RESUMEN
Leishmania parasites must survive and proliferate in two vastly different environments - the guts of poikilothermic sandflies and the antigen-presenting cells of homeothermic mammals. The change of temperature during the transmission from sandflies to mammals is both a key trigger for the progression of their life cycle and for elevated synthesis of heat shock proteins, which have been implicated in their survival at higher temperatures. Although the functions of the main heat shock protein families in the Leishmania life cycle have been studied, nothing is known about the roles played by small heat shock proteins. Here, we present the first evidence for the pivotal role played by the Leishmania donovani 23-kDa heat shock protein (which we called HSP23), which is expressed preferentially during the mammalian stage where it assumes a perinuclear localisation. Loss of HSP23 causes increased sensitivity to chemical stressors and renders L. donovani non-viable at 37°C. Consequently, HSP23-null mutants are non-infectious to primary macrophages in vitro. All phenotypic effects could be abrogated by the introduction of a functional HSP23 transgene into the null mutant, confirming the specificity of the mutant phenotype. Thus, HSP23 expression is a prerequisite for L. donovani survival at mammalian host temperatures and a crucial virulence factor.
Asunto(s)
Proteínas de Choque Térmico Pequeñas/metabolismo , Leishmania donovani/metabolismo , Leishmania donovani/fisiología , Proteínas Protozoarias/metabolismo , Animales , Células Cultivadas , Proteínas de Choque Térmico Pequeñas/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Protozoarias/genética , TemperaturaRESUMEN
The heat shock protein 90 plays a pivotal role in the life cycle control of Leishmania donovani promoting the fast-growing insect stage of this parasite. Equally important for insect stage growth is the co-chaperone Sti1. We show that replacement of Sti1 is only feasible in the presence of additional Sti1 transgenes indicating an essential role. To better understand the impact of Sti1 and its interaction with Hsp90, we performed a mutational analysis of Hsp90. We established that a single amino acid exchange in the Leishmaniaâ Hsp90 renders that protein resistant to the inhibitor radicicol (RAD), yet does not interfere with its functionality. Based on this RAD-resistant Hsp90, we established a combined chemical knockout/gene complementation (CKC) approach. We can show that Hsp90 function is required in both insect and mammalian life stages and that the Sti1-binding motif of Hsp90 is crucial for proliferation of insect and mammalian stages of the parasite. The Sti1-binding motif in Leishmaniaâ Hsp90 is suboptimal - optimizing the motif increased initial intracellular proliferation underscoring the importance of the Hsp90-Sti1 interaction for this important parasitic protozoan. The CKC strategy we developed will allow the future analysis of more Hsp90 domains and motifs in parasite viability and infectivity.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Interacciones Huésped-Patógeno , Leishmania donovani/fisiología , Estadios del Ciclo de Vida , Animales , Insectos , Leishmania donovani/crecimiento & desarrollo , Mamíferos , Mapeo de Interacción de ProteínasRESUMEN
Leishmania is exposed to a sudden increase in environmental temperature during the infectious cycle that triggers stage differentiation and adapts the parasite phenotype to intracellular survival in the mammalian host. The absence of classical promoter-dependent mechanisms of gene regulation and constitutive expression of most of the heat-shock proteins (HSPs) in these human pathogens raise important unresolved questions as to regulation of the heat-shock response and stage-specific functions of Leishmania HSPs. Here we used a gel-based quantitative approach to assess the Leishmania donovani phosphoproteome and revealed that 38% of the proteins showed significant stage-specific differences, with a strong focus of amastigote-specific phosphoproteins on chaperone function. We identified STI1/HOP-containing chaperone complexes that interact with ribosomal client proteins in an amastigote-specific manner. Genetic analysis of STI1/HOP phosphorylation sites in conditional sti1(-/-) null mutant parasites revealed two phosphoserine residues essential for parasite viability. Phosphorylation of the major Leishmania chaperones at the pathogenic stage suggests that these proteins may be promising drug targets via inhibition of their respective protein kinases.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Leishmania donovani/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Leishmania donovani/crecimiento & desarrollo , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosforilación , Unión Proteica , Proteoma/química , Proteínas Protozoarias/química , Alineación de SecuenciaRESUMEN
In Leishmania donovani, the HSP90 chaperone complex plays an essential role in the control of the parasite's life cycle, general viability and infectivity. Several of the associated co-chaperones were also shown to be essential for viability and/or infectivity to mammalian cells. Here, we identify and describe the co-chaperone P23 and distinguish its function from that of the structurally related small heat shock protein HSP23. P23 is expressed constitutively and associates itself with members of the HSP90 complex, i.e. HSP90 and Sti1. Viable P23 gene replacement mutants could be raised and confirmed as null mutants without deleterious effects on viability under a variety of physiological growth conditions. The null mutant also displays near-wild-type infectivity, arguing against a decisive role played by P23 in laboratory settings. However, the P23 null mutant displays a marked hypersensitivity against HSP90 inhibitors geldanamycin and radicicol. P23 also appears to affect the radicicol resistance of a HSP90 Leu33-Ile mutant described previously. Therefore, the annotation of L. donovani P23 as HSP90-interacting co-chaperone is confirmed.
Asunto(s)
Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Leishmania donovani/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Benzoquinonas/farmacología , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Lactamas Macrocíclicas/farmacología , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/patogenicidad , Estadios del Ciclo de Vida/efectos de los fármacos , Macrólidos/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Chaperonas Moleculares/genética , Proteínas Protozoarias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Transcriptoma , TransfecciónRESUMEN
Molecular chaperone proteins play a pivotal role in the protozoan parasite Leishmania donovani, controlling cell fate and ensuring intracellular survival. In higher eukaryotes, the so-called co-chaperone proteins are required for client protein recognition and proper function of chaperones, among them the small glutamine-rich tetratricopeptide repeat proteins (SGT) which interact with both HSP70 and HSP90 chaperones. An atypical SGT homolog is found in the L. donovani genome, encoding a protein lacking the C-terminal glutamine-rich region, normally typical for SGT family members. The gene is expressed constitutively during the life cycle and is essential for survival and/or growth of the parasites. LdSGT forms large, stable complexes that also include another putative co-chaperone, HSC70 interacting protein (HIP). The gene product forms cytoplasmic clusters, matching the subcellular distribution of HIP and partly that of the major cytoplasmic chaperones, HSP70 and HSP90, reflecting a direct molecular interaction with both chaperones.
Asunto(s)
Leishmania donovani/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Due to the apparent lack of sexual recombination in Leishmania spp., gene replacement strategies in this diploid organism necessitate the subsequent targeting of two gene alleles by using two constructs, bearing different antibiotic resistance markers. This approach is time consuming and often gives rise to spontaneous amplification of the targeted gene, nullifying efforts to create functional null mutants. Here, we show that by using two homologous recombination constructs in a co-transfection of Leishmania donovani promastigotes, we can obtain double-allele gene replacement mutants. This approach reduces the time required for the generation of functional null mutants and the number of in vitro passage cycles, potentially limiting culture-associated artefacts.