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Molecular & Cellular Proteomics : MCP Sep 2008Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also...
Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to approximately 6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3'-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens.
Topics: 3' Untranslated Regions; Animals; Animals, Genetically Modified; Antigens, Protozoan; Cell Separation; Centrifugation, Isopycnic; Codon; Flow Cytometry; Fluorescence; Genome, Protozoan; Leishmania mexicana; Leishmaniasis Vaccines; Macrophages; Mice; Open Reading Frames; Proteome; Proteomics; Protozoan Proteins; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 18474515
DOI: 10.1074/mcp.M700343-MCP200 -
Acta Tropica Feb 2019Leishmaniasis is a neglected tropical disease caused by different species of protozoan parasites of the genus Leishmania. Dogs have been proven as primary hosts of the...
Leishmaniasis is a neglected tropical disease caused by different species of protozoan parasites of the genus Leishmania. Dogs have been proven as primary hosts of the parasite. Cases of cutaneous leishmaniasis in humans caused by Leishmania mexicana have been reported in Sinaloa; however, the vectors and hosts involved in the epidemiology of the parasite in northwestern Mexico are still unknown. Given the public health implications of this parasite's domestic hosts regarding the permanence and transmission of the disease to humans, the objective of the present study was to detect and determine the species of Leishmania that caused the first three cases of autochthonous canine leishmaniasis in the state of Sinaloa, Mexico. Three domestic dogs showing symptoms similar to canine leishmaniasis were identified, including chronic eye inflammation, corneal opacity, ocular exudate, emaciation and hyporexia. DNA was extracted from venous blood of the infected animals using a commercial kit. The internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was amplified by specific primers for Leishmania from the extracted DNA, and the PCR products were digested with the restriction enzyme HaeIII. In addition, PCR products were subjected to automated sequencing. Molecular analysis showed that the infecting species was L. mexicana. This is the first report of autochthonous canine leishmaniasis caused by L. mexicana in Sinaloa, Mexico. Further studies are required to identify the species that serve as vectors and other wild and domestic hosts of the parasite, as well as to determine if there are more species of Leishmania circulating in Sinaloa.
Topics: Animals; Dog Diseases; Dogs; Leishmania mexicana; Leishmaniasis, Cutaneous; Polymerase Chain Reaction
PubMed: 30500369
DOI: 10.1016/j.actatropica.2018.11.027 -
Molecular and Biochemical Parasitology Nov 2020Leishmania parasites are of great relevance to public health because they are the causative agents of various long-term and health-threatening diseases in humans....
Two phylogenetically divergent isocitrate dehydrogenases are encoded in Leishmania parasites. Molecular and functional characterization of Leishmania mexicana isoenzymes with specificity towards NAD and NADP.
Leishmania parasites are of great relevance to public health because they are the causative agents of various long-term and health-threatening diseases in humans. Dependent on the manifestation, drugs either require difficult and lengthy administration, are toxic, expensive, not very effective or have lost efficacy due to the resistance developed by these pathogens against clinical treatments. The intermediary metabolism of Leishmania parasites is characterized by several unusual features, among which whether the Krebs cycle operates in a cyclic and/or in a non-cyclic mode is included. Our survey of the genomes of Leishmania species and monoxenous parasites such as those of the genera Crithidia and Leptomonas (http://www.tritrypdb.org) revealed that two genes encoding putative isocitrate dehydrogenases (IDHs) -with distantly related sequences- are strictly conserved among these parasites. Thus, in this study, we aimed to functionally characterize the two leishmanial IDH isoenzymes, for which we selected the genes LmxM10.0290 (Lmex_IDH-90) and LmxM32.2550 (Lmex_IDH-50) from L. mexicana. Phylogenetic analysis showed that Lmex_IDH-50 clustered with members of Subfamily I, which contains mainly archaeal and bacterial IDHs, and that Lmex_IDH-90 was a close relative of eukaryotic enzymes comprised within Subfamily II IDHs. 3-D homology modeling predicted that both IDHs exhibited the typical folding motifs recognized as canonical for prokaryotic and eukaryotic counterparts, respectively. Both IDH isoforms displayed dual subcellular localization, in the cytosol and the mitochondrion. Kinetic studies showed that Lmex_IDH-50 exclusively catalyzed the reduction of NAD, while Lmex_IDH-90 solely used NADP as coenzyme. Besides, Lmex_IDH-50 differed from Lmex_IDH-90 by exhibiting a nearly 20-fold lower apparent K value towards isocitrate (2.0 μM vs 43 μM). Our findings showed, for the first time, that the genus Leishmania differentiates not only from other trypanosomatids such as Trypanosoma cruzi and Trypanosoma brucei, but also from most living organisms, by exhibiting two functional homo-dimeric IDHs, highly specific towards NAD and NADP, respectively. It is tempting to argue that any or both types of IDHs might be directly or indirectly linked to the Krebs cycle and/or to the de novo synthesis of glutamate. Our results about the biochemical and structural features of leishmanial IDHs show the relevance of deepening our knowledge of the metabolic processes in these pathogenic parasites to potentially identify new therapeutic targets.
