Genetic dissection of the Leishmania paraflagellar rod, a unique flagellar cytoskeleton structure.
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The paraflagellar rod (PFR) is a unique network of cytoskeletal filaments that lies alongside the axoneme in the flagella of most trypanosomatids. While little is known about how two major Leishmania mexicana PFR protein components, PFR1 and PFR2, assemble into this complex structure, previous analysis of PFR2 null mutants demonstrated that the PFR is essential for proper cell motility. The structural roles of PFR1 and PFR2 are now examined through comparison of PFR2 null mutants with new PFR1 null mutant and PFR1/PFR2 double null mutant parasites. Both PFR1 and PFR2 were essential for PFR formation and cell motility. When elimination of one PFR gene prevented assembly of a native PFR structure, the other PFR protein accumulated at the distal flagellar tip. Comparison of PFR substructures remaining in each mutant revealed that: (1) fibers that attach the PFR to the axoneme did not contain PFR1 or PFR2, and assemble in the absence of a PFR. (2) PFR1 was synthesized and transported to the flagella in the absence of PFR2, where it formed a stable association with the axoneme attachment fibers. (3) PFR2 was synthesized and transported to the flagella in the absence of PFR1, though it was not found associated with the axoneme attachment fibers. (4) PFR1 and PFR2 were located throughout the subdomains of the PFR. These data suggest that while PFR filaments contain both PFR1 and PFR2, the PFR is attached to the axoneme by interaction of PFR1 with the axoneme attachment fibers. (+info)
Elucidation of carbon sources used for the biosynthesis of fatty acids and sterols in the trypanosomatid Leishmania mexicana.
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Sterols are necessary for the growth of trypanosomatid protozoans; sterol biosynthesis is a potential target for the use and development of drugs to treat the diseases caused by these organisms. This study has used (14)C-labelled substrates to investigate the carbon sources utilized by promastigotes and amastigotes of Leishmania mexicana for the production of sterol [mainly ergosta-5,7,24(24(1))-trien-3beta-ol] and the fatty acid moieties of the triacylglycerol (TAG) and phospholipid (PL) of the organism. The isoprenoid precursor mevalonic acid (MVA) was incorporated into the sterols, and the sterol precursor squalene, by the promastigotes of L. mexicana. However, acetate (the precursor to MVA in most organisms) was a very poor substrate for sterol production but was readily incorporated into the fatty acids of TAG and PL. Other substrates (glucose, palmitic acid, alanine, serine and isoleucine), which are metabolized to acetyl-CoA, were also very poor precursors to sterol but were incorporated into TAG and PL and gave labelling patterns of the lipids similar to those of acetate. In contrast, the amino acid leucine was the only substrate to be incorporated efficiently into the squalene and sterol of L. mexicana promastigotes. Quantitative measurements revealed that at least 70-80% of the sterol synthesized by the promastigotes of L. mexicana is produced from carbon provided by leucine metabolism. Studies with the amastigote form of L. mexicana showed that in this case leucine was again the major sterol precursor, whereas acetate was utilized for fatty acid production. (+info)
The fucose-mannose ligand-ELISA in the diagnosis and prognosis of canine visceral leishmaniasis in Brazil.
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The fucose-mannose ligand (FML)-ELISA assay showed a sensitivity of 100% and a specificity of 100% in diagnosis of canine visceral leishmaniasis (CVL) (kala-azar) in sera from naturally infected dogs from Sao Goncalo do Amaranto, Rio Grande de Norte, Brazil. The overall prevalence of antibodies to Leishmania in the endemic area was 23% (79 of 343). Seroreactivity detected by a Leishmania chagasi immunofluorescent (IF) assay was much lower (2.9%) and similar to the percentage of dogs with kala-azar symptoms (2.6%). Twenty-one of 21 asymptomatic, FML-seropositive animals died of kala-azar in a period ranging from 0 to 6 months after diagnosis. The predictive value was 100% for the FML-ELISA, 43% for an L. mexicana ELISA, and 24% for the L. mexicana and L. chagasi IF assays, respectively. In experimentally infected dogs, all assays detected seropositivity between 90 and 120 days after infection. Since the current strategy for control of CVL is based on detection and destruction of infected dogs, the highly predictive, sensitive, and specific FML-ELISA represents a useful tool for field control of the disease. (+info)
Experimental infection of canine peritoneal macrophages with visceral and dermotropic Leishmania strains.
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A study was carried out using macrophages cultured from the peritoneal exudate of dogs infected in vitro with three species of Leishmania: L. (L.) chagasi, L. (Viannia) braziliensis and L. (L.) amazonensis with the aim of investigating the growth kinetics and infectivity of these species in the host cell. Results were expressed as the percentage of macrophages infected measured at 24 hr intervals over six days in RPMI - 1640 culture medium at a temperature of 34-35 degrees C. The findings open the possibility of using canine peritoneal cells as a model for the screening of leishmanicide drugs and to study the pathogenesis of these species. (+info)
Proteophosphoglycan, a major secreted product of intracellular Leishmania mexicana amastigotes, is a poor B-cell antigen and does not elicit a specific conventional CD4+ T-cell response.
