Synthesis and biological function of fucose in Plasmodium falciparum

Abstract

Malaria is a parasitic disease caused by Plasmodium parasites and it is transmitted by female Anopheles mosquito. P. falciparum has a complex life cycle that includes important stages in two different hosts: a mosquito and a human. The transmission between the human and the mosquito host also involves the transition between asexual and sexual forms of the parasites. Glycobiology includes the study of carbohydrate metabolism and glycoconjugate (glycoprotein and glycolipid) structures. Protozoan parasites synthesize different glycoconjugates for protection and to respond to changes in the environment. Glycoconjugates coat the parasite surface with carbohydrates generally different from the host ones. They are crucial for parasite virulence and survival. Until very recently the only glycan structures described in P. falciparum were the GPI-anchors, however other glycan structures have been found in the past few years as the N-glycans or C-mannosylation. The glycome consists in the complete set of glycosylations that an organism or a cell produces at a certain time point, therefore the description of the parasite glycome may help to understand better the host- pathogen interactions in parasitic diseases. Sugar nucleotides are activated forms of monosaccharides that are the donors of glycosyltransferases to form glyconjugates. They can be synthesized by a de novo pathway that consists in the bioconversion of an existing sugar or sugar nucleotide; or by a salvage pathway that involves an activation and a further pyrophosphorylation. The identification and quantification of the sugar nucleotides present in malaria parasites may help to describe its glycosylation profile. The first paper presented in the thesis describes the identification and quantification of the sugar nucleotides present in the parasite, among which we found: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. We also investigated the salvage pathways present in the parasite but we couldn’t elucidate the presence of a fucose salvage pathway. Plasmodium parasites conserve homolog genes for the de novo biosynthetic pathway of GDP-Fuc: GMD and FS. We were able to prove the in vitro activity of GMD and FS enzymes and to show that both enzymes are required for the synthesis of fucose. GMD and FS are expressed along the intraerythrocytic life cycle and both enzymes localize in the cytoplasm of the parasite, as well as other parasite enzymes related with carbohydrate metabolism. The expression of a putative O-fucosyltransferase (PoFUT2) present in the parasite genome, together with the uptake of GDP-Fuc by parasite extracts suggested the presence of a fucose containing glycan. In the second paper, we characterized the enzymes responsible for the synthesis of GDP-Fuc, GMD and FS. We disrupted both genes in the parasite and analyzed the sugar nucleotide content present in the parasite. After GMD and FS disruption, GDP-Fuc was still detected in the parasite and no evidence of salvage mechanism was found. We described indirect evidence of a fucose containing glycan that was abrogated after the disruption of GMD. The last work here presented (not yet published) tries to characterize the enzyme that is probably responsible for the transfer of fucose to the glycoconjugate. We disrupted, by double crossover recombination, PoFUT2 gene in the human and in the rodent malaria parasite. The disruption of PoFUT2 does not have any significant effect for the viability and growth of the parasite along the parasite cycle in the human and in the mosquito host. These works open the door to new research lines to find an alternative pathway for obtaining fucose or GDP-Fuc. Obtaining evidence of the glycosylation state of PoFUT2 mutants and the characterization of other possible glycosylation reaction present in the parasite are other research topics to investigate.

La malaria está causada por el parásito Plasmodium y se transmite mediante hembras del mosquito Anopheles. La glicobiología es el estudio de los procesos relacionados con los carbohidratos y las estructuras glicoconjugadas que forman. Los parásitos sintetizan glicoconjugados o proteínas de unión a glicanos, y muchas veces se encargan de mediar las interacciones huésped-patógeno. Los azúcares nucleótidos son formas activadas de monosacáridos que son usados por glicosiltransferasas para formar glicoconjugados. La identificación y cuantificación de estos azúcares nucleótidos en el parásito de la malaria puede contribuir a la definición de su perfil de glicosilación. El primer trabajo presentado en la tesis permitió identificar los azúcares nucleótidos presentes en el parásito, así como la posible presencia de un glicoconjugado que contenga fucosa, sintetizado a partir de la actividad O-fucosiltransferasa de una proteína homóloga anotada en el genoma del parásito, PoFUT2. El siguiente trabajo permitió caracterizar las enzimas implicadas en la síntesis de la GDP-fucosa, GMD y FS. Este artículo nos permitió mostrar evidencias indirectas de la presencia del glicococonjugado que contiene fucosa. A partir de la disrupción de los genes de biosíntesis descubrimos que el contenido de GDP-fucosa en parásitos mutantes no variaba con respecto a los parásitos salvajes. Sin embargo, la síntesis del gliconjugado sí que se reducía. El tercer trabajo (sin publicar) se centra en la caracterización de la enzima encargada de transferir la fucosa al glicoconjugado. La disrupción de PoFUT2 no parece tener ningún efecto en la viabilidad y crecimiento del parásito a lo largo del ciclo de éste en el humano y en el mosquito. Estos trabajos abren la puerta a nuevas investigaciones para descubrir una vía alternativa de obtención de GDP-Fucosa. La obtención de evidencias del estado de glicosilación de los mutantes de PoFUT2 y la caracterización de otras posibles glicosilaciones son otros temas a investigar.