RESUMEN
Active host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface.
Asunto(s)
Membrana Celular/parasitología , Proteínas Protozoarias/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Toxoplasma/metabolismo , Toxoplasmosis/patología , Transporte Biológico/fisiología , Células Cultivadas , Interacciones Huésped-Parásitos , Humanos , Vías Secretoras/fisiología , Factores de VirulenciaRESUMEN
Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolate and characterize anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies block PvRBP2b binding to reticulocytes and inhibit complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.
Asunto(s)
Anticuerpos Bloqueadores , Anticuerpos Monoclonales/inmunología , Proteínas de la Membrana/metabolismo , Plasmodium vivax/metabolismo , Proteínas Protozoarias/metabolismo , Reticulocitos/metabolismo , Anticuerpos Antiprotozoarios/inmunología , Antígenos CD , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Cambodia , Cristalografía por Rayos X , Humanos , Estudios Longitudinales , Malaria Vivax/inmunología , Malaria Vivax/parasitología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Modelos Moleculares , Plasmodium vivax/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Receptores de TransferrinaRESUMEN
Proteomics is a key tool in the identification of new bile biomarkers for differentiating malignant and nonmalignant biliary stenoses. Unfortunately, the complexity of bile and the presence of molecules interfering with protein analysis represent an obstacle for quantitative proteomic studies in bile samples. The simultaneous need to introduce purification steps and minimize the use of pre-fractionation methods inevitably leads to protein loss and limited quantifications. This dramatically reduces the chance of identifying new potential biomarkers. In the present study, we included differential centrifugation as a preliminary step in a quantitative proteomic workflow involving iTRAQ labeling, peptide fractionation by OFFGEL electrophoresis and LC-MS/MS, to compare protein expression in bile samples collected from patients with malignant or nonmalignant biliary stenoses. A total of 1267 proteins were identified, including a set of 322 newly described bile proteins, mainly belonging to high-density cellular fractions. The subsequent comparative analysis led to a 5-fold increase in the number of quantified proteins over previously published studies and highlighted 104 proteins overexpressed in malignant samples. Finally, immunoblot verifications performed on a cohort of 8 malignant (pancreatic adenocarcinoma, n=4; cholangiocarcinoma, n=4) and 5 nonmalignant samples (chronic pancreatitis, n=3; biliary stones, n=2) confirmed the results of proteomic analysis for three proteins: olfactomedin-4, syntenin-2 and Ras-related C3 botulinum toxin substrate 1. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.