Topics: Amino Acid Sequence; Cloning, Molecular; Enzyme Activation; Gene Expression; Humans; Isocitrate Dehydrogenase; Isoenzymes; Kinetics; Leishmania mexicana; Leishmaniasis, Cutaneous; NAD; NADP; Phylogeny; Protein Transport; Sequence Analysis, DNA; Structure-Activity Relationship; Substrate Specificity
PubMed: 32980452
DOI: 10.1016/j.molbiopara.2020.111320 -
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 -
Antimicrobial Agents and Chemotherapy Oct 2016We report that the tuberculosis drug SQ109 [N-adamantan-2-yl-N'-((E)-3,7-dimethyl-octa-2,6-dienyl)-ethane-1,2-diamine] has potent activity against the intracellular...
We report that the tuberculosis drug SQ109 [N-adamantan-2-yl-N'-((E)-3,7-dimethyl-octa-2,6-dienyl)-ethane-1,2-diamine] has potent activity against the intracellular amastigote form of Leishmania mexicana (50% inhibitory concentration [IC50], ∼11 nM), with a good selectivity index (>500). It is also active against promastigotes (IC50, ∼500 nM) and acts as a protonophore uncoupler, in addition to disrupting Ca(2+) homeostasis by releasing organelle Ca(2+) into the cytoplasm, and as such, it is an interesting new leishmaniasis drug hit candidate.
Topics: Adamantane; Animals; Antiprotozoal Agents; Antitubercular Agents; Calcium; Cell Line; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Ethylenediamines; Inhibitory Concentration 50; Leishmania mexicana; Macrophages; Mice
PubMed: 27458218
DOI: 10.1128/AAC.00945-16 -
Experimental Parasitology Oct 2002During the insect phase of the parasite lifecycle, Leishmania promastigotes move from the midgut to the anterior regions of the alimentary tract of their sandfly vector....
During the insect phase of the parasite lifecycle, Leishmania promastigotes move from the midgut to the anterior regions of the alimentary tract of their sandfly vector. Chemotaxis of Leishmania promastigotes towards sugars has been reported, and the putative presence of sugar gradient in the insect foregut has been suggested to play a role in promastigote development in the insect. We have further investigated the potential of Leishmania mexicana promastigotes to respond to chemical stimulii. We find that promastigotes move towards concentrations of all substances tested and that this taxis requires the presence of an osmotic gradient. Our results indicate that behaviour that has previously been interpreted as chemotaxis is better understood as osmotaxis. The implications of this observation are discussed in the context of promastigote development.
Topics: Animals; Carbohydrates; Chemotaxis; Insect Vectors; Leishmania mexicana; Movement; Osmolar Concentration; Psychodidae
PubMed: 12706748
DOI: 10.1016/s0014-4894(03)00031-6 -
Journal of Molecular Microbiology and... 20106-Phosphogluconate dehydrogenase (6PGDH) is a key enzyme of the oxidative branch involved in the generation of NADPH and ribulose 5-phosphate. In the present work, we...
