RESUMEN
The observation that Plasmodium falciparum possesses cyanide insensitive respiration that can be inhibited by salicylhydroxamic acid (SHAM) and propyl gallate is consistent with the presence of an alternative oxidase (AOX). However, the completion and annotation of the P. falciparum genome project did not identify any protein with convincing similarity to the previously described AOXs from plants, fungi or protozoa. We undertook a survey of the available apicomplexan genome projects in an attempt to address this anomaly. Putative AOX sequences were identified and sequenced from both type 1 and 2 strains of Cryptosporidium parvum. The gene encodes a polypeptide of 336 amino acids and has a predicted N-terminal transit sequence similar to that found in proteins targeted to the mitochondria of other species. The potential of AOX as a target for new anti-microbial agents for C. parvum is evident by the ability of SHAM and 8-hydroxyquinoline to inhibit in vitro growth of C. parvum. In spite of the lack of a good candidate for AOX in either the P. falciparum or Toxoplasma gondii genome projects, SHAM and 8-hydroxyquinoline were found to inhibit the growth of these parasites. Phylogenetic analysis suggests that AOX and the related protein immutans are derived from gene transfers from the mitochondrial endosymbiont and the chloroplast endosymbiont, respectively. These data are consistent with the functional localisation studies conducted thus far, which demonstrate mitochondrial localisation for some AOX and chloroplastidic localization for immutans. The presence of a mitochondrial compartment is further supported by the prediction of a mitochondrial targeting sequence at the N-terminus of the protein and MitoTracker staining of a subcellular compartment in trophozoite and meront stages. These results give insight into the evolution of AOX and demonstrate the potential of targeting the alternative pathway of respiration in apicomplexans.
Asunto(s)
Coccidiostáticos/farmacología , Cryptosporidium parvum/efectos de los fármacos , Mitocondrias/enzimología , Oxidorreductasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Cryptosporidium parvum/enzimología , Cryptosporidium parvum/crecimiento & desarrollo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Genes Protozoarios , Genoma , Proteínas Mitocondriales , Datos de Secuencia Molecular , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Oxiquinolina/farmacología , Filogenia , Proteínas de Plantas , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo , Salicilamidas/farmacología , Alineación de Secuencia , Toxoplasma/efectos de los fármacos , Toxoplasma/crecimiento & desarrolloRESUMEN
Infection of human erythrocytes by the apicomplexan malaria parasite Plasmodium falciparum results in endovacuolar uptake of 4 host proteins that reside in erythrocyte detergent-resistant membranes (DRMs). Whether this vacuolar transport reflects selective uptake of host DRM proteins remains unknown. A further complication is that DRMs of vastly different protein and cholesterol contents have been isolated from erythrocytes. Here we show that isolated DRMs containing the highest cholesterol-to-protein ratio have low protein mass. Liquid chromatography, mass spectrometry, and antibody-based studies reveal that the major DRM proteins are band 3, flotillin-1 and -2, peroxiredoxin-2, and stomatin. Band 3 and stomatin, which reflect the bulk mass of erythrocyte DRM proteins, and all tested non-DRM proteins are excluded from the vacuolar parasite. In contrast, flotillin-1 and -2 and 8 minor DRM proteins are recruited to the vacuole. These data suggest that DRM association is necessary but not sufficient for vacuolar recruitment and there is active, vacuolar uptake of a subset of host DRM proteins. Finally, the 10 internalized DRM proteins show varied lipid and peptidic anchors indicating that, contrary to the prevailing model of apicomplexan vacuole formation, DRM association, rather than lipid anchors, provides the preferred criteria for protein recruitment to the malarial vacuole.
Asunto(s)
Detergentes/farmacología , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/parasitología , Malaria/sangre , Malaria/patología , Animales , Proteínas Sanguíneas , Western Blotting , Colesterol/metabolismo , Cromatografía Liquida , Citoplasma/metabolismo , Eritrocitos/metabolismo , Humanos , Immunoblotting , Lípidos/química , Espectrometría de Masas , Microdominios de Membrana , Proteínas de la Membrana/sangre , Microscopía Fluorescente , Modelos Biológicos , Péptidos/química , Peroxidasas/sangre , Peroxirredoxinas , Plasmodium falciparum/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Erythrocytic mechanisms involved in malarial infection are poorly understood. We have found that signaling via the erythrocyte beta2-adrenergic receptor and heterotrimeric guanine nucleotide-binding protein (Galphas) regulated the entry of the human malaria parasite Plasmodium falciparum. Agonists that stimulate cyclic adenosine 3',5'-monophosphate production led to an increase in malarial infection that could be blocked by specific receptor antagonists. Moreover, peptides designed to inhibit Galphas protein function reduced parasitemia in P. falciparum cultures in vitro, and beta-antagonists reduced parasitemia of P. berghei infections in an in vivo mouse model. Thus, signaling via the erythrocyte beta2-adrenergic receptor and Galphas may regulate malarial infection across parasite species.
