RESUMEN
Phosphomannomutase (PMM) catalyses the conversion of mannose-6-phosphate to mannose-1-phosphate, an essential step in mannose activation and the biosynthesis of glycoconjugates in all eukaryotes. Deletion of PMM from Leishmania mexicana results in loss of virulence, suggesting that PMM is a promising drug target for the development of anti-leishmanial inhibitors. We report the crystallization and structure determination to 2.1 A of L. mexicana PMM alone and in complex with glucose-1,6-bisphosphate to 2.9 A. PMM is a member of the haloacid dehalogenase (HAD) family, but has a novel dimeric structure and a distinct cap domain of unique topology. Although the structure is novel within the HAD family, the leishmanial enzyme shows a high degree of similarity with its human isoforms. We have generated L. major PMM knockouts, which are avirulent. We expressed the human pmm2 gene in the Leishmania PMM knockout, but despite the similarity between Leishmania and human PMM, expression of the human gene did not restore virulence. Similarities in the structure of the parasite enzyme and its human isoforms suggest that the development of parasite-selective inhibitors will not be an easy task.
Asunto(s)
Leishmania mexicana/enzimología , Fosfotransferasas (Fosfomutasas)/química , Animales , Cristalografía por Rayos X , Humanos , Isoenzimas/química , Isoenzimas/genética , Leishmania mexicana/genética , Leishmania mexicana/patogenicidad , Fosfotransferasas (Fosfomutasas)/genética , Fosfotransferasas (Fosfomutasas)/aislamiento & purificación , Homología Estructural de Proteína , Virulencia/genéticaRESUMEN
In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDP-MP) is essential for the formation of GDP-mannose, the donor of activated mannose for all glycosylation reactions. Unlike other eukaryotes, where deletion of GDP-mannose pyrophosphorylase is lethal, deletion of this gene in Leishmania mexicana has no effect on viability, but leads to the generation of avirulent parasites. In this study, we show that the null mutants have a perturbed morphology and cytokinesis, retarded growth and increased adherence to the substratum where they form large colonies. The null mutants attach avidly to mouse macrophages, but unlike the wild type organisms, they do not bind to the complement receptor 3 and are slow to induce phagocytosis. Once internalised, they localise to the phagolysosome, but in contrast to wild type organisms which transform into the intracellular amastigote and establish in the macrophage, they are cleared by 24 h in culture and by 5 h in vivo. The null mutants are hypersensitive to human but not mouse complement and to temperature and acidic pH. Surprisingly, in view of the lack of several known host-protective antigens, injection of the mutant parasites into BALB/c mice confers significant and long lasting protection against infection, suggesting that these temperature sensitive mutants are an attractive candidate for a live attenuated vaccine.
Asunto(s)
Leishmania mexicana/fisiología , Animales , Anticuerpos/inmunología , Adhesión Celular/fisiología , Línea Celular , Citocinesis/fisiología , Femenino , Guanosina Difosfato Manosa/genética , Interacciones Huésped-Parásitos , Humanos , Concentración de Iones de Hidrógeno , Leishmania mexicana/genética , Leishmania mexicana/crecimiento & desarrollo , Antígeno de Macrófago-1/inmunología , Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Mutación , Fenotipo , Temperatura , Vacunación/métodos , VirulenciaRESUMEN
Recognition of pathogen-associated molecular patterns (PAMP) influences the response of dendritic cells (DC) and therefore development of innate and adaptive immunity. Different forms of Leishmania mexicana have distinct effects on DC, with promastigotes and amastigotes being activating and apparently neutral, respectively. We investigated whether stage-specific differences in surface composition might account for these distinct effects. Amastigotes and promastigotes lacking the lpg1 gene needed for lipophosphoglycan (LPG) biosynthesis could not activate DC in vitro. Genome-wide transcriptional profiling of DC infected with wild-type or mutant promastigotes or wild-type amastigotes revealed that wild-type promastigotes induce an inflammatory signature that is lacking in DC exposed to the other parasite forms. The proinflammatory response pattern was partly recovered by reconstitution of lpg1 expression in lpg1-/- parasites, and exposure to purified LPG increased the expression of MHC class II and CD86 on DC. Infection with wild-type but not lpg1-/- promastigotes increased the number of activated DC in draining lymph nodes, and this was correlated with lower early parasite burdens in wild-type-infected animals. These in vivo and in vitro results suggest an LPG-dependent activation of DC that contributes to host defense and agree with the notion that the parasites evolved under immune pressure to down-regulate PAMP expression in mammalian hosts.
Asunto(s)
Células Dendríticas/inmunología , Glicoesfingolípidos/inmunología , Leishmania mexicana/inmunología , Leishmaniasis Cutánea/inmunología , Animales , Células Dendríticas/metabolismo , Galactosiltransferasas/metabolismo , Perfilación de la Expresión Génica , Glicoesfingolípidos/biosíntesis , Interleucina-12/metabolismo , Subunidad p40 de la Interleucina-12 , Ratones , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/metabolismoRESUMEN
Leishmania parasites synthesize a range of mannose-containing glycoconjugates thought to be essential for virulence in the mammalian host and sandfly vector. A prerequisite for the synthesis of these molecules is the availability of the activated mannose donor, GDP-Man, the product of the catalysis of mannose-1-phosphate and GTP by GDP-mannose pyrophosphorylase (GDP-MP). In contrast to the lethal phenotype in fungi, the deletion of the gene in Leishmania mexicana did not affect parasite viability but led to a total loss of virulence, making GDP-MP an ideal target for anti-Leishmania drug development. We show by immunofluorescence and subcellular fractionation that GDP-MP is a cytoplasmic protein, and we describe a colorimetric activity assay suitable for the high throughput screening of small molecule inhibitors. We expressed recombinant GDP-MP as a fusion with maltose-binding protein and separated the enzyme from maltose-binding protein by thrombin cleavage, ion-exchange, and size exclusion chromatography. Size exclusion chromatography and analytical ultracentrifugation studies demonstrate that GDP-MP self-associates to form an enzymatically active and stable hexamer. However, sedimentation studies show that the GDP-MP hexamer dissociates to trimers and monomers in a time-dependent manner, at low protein concentrations, at low ionic strength, and at alkaline pH. Circular dichroism spectroscopy reveals that GDP-MP is comprised of mixed alpha/beta structure, similar to its closest related homologue, N-acetyl-glucoseamine-1-phosphate uridyltransferase (Glmu) from Streptococcus pneumoniae. Our studies provide insight into the structure of a novel target for the development of anti-Leishmania drugs.