: 10.1016/j.jprot.2016.12.009 -
Experimental Parasitology Mar 2012Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas' disease and African sleeping sickness. Arginase, which is a...
Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas' disease and African sleeping sickness. Arginase, which is a metallohydrolase, is the first enzyme involved in polyamine biosynthesis and converts arginine into ornithine and urea. Ornithine is used in the polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. The flavonols quercetin and quercitrin have been described as antitrypanosomal and antileishmanial compounds, and their ability to inhibit arginase was tested in this work. We characterized the inhibition of recombinant arginase from Leishmania (Leishmania) amazonensis by quercetin, quercitrin and isoquercitrin. The IC(50) values for quercetin, quercitrin and isoquercitrin were estimated to be 3.8, 10 and 4.3 μM, respectively. Quercetin is a mixed inhibitor, whereas quercitrin and isoquercitrin are uncompetitive inhibitors of L. (L.) amazonensis arginase. Quercetin interacts with the substrate l-arginine and the cofactor Mn(2+) at pH 9.6, whereas quercitrin and isoquercitrin do not interact with the enzyme's cofactor or substrate. Docking analysis of these flavonols suggests that the cathecol group of the three compounds interact with Asp129, which is involved in metal bridge formation for the cofactors Mn(A)(2+) and Mn(B)(2+) in the active site of arginase. These results help to elucidate the mechanism of action of leishmanicidal flavonols and offer new perspectives for drug design against Leishmania infection based on interactions between arginase and flavones.
Topics: Antiprotozoal Agents; Arginase; Arginine; Dose-Response Relationship, Drug; Inhibitory Concentration 50; Leishmania mexicana; Manganese; Quercetin
PubMed: 22327179
DOI: 10.1016/j.exppara.2012.01.015 -
BioMed Research International 2022Endogenous nucleotides produced by various group of cells under inflammatory conditions act as potential danger signals . Extracellularly released nucleotides such as...
Endogenous nucleotides produced by various group of cells under inflammatory conditions act as potential danger signals . Extracellularly released nucleotides such as ATP are rapidly hydrolyzed to adenosine by the coordinated ectonucleotidase activities of CD39 and CD73. is an obligate intracellular parasite of macrophages and capable of modulating host immune response in order to survive and multiply within host cells. In this study, the activity of CD73 induced by in infected macrophages has been investigated and correlated with parasite survival and infection . For this, the expression of CD39 and CD73, by flow cytometry, in murine peritoneal macrophages infected with metacyclic promastigotes of has been analyzed. Our results showed that infected macrophages, unlike LPS-treated macrophages, increased CD73 expression. It was also noted that when CD73 enzymatic activity was blocked by , -methyleneadenosine 5'-diphosphate sodium salt (APCP), macrophage parasitism was significantly decreased. Interestingly, these effects were not associated with the production of TNF-, IL-10, or nitric oxide (NO). Together, these data demonstrate that induces a regulatory phenotype in macrophages, which by activating the CD39/CD73 pathway allows parasite survival through the action of immunomodulatory adenosine receptors.
Topics: 5'-Nucleotidase; Adenosine Triphosphatases; Animals; Antigens, CD; Apyrase; Leishmania mexicana; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL
PubMed: 35187176
DOI: 10.1155/2022/9928362