RESUMEN
In August 2017, the National Institute of Allergy and Infectious Diseases convened a meeting, entitled "Understanding the Liver-Stage Biology of Malaria Parasites to Enable and Accelerate the Development of a Highly Efficacious Vaccine," to discuss the needs and strategies to develop a highly efficacious, whole organism-based vaccine targeting the liver stage of malaria parasites. It was concluded that attenuated sporozoite platforms have proven to be promising approaches, and that late-arresting sporozoites could potentially offer greater vaccine performance than early-arresting sporozoites against malaria. New knowledge and emerging technologies have made the development of late-arresting sporozoites feasible. Highly integrated approaches involving liver-stage research, "omics" studies, and cutting-edge genetic editing technologies, combined with in vitro culture systems or unique animal models, are needed to accelerate the discovery of candidates for a late-arresting, genetically attenuated parasite vaccine.
Asunto(s)
Hígado/inmunología , Vacunas contra la Malaria/genética , Malaria Falciparum/prevención & control , Malaria Vivax/prevención & control , Plasmodium falciparum/inmunología , Plasmodium vivax/inmunología , Esporozoítos/inmunología , Animales , Modelos Animales de Enfermedad , Rayos gamma , Ingeniería Genética/métodos , Humanos , Hígado/parasitología , Malaria/inmunología , Malaria/parasitología , Malaria/prevención & control , Vacunas contra la Malaria/administración & dosificación , Vacunas contra la Malaria/metabolismo , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Malaria Vivax/inmunología , Malaria Vivax/parasitología , Ratones , Plasmodium berghei/química , Plasmodium berghei/genética , Plasmodium berghei/inmunología , Plasmodium berghei/efectos de la radiación , Plasmodium falciparum/química , Plasmodium falciparum/genética , Plasmodium falciparum/efectos de la radiación , Plasmodium vivax/química , Plasmodium vivax/genética , Plasmodium vivax/efectos de la radiación , Plasmodium yoelii/química , Plasmodium yoelii/genética , Plasmodium yoelii/inmunología , Plasmodium yoelii/efectos de la radiación , Esporozoítos/química , Esporozoítos/genética , Esporozoítos/efectos de la radiación , Vacunas AtenuadasRESUMEN
Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a approximately 33 kDa protein, with a well-conserved substrate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3::HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immuno-fluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3::HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.
Asunto(s)
Leishmania donovani/enzimología , Lipasa , Animales , Genes Protozoarios , Humanos , Estadios del Ciclo de Vida , Lipasa/química , Lipasa/genética , Lipasa/fisiología , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
The ancient eukaryotic human pathogen, Entamoeba histolytica, is a nucleo-base auxotroph (i.e. lacks the ability to synthesize purines or pyrimidines de novo) and therefore is totally dependent upon its host for the supply of these essential nutrients. In this study, we identified two unique 28-kDa, dithiothreitol-sensitive nucleases and showed that they are constitutively released/secreted by parasites during axenic culture. Using several different molecular approaches, we identified and characterized the structure of EhNucI and EhNucII, genes that encode ribonuclease T2 family proteins. Homologous episomal expression of epitope-tagged EhNucI and EhNucII chimeric constructs was used to define the functional and biochemical properties of these released/secreted enzymes. Results of coupled immunoprecipitation-enzyme activity analyses demonstrated that these "secretory" enzymes could hydrolyze a variety of synthetic polynucleotides, as well as the natural nucleic acid substrate RNA. Furthermore, our results demonstrated that sera from acutely infected amebiasis patients recognized and immunoprecipitated these parasite secretory enzymes. Based on these observations, we hypothesize that within its host, these secretory nucleases could function, at a distance away from the parasite, to harness (i.e. hydrolyze/access) host-derived nucleic acids to satisfy the essential purine and pyrimidine requirements of these organisms. Thus, these enzymes might play an important role in facilitating the survival, growth, and development of this important human pathogen.
