RESUMO
Transmission of leishmaniasis is effected by a specific developmental stage, the metacyclic promastigote. The precursors of metacyclic promastigotes were a distinct subpopulation of parasites, identified for the first time as a new stage in the life-cycle and named leptomonad promastigotes. Microdissection of infected sandflies into 4 midgut regions and foregut allowed precursor-product relationships to be established for amastigote-procyclic promastigote, procyclic-nectomonad promastigote, nectomonad-leptomonad promastigote and leptomonad-metacyclic promastigote developmental switches. Metacyclic promastigotes occurred mainly in the thoracic midgut and cardia, coincident with the accumulation of a promastigote secretory gel (PSG) plug in these anterior regions. The gel-like plug was isolated from flies with mature infections and found to contain predominantly leptomonad promastigotes. The PSG plug also contained the majority (75%) of the total metacyclic promastigote population in the sandflies, which were concentrated at the anterior pole. The PSG plug was found to be the main site of metacyclogenesis, and acted as a reservoir of leptomonad promastigotes from which metacyclic forms differentiated and migrated forward to promote the infective potential of the fly. The PSG plug occluded and distorted the midgut, forcing the stomodeal valve open and affecting the feeding success of the sandflies, such that they experienced difficulty in taking a full meal. Collectively, these data support the role of the PSG in the transmission of leishmaniasis, by conditioning the midgut environment for metacyclogenesis and altering the feeding ability of infected sandflies.
Assuntos
Leishmania mexicana/crescimento & desenvolvimento , Leishmaniose Cutânea/transmissão , Proteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Proteínas de Protozoários , Psychodidae/parasitologia , Animais , Sistema Digestório/metabolismo , Sistema Digestório/parasitologia , Comportamento Alimentar , Géis , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/parasitologia , Psychodidae/fisiologiaRESUMO
Two cases of skin lesions similar to those caused by Leishmania parasites have been reported from Martinique. Parasites isolated from these lesions were unlike Leishmania reference strains by isoenzyme analysis and electron microscopy and were assumed to be monoxenous trypanosomatids which normally only infect invertebrates. Both strains have now been retyped by isoenzyme analysis and found to be identical to each other and distantly related to all other Leishmania species. The sequence of the 18S ribosomal RNA gene and partial sequences of the DNA polymerase alpha and RNA polymerase II largest subunit genes were obtained. These sequences indicated that the Martinique parasites clustered with L. enriettii and were basal to all other euleishmania. However, support for both the position basal to all euleishmania and the clustering with L. enriettii was low. The Martinique parasites may cluster with L. (Leishmania) or L. (Viannia) or form a novel clade within the euleishmania either with or without L. enriettii.
Assuntos
Leishmania/classificação , Leishmaniose Cutânea/parasitologia , Animais , DNA Polimerase I/química , DNA Polimerase I/genética , DNA de Protozoário/química , DNA de Protozoário/genética , DNA de Protozoário/isolamento & purificação , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Ribossômico/isolamento & purificação , Eletroforese em Gel de Amido , Humanos , Isoenzimas/metabolismo , Leishmania/enzimologia , Leishmania/genética , Leishmaniose Cutânea/patologia , Martinica , Filogenia , Reação em Cadeia da Polimerase , RNA Polimerase II/química , RNA Polimerase II/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2-(13)C]leucine with L. mexicana promastigotes in the presence of ketoconazole gave 14alpha-methylergosta-8,24(24(1))-3beta-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of (13)C per molecule. (13)C NMR analysis of the 14alpha-methylergosta-8,24(24(1))-3beta-ol revealed that it was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and C-23. This established that the leucine skeleton is incorporated intact into the isoprenoid pathway leading to sterol; it is not converted first to acetyl-CoA, as in animals and plants, with utilization of the acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of [1-(14)C]acetate into sterol but had no inhibitory effect on [U-(14)C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-(14)C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-(14)C]leucine into sterol. These results are compatible with two routes by which the leucine skeleton may enter intact into the isoprenoid pathway. The catabolism of leucine could generate HMG-CoA that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA.