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Secreted and surface-exposed antigens of intracellular pathogens are thought to provide target structures for detection by the host immune system. The major secreted product of intracellular Leishmania mexicana amastigotes, a proteophosphoglycan (aPPG), is known to contribute to the establishment of the parasitophorous vacuole and is able to activate complement. aPPG belongs to a novel class of serine- and threonine-rich Leishmania proteins that are extensively modified by phosphodiester-linked phosphooligosaccharides and terminal mannooligosaccharides. Here we show that mice chronically infected with L. mexicana generally do not produce antibodies or Th cells specific for aPPG. Similarly, antibody titers are very low in mice vaccinated with aPPG, and specific CD4+ T cells are undetectable. Comparative analyses of other Leishmania glycoconjugates indicate that L. mexicana-specific carbohydrate structures are poorly immunogenic in mice and that the proteophosphoglycan aPPG behaved immunologically like a carbohydrate. The latter observation is explained by the lack of induction of aPPG-specific CD4+ T cells. In contrast, recombinant aPPG peptides stimulate CD4+ T-cell responses and high titers of specific antibodies are found in the sera of mice vaccinated with these peptides. Native aPPG is highly resistant to proteinases and apparently cannot be degraded by macrophages. It is concluded that conventional CD4+ T cells against the polypeptide backbone of aPPG are not induced because the molecule resists antigen processing due to its extensive and complex carbohydrate modification. The complex glycan chains of aPPG, which exhibit important biological functions for the parasite, may therefore also have evolved to evade detection by the immune system of the host organism. (+info)
Short report: a focus of Leishmania mexicana near Tucson, Arizona.
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Twenty-eight white-throated woodrats (Neotoma albigula) collected in Pima County, Arizona were screened for Leishmania using culture and the polymerase chain reaction (PCR). Two rodents were culture positive. Isoenzyme analysis determined the isolates to be Leishmania mexicana. The two culture-positive and four additional rodents were determined to be Leishmania-positive by the PCR. These isolates extend the geographic and ecologic range of enzootic leishmaniasis in the United States and represent a new host record. (+info)
Protection of C57BL/10 mice by vaccination with association of purified proteins from Leishmania (Leishmania) amazonensis.
(15/463)
In the past few years, induction of protective immunity to cutaneous leishmaniasis has been attempted by many researchers using a variety of antigenic preparations, such as living promastigotes or promastigote extracts, partially purified, or defined proteins. In this study, eleven proteins from Leishmania (Leishmania) amazonensis (LLa) with estimated molecular mass ranging from 97 to 13.5kDa were isolated by polyacrylamide gel electrophoresis and electro-elution. The proteins were associated as vaccine in different preparations with gp63 and BCG (Bacilli Calmette-Guerin). The antigenicity of these vaccines was measured by their ability to induce the production of IFN-gamma by lymphocyte from subjects vaccinated with Leishvacinregister mark or target. The immunogenicity was evaluated in vaccinated mice. C57BL/10 mice were vaccinated with three doses of each vaccine consisting of 30 microg of each protein at 15 days interval. One hundred microg of live BCG was only used in the first dose. Seven days after the last dose, they received a first challenge infection with 10(5) infective promastigotes and four months later, a second challenge was done. Two months after the second challenge, 42.86% of protection was obtained in the group of mice vaccinated with association of proteins of gp63+46+22kDa, gp63+13.5+25+42kDa, gp63+46+42kDa, gp63+66kDa, and gp63+97kDa; 57. 14% of protection was demonstrated with gp63+46+97+13.5kDa, gp63+46+97kDa, gp63+46+33kDa, and 71.43% protection for gp63 plus all proteins. The vaccine of gp63+46+40kDa that did not protect the mice, despite the good specific stimulation of lymphocytes (LSI = 7. 60) and 10.77UI/ml of IFN-gamma production. When crude extract of L. (L.) amazonensis was used with BCG a 57.14% of protection was found after the first challenge and 28.57% after the second, the same result was observed for gp63. The data obtained with the vaccines can suggest that the future vaccine probably have to contain, except the 40kDa, a cocktail of proteins that would protect mice against cutaneous leishmaniasis. (+info)
Proteophosphoglycans of Leishmania mexicana. Identification, purification, structural and ultrastructural characterization of the secreted promastigote proteophosphoglycan pPPG2, a stage-specific glycoisoform of amastigote aPPG.
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Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans that appear to be important for successful colonization of the sandfly and for virulence in the mammalian host. A hallmark of these molecules is extensive phosphoglycosylation by phosphoglycan chains via the unusual linkage Manalpha1-PO(4)-Ser. In this study we have identified and purified to apparent homogeneity a novel proteophosphoglycan (pPPG2) which is secreted by Leishmania mexicana promastigotes (sandfly stage). Amino acid analysis and immunoblots using polypeptide-specific antisera suggest that pPPG2 shares a common protein backbone with a proteophosphoglycan (aPPG) secreted by Leishmania mexicana amastigotes (mammalian stage). Both pPPG2 and aPPG show a similar degree of Ser phosphoglycosylation (50. 5 mol% vs. 44.6 mol%), but the structure of their phosphoglycan chains is developmentally regulated: in contrast to aPPG which displays unique, complex and highly branched glycan chains [Ilg, Craik, Currie, Multhaup, and Bacic (1998) J. Biol. Chem. 273, 13509-13523], pPPG2 contains short unbranched structures consisting of >60 mol% neutral glycans, most likely (Manalpha1-2)(0-5)Man and Galbeta1-4Man, as well as about 40 mol% monophosphorylated glycans of the proposed structures PO(4)-6Galbeta1-4Man and PO(4)-6(Glcbeta1-3)Galbeta1-4Man. The major differences between pPPG2 and aPPG with respect to their apparent molecular mass, their ultrastructure and their proteinase sensitivity are most likely a consequence of this stage-specific glycosylation of their common protein backbone. (+info)