Asunto(s)
Eritrocitos/parasitología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Malaria/parasitología , Plasmodium berghei/fisiología , Plasmodium falciparum/fisiología , Receptores Adrenérgicos beta 2/metabolismo , Agonistas de Receptores Adrenérgicos beta 2 , Antagonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Alprenolol/farmacología , Animales , Catecolaminas/metabolismo , AMP Cíclico/metabolismo , Membrana Eritrocítica/metabolismo , Eritrocitos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/química , Humanos , Malaria/metabolismo , Microdominios de Membrana/metabolismo , Ratones , Parasitemia , Fragmentos de Péptidos/farmacología , Plasmodium falciparum/crecimiento & desarrollo , Propranolol/farmacología , Agonistas del Receptor Purinérgico P1 , Antagonistas de Receptores Purinérgicos P1 , Receptores Purinérgicos P1/metabolismo , Transducción de Señal , Estereoisomerismo , Vacuolas/parasitologíaRESUMEN
The human malaria parasite Plasmodium falciparum develops in a parasitophorous vacuolar membrane (PVM) within the mature red cell and extensively modifies structural and antigenic properties of this host cell. Recent studies shed significant new, mechanistic perspective on the underlying processes. There is finally, definitive evidence that despite the absence of endocytosis, transmembrane proteins in the host red cell membrane are imported in to the PVM. These are not major erythrocyte proteins but components that reside in detergent resistant membrane (DRM) rafts in red cell membrane and are detected in rafts in the PVM. Disruption of either erythrocyte or vacuolar rafts is detrimental to infection suggesting that raft proteins and lipids are essential for the parasitization of the red cell. On secretory export of parasite proteins: an ER secretory signal (SS) sequence is required for protein secretion to the PV. Proteins carrying an additional plastid targeting sequence (PTS) are also detected in the PV but subsequently delivered to the plastid organelle within the parasite, suggesting that the PTS may have a second function as an endocytic sorting signal. A distinct but yet undefined peptidic motif underlies protein transport across the PVM to the red cell (although all of the published data does not yet fit this model). Further multiple exported proteins transit through secretory 'cleft' structures, suggesting that clefts may be sorting compartments assembled by the parasite in the red cell.
Asunto(s)
Eritrocitos/metabolismo , Eritrocitos/parasitología , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/sangre , Animales , Transporte Biológico Activo , Proteínas Sanguíneas/metabolismo , Interacciones Huésped-Parásitos , Humanos , Malaria Falciparum/sangre , Malaria Falciparum/parasitología , Microdominios de Membrana/metabolismo , Modelos Biológicos , Transducción de Señal , Vacuolas/metabolismo , Vacuolas/parasitologíaRESUMEN
The heme polymer hemozoin is produced in the food vacuole (fv) of the parasite after hemoglobin proteolysis and is the target of the drug chloroquine. A candidate heme polymerase, the histidine-rich protein II (HRPII), is proposed to be delivered to the fv by ingestion of the infected-red cell cytoplasm. Here we show that 97% of endogenous Plasmodium falciparum (Pf) HRPII (PfHRPII) is secreted as soluble protein in the periphery of the red cell and avoids endocytosis by the parasite, and 3% remains membrane-bound within the parasite. Transfected cells release 90% of a soluble transgene PfHRPIImyc into the red cell periphery and contain 10% membrane bound within the parasite. Yet these cells show a minor reduction in hemozoin production and IC(50) for chloroquine. They also show decreased transport of resident fv enzyme PfPlasmepsin I, the endoplasmic reticulum (ER) marker PfBiP, and parasite-associated HRPII to fvs. Instead, all three proteins accumulate in the ER, although there is no defect in protein export from the parasite. The data suggest that novel mechanisms of sorting (i) soluble antigens like HRPII in the red cell cytoplasm and (ii) fv-bound membrane complexes in the ER regulate parasite digestive processes.