6-Phosphogluconate dehydrogenase (6PGDH) is a key enzyme of the oxidative branch involved in the generation of NADPH and ribulose 5-phosphate. In the present work, we describe the cloning, sequencing and characterization of a 6PGDH gene from Leishmania (Leishmania) mexicana. The gene encodes a polypeptide chain of 479 amino acid residues with a predicted molecular mass of 52 kDa and a pI of 5.77. The recombinant protein possesses a dimeric quaternary structure and displays kinetic parameter values intermediate between those reported for Trypanosoma brucei and T. cruzi with apparent K(m) values of 6.93 and 5.2 μM for 6PG and NADP(+), respectively. The three-dimensional structure of the enzymes of Leishmania and T. cruzi were modelled from their amino acid sequence using the crystal structure of the enzyme of T. brucei as template. The amino acid residues located in the 6PGDH C-terminal region, which are known to participate in the salt bridges maintaining the protein dimeric structure, differed significantly among the enzymes of Leishmania, T. cruzi, and T. brucei. Our results strongly suggest that 6PGDH can be selected as a potential target for the development of new therapeutic drugs in order to improve existing chemotherapeutic treatments against these parasites.
Topics: Amino Acid Sequence; Cloning, Molecular; Leishmania mexicana; Models, Molecular; Molecular Sequence Data; Phosphogluconate Dehydrogenase; Protein Structure, Secondary; Recombinant Proteins; Sequence Alignment
PubMed: 21160204
DOI: 10.1159/000320697 -
Parasitology Research Apr 2013Macrophages (Mφ) and dendritic cells are the major target cell populations of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a...
Macrophages (Mφ) and dendritic cells are the major target cell populations of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a strategy employed by multiple pathogens to ensure their survival in the infected cell. Leishmania promastigotes have been shown to protect Mφ, neutrophils, and dendritic cells from both natural and induced apoptosis. Nevertheless, the effect of the infection with Leishmania amastigotes in the apoptosis of these cell populations has not been established, which results are very important since amastigotes persist in cells for many days and are responsible for sustaining infection in the host. As shown in this study, apoptosis of monocyte-derived dendritic cells (moDC) induced by treatment with camptothecin was downregulated by infection with L. mexicana amastigotes from 42.48 to 36.92% as detected by Annexin-V binding to phosphatidylserine. Also, the infection of moDC with L. mexicana amastigotes diminished the fragmentation of DNA as detected by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay, and changes in cell morphology were analyzed by electron microscopy. The observed antiapoptotic effect was found to be associated with an 80% reduction in the presence of active caspase-3 in infected moDC. The capacity of L. mexicana amastigotes to delay apoptosis induction in the infected moDC may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.
Topics: Animals; Annexin A5; Apoptosis; Dendritic Cells; Electrons; Humans; Immune Evasion; In Situ Nick-End Labeling; Leishmania mexicana
PubMed: 23420408
DOI: 10.1007/s00436-013-3334-2 -
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 -
Memorias Do Instituto Oswaldo Cruz 1987Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell...
Inhibition of one Leishmania subspecies by exometabolites of another subspecies, a phenomenon not previously reported, is suggested by our recent observations in cell cloning experiments with Leishmania mexicana mexicana and Leishmania mexicana amazonensis. Clones were identified using the technique of schizodeme analysis. The phenomenon observed is clearly relevant to studies of parasite isolation, leishmanial metabolism, cross-immunity and chemotherapy.
Topics: Animals; Culture Media; DNA Restriction Enzymes; DNA, Circular; DNA, Kinetoplast; Electrophoresis, Polyacrylamide Gel; Growth Inhibitors; In Vitro Techniques; Leishmania mexicana; Species Specificity
PubMed: 2853814
DOI: 10.1590/s0074-02761987000400011