Assuntos
Leishmania mexicana/metabolismo , Leucina/metabolismo , Esteróis/biossíntese , Animais , Antiprotozoários/farmacologia , Isótopos de Carbono , Inibidores Enzimáticos/farmacologia , Ácidos Graxos Insaturados/farmacologia , Hidroximetilglutaril-CoA Sintase/antagonistas & inibidores , Lactonas/farmacologia , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/enzimologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Ressonância Magnética Nuclear Biomolecular , Esteróis/químicaRESUMO
Contradictory biogeographic hypotheses for either a Neotropical or a Palaearctic origin of the genus Leishmania have been proposed. Hypotheses constructed on the basis of biogeographic data must be tested against an independent dataset and cannot be supported by biogeographic data alone. In the absence of a fossil record for the Leishmania these two hypotheses were tested against a combined dataset of sequences from the DNA polymerase A catalytic subunit and the RNA polymerase II largest subunit. The phylogeny obtained provided considerable support for a Neotropical origin of the genus Leishmania and leads us to reject the hypothesis for a Palaearctic origin.
Assuntos
Leishmania/genética , Filogenia , Animais , DNA Polimerase III/análise , Leishmania/classificação , RNA Polimerase II/análiseRESUMO
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.
Assuntos
Ácidos Graxos/biossíntese , Leishmania mexicana/metabolismo , Esteróis/biossíntese , Ácido Acético/metabolismo , Aminoácidos/metabolismo , Animais , Carbono/metabolismo , Radioisótopos de Carbono , Glucose/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Leucina/metabolismo , Ácido Mevalônico/metabolismo , Ácido Palmítico/metabolismo , Fosfolipídeos/biossíntese , Esqualeno/metabolismo , Triglicerídeos/biossínteseRESUMO
Promastigotes of Leishmania braziliensis, L. donovani, L. major and L. mexicana recently derived from tissue amastigotes were cultured in Schneider's Drosophila medium supplemented with 20% (v/v) heat-inactivated foetal calf serum and 25 micrograms gentamicin sulfate/ml at pH 5.5. These cultures produced more metacyclic promastigotes in their stationary-phase populations than others cultured at pH 7.0. Metacyclic promastigotes possessed a short (< or = 8 microns) and narrow (< or = 1.5 microns) cell body with a flagellum twice or more the length of the cell body. Promastigotes from acidic cultures were more resistant to complement-mediated lysis and more infective in vivo than those grown at neutral pH. These results demonstrate that induction of metacyclogenesis by acidic pH is a response conserved across a variety of species of Leishmania.
Assuntos
Leishmania/crescimento & desenvolvimento , Animais , Proteínas do Sistema Complemento/imunologia , Cricetinae , Interações Hospedeiro-Parasita , Concentração de Íons de Hidrogênio , Leishmania/citologia , Leishmania/imunologia , Leishmania/patogenicidade , Leishmania braziliensis/crescimento & desenvolvimento , Leishmania donovani/crescimento & desenvolvimento , Leishmania major/crescimento & desenvolvimento , Leishmania mexicana/crescimento & desenvolvimento , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Estágios do Ciclo de Vida , Camundongos , Camundongos Endogâmicos BALB C , Especificidade da EspécieRESUMO
In the Mediterranean region Leishmania infantum causes both visceral and cutaneous leishmaniasis. These two pathologies tend to be caused by distinct parasite zymodemes. We have studied 33 isolates of Leishmania, 2 from sandflies, 5 from visceral cases, and 26 from cutaneous cases in Honduras, to determine if there is a correlation between pathology and parasite type in the New World similar to that in the Mediterranean region. Nine of the 26 cutaneous cases were caused by L. mexicana parasites, which have not been previously reported from Honduras; the remaining 17 cutaneous cases were due to L. chagasi. Only minor differences were found between the Honduran L. chagasi parasites by random amplified polymorphic DNA, differential display, pulsed-field gel electrophoresis, and schizodemes. This suggests that in Honduras the parasite type may not be the only factor determining the clinical outcome of L. chagasi infections.
Assuntos
Leishmania infantum/genética , Leishmaniose Cutânea/parasitologia , Leishmaniose Visceral/parasitologia , Adolescente , Animais , Criança , Pré-Escolar , DNA de Cinetoplasto/análise , Eletroforese em Gel de Campo Pulsado , Feminino , Genótipo , Honduras/epidemiologia , Humanos , Lactente , Insetos Vetores/parasitologia , Cariotipagem , Leishmania infantum/classificação , Leishmaniose Cutânea/epidemiologia , Leishmaniose Visceral/epidemiologia , Masculino , Região do Mediterrâneo/epidemiologia , Reação em Cadeia da Polimerase , Polimorfismo Genético , Psychodidae/parasitologia , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
Cutaneous and mucocutaneous leishmaniases are widely spread in the mountainous Andean regions of South America. In Venezuela, these regions consist of the coffee-growing states of Trujillo, Merida and Tachira. Entomological and parasitological investigations in three geographically different climatic zones (Lomas Bajas, Delicias and La Grita) in Tachira state have shown a predominance of the sandfly species Lutzomyia spinicrassa (verrucarum group) and two Leishmania species, Leishmania mexicana and Leishmania braziliensis. Two transmission cycles appear to occur: a peridomestic cycle in Lomas Bajas and a sylvatic one in Delicias and La Grita.