Asunto(s)
Entamoeba histolytica/enzimología , Ribonucleasas/química , Ribonucleasas/fisiología , Secuencia de Aminoácidos , Animales , Catálisis , Sistema Libre de Células/enzimología , Cisteína/química , Disulfuros/química , Entamoeba histolytica/patogenicidad , Epítopos , Genes Protozoarios , Histidina/química , Humanos , Hidrólisis , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , Pruebas de Precipitina , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Ribonucleasas/clasificación , Ribonucleasas/genética , Ribonucleasas/metabolismoRESUMEN
Lipid rafts are highly ordered, cholesterol-rich, and detergent-resistant microdomains found in the plasma membrane of many eukaryotic cells. These domains play important roles in endocytosis, secretion, and adhesion in a variety of cell types. The parasitic protozoan Entamoeba histolytica, the causative agent of amoebic dysentery, was determined to have raft-like plasma membrane domains by use of fluorescent lipid analogs that specifically partition into raft and nonraft regions of the membrane. Disruption of raft-like membrane domains in Entamoeba with the cholesterol-binding agents filipin and methyl-beta-cyclodextrin resulted in the inhibition of several important virulence functions, fluid-phase pinocytosis, and adhesion to host cell monolayers. However, disruption of raft-like domains did not inhibit constitutive secretion of cysteine proteases, another important virulence function of Entamoeba. Flotation of the cold Triton X-100-insoluble portion of membranes on sucrose gradients revealed that the heavy, intermediate, and light subunits of the galactose-N-acetylgalactosamine-inhibitible lectin, an important cell surface adhesion molecule of Entamoeba, were enriched in cholesterol-rich (raft-like) fractions, whereas EhCP5, another cell surface molecule, was not enriched in these fractions. The subunits of the lectin were also observed in high-density, actin-rich fractions of the sucrose gradient. Together, these data suggest that pinocytosis and adhesion are raft-dependent functions in this pathogen. This is the first report describing the existence and physiological relevance of raft-like membrane domains in E. histolytica.
Asunto(s)
Adhesión Celular , Entamoeba histolytica/patogenicidad , Microdominios de Membrana/metabolismo , Pinocitosis , Animales , Células CHO , Membrana Celular/química , Cricetinae , Entamoeba histolytica/fisiología , Humanos , Microdominios de Membrana/químicaRESUMEN
The Entamoeba histolytica Rab11 family of small molecular weight GTPases consists of three members, EhRab11, EhRab11B, and EhRab11C. The functions of these Rabs in Entamoeba have not been determined. Therefore, as an approach to elucidate the role of the Rab11 family of GTPases in Entamoeba, immunofluorescence microscopy was undertaken to define the subcellular localization of one member of this family, EhRab11. Under conditions of growth, EhRab11 displayed a punctate pattern in the cytoplasm of trophozoites. EhRab11 did not colocalize with markers for the Golgi apparatus, endoplasmic reticulum, pinosomes, phagosomes, or compartments formed by receptor-mediated endocytosis, suggesting that this Rab may not play a role in vesicle trafficking between these organelles. Under conditions of iron and serum starvation, EhRab11 was translocated to the periphery of the cell. The altered cellular localization was accompanied by multinucleation of the cells as well as the acquisition of detergent resistance by the cells, features that are characteristic of Entamoeba cysts. The translocation of EhRab11 to the periphery of the cell during iron and serum starvation was specific as the subcellular localizations of two other Rab GTPases, EhRab7 and EhRabA, were not altered under the same conditions. In addition, the formation of multinucleated cells by inhibition of cytokinesis was not sufficient to induce the translocation of EhRab11 to the cell periphery. Taken together, the data suggest that iron and serum starvation may induce encystation in E. histolytica and that EhRab11 may play a role in this process. Moreover, these studies are the first to describe a putative role for a Rab GTPase in encystation in Entamoeba sp.