Assuntos
Leishmaniose Cutânea/epidemiologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Animais , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Leishmania braziliensis/isolamento & purificação , Leishmania mexicana/isolamento & purificação , Leishmaniose Cutânea/parasitologia , Leishmaniose Mucocutânea/epidemiologia , Leishmaniose Mucocutânea/parasitologia , Pessoa de Meia-Idade , Prevalência , Chuva , Venezuela/epidemiologiaRESUMO
Studies of cutaneous leishmaniasis in 3 endemic foci in Tachira state, western Venezuela have revealed sympatric populations of parasites causing both cutaneous and mucocutaneous disease. Immunological techniques and measurement of protease/acid phosphatase activities have been used to detect species-specific parasite antigens from 3 isolates from Tachira. Identified antigens of particular interest had molecular masses of 100, 82, 66, 50 and 27 kDa, but there was a high degree of heterogeneity between the antigens of the Tachira isolates and other Venezuelan strains of Leishmania braziliensis and L. mexicana. This heterogeneity has implications concerning the selection of antigens for use in serodiagnosis of leishmaniasis.
Assuntos
Antígenos de Protozoários/análise , Leishmania/imunologia , Leishmaniose Cutânea/parasitologia , Fosfatase Ácida/metabolismo , Animais , Antígenos de Protozoários/química , Western Blotting , Humanos , Leishmania/química , Leishmania braziliensis/imunologia , Leishmania mexicana/imunologia , Peso Molecular , Peptídeo Hidrolases/metabolismo , Testes de Precipitina , Biossíntese de Proteínas , Especificidade da EspécieRESUMO
Leishmaniasis is on the increase in Venezuela (ca 30,000 new cases per year) due to deterioration in health management, increased risk groups among inmunosuppressed individuals and increased human penetration into the ecological habitats of sandfly vectors. An STD2-funded project (1989-1992) focused on the Andean state of Táchira, which showed the highest annual index of new cases (ca 200-250). The project aimed at contributing to vector/parasite identification through a combination of molecular and well established field techniques: Newly developed molecular methods distinguished among Lu. spinicrassa, Lu. youngi and Lu. townsendi. These three species of the Verrucarum group are sympatric in the Northeast of the state and could be successfully identified by CHA, DNA probes and RAPD. A Le. braziliensis specific KDNA probe used with squash blots indicated that Lu. spinicrassa is the main vector and that Le. braziliensis is the main parasite species in Táchira state, Venezuela. PCR and the Le. brasiliensis specific DNA probe, schizodemes, isoenzymes and polyclonal antibodies agreed as taxonomic criteria for classification of Leishmania isolated from parasitologically confirmed cases in Tachira. Considerable degree of antigen heterogeneity in Venezuelan Le. braziliensis complex and Le. mexicana complex isolates from Tachira suggests multiple candidate antigens for improving the specificity of immunological diagnosis. The methods developed and tested in Táchira state should be valuable in order to help solving other outstanding epidemiological problems such as following of the epidemiological impact of intervention and vector control measures in highly endemic areas. Future work (STD3 funded, 1993-1996) aims to apply these molecular techniques to a vector control pilot study in Lara state, an area showing the highest incidence of new cases in the country.
Assuntos
Insetos Vetores , Leishmania/genética , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/genética , Epidemiologia Molecular/métodos , Vigilância da População , Psychodidae , Animais , Impressões Digitais de DNA/métodos , Sondas de DNA , Estudos de Avaliação como Assunto , Heterogeneidade Genética , Humanos , Immunoblotting/métodos , Incidência , Controle de Insetos , Leishmania/classificação , Leishmania/imunologia , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/prevenção & controle , Sondas Moleculares , Reação em Cadeia da Polimerase/métodos , Fatores de Risco , Sensibilidade e Especificidade , Venezuela/epidemiologiaRESUMO
To understand the epidemiology of cutaneous and mucocutaneous leishmaniasis in three distinct endemic foci of Tachira state, Western Venezuela, we aim to improve vector identification methods by developing species-specific sandfly DNA probes. These probes will be able to distinguish between sympatric sandfly populations thereby providing epidemiological data for determining the significance of individual sandfly groups related to their vectorial capacity.