Synthesis and biological function of fucose in Plasmodium falciparum

[eng] 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.[spa] 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

Diabetic Retinopathy: Two Faces Of The Same Coin

We report two contrasting cases of diabetic retinopathy to illustrate vascular and retinal abnormalities in this condition. In the first case, mild diabetic retinopathy is diagnosed in an asymptomatic patient with diabetes mellitus of recent onset. The second patient has severe diabetic retinopathy, causing vision loss as a consequence of poor metabolic control

Reversibility of cataracts in diabetes

[Letter to the Editor ] The article by Ramkumar and Basti[1] describes the acute onset of a bilateral cataract related to the onset of diabetes, and the eventual reversal of the lens opacities after accomplishing a fall in blood glucose levels with insulin therapy. Reports of this ocular manifestation of diabetes, also called "acute sugar cataract", are scarce[2]. The development of a reversible lens opacification in human diabetics is associated with changes in the lens hydration producing oedema, vacuole formation, and increased membrane permeability[3]. In addition, other mechanisms, such as oxidative stress, are involved...

Protein O-Fucosyltransferase 2 Is Not Essential for Plasmodium berghei Development

Thrombospondin type I repeat (TSR) domains are commonly O-fucosylated by protein O-fucosyltransferase 2 (PoFUT2), and this modification is required for optimal folding and secretion of TSR-containing proteins. The human malaria parasite Plasmodium falciparum expresses proteins containing TSR domains, such as the thrombospondin-related anonymous protein (TRAP) and circumsporozoite surface protein (CSP), which are O-fucosylated. TRAP and CSP are present on the surface of sporozoites and play essential roles in mosquito and human host invasion processes during the transmission stages. Here, we have generated PoFUT2 null-mutant P. falciparum and Plasmodium berghei (rodent) malaria parasites and, by phenotyping them throughout their complete life cycle, we show that PoFUT2 disruption does not affect the growth through the mosquito stages for both species. However, contrary to what has been described previously by others, P. berghei PoFUT2 null mutant sporozoites showed no deleterious motility phenotypes and successfully established blood stage infection in mice. This unexpected result indicates that the importance of O-fucosylation of TSR domains may differ between human and RODENT malaria parasites; complicating our understanding of glycosylation modifications in malaria biology

Treatment of adult chronic indeterminate Chagas disease with benznidazole and three E1224 dosing regimens: a proof-of-concept, randomised, placebo-controlled trial

Background: Chagas disease is a major neglected vector-borne disease. In this study, we investigated the safety and efficacy of three oral E1224 (a water-soluble ravuconazole prodrug) regimens and benznidazole versus placebo in adult chronic indeterminate Chagas disease. Method: In this proof-of-concept, double-blind, randomised phase 2 clinical trial, we recruited adults (18–50 years) with confirmed diagnosis of Trypanosoma cruzi infection from two outpatient units in Bolivia. Patients were randomised with a computer-generated randomisation list, which was stratified by centre and used a block size of ten. Patients were randomly assigned (1:1:1:1:1) to five oral treatment groups: high-dose E1224 (duration 8 weeks, total dose 4000 mg), low-dose E1224 (8 weeks, 2000 mg), short-dose E1224 (4 weeks + 4 weeks placebo, 2400 mg), benznidazole (60 days, 5 mg/kg per day), or placebo (8 weeks, E1224-matched tablets). Double-blinding was limited to the E1224 and placebo arms, and assessors were masked to all treatment allocations. The primary efficacy endpoint was parasitological response to E1224 at the end of treatment, assessed by PCR. The secondary efficacy endpoints were parasitological response to benznidazole at end of treatment, assessed by PCR; sustainability of parasitological response until 12 months; parasite clearance and changes in parasite load; incidence of conversion to negative response in conventional and non-conventional (antigen trypomastigote chemiluminescent ELISA [AT CL-ELISA]) serological response; changes in levels of biomarkers; and complete response. The primary analysis population consisted of all randomised patients by their assigned treatment arms. This trial is registered with ClinicalTrials.gov, number NCT01489228. Findings: Between July 19, 2011, and July 26, 2012, we screened 560 participants with confirmed Chagas disease, of whom 231 were enrolled and assigned to high-dose E1224 (n=45), low-dose E1224 (n=48), short-dose E1224 (n=46), benznidazole (n=45), or placebo (n=47). Parasite clearance was observed with E1224 during the treatment phase, but no sustained response was seen with low-dose and short-dose regimens, whereas 13 patients (29%, 95% CI 16·4–44·3) had sustained response with the high-dose regimen compared with four (9%, 2·4–20·4) in the placebo group (p<0·0001). Benznidazole had a rapid and sustained effect on parasite clearance, with 37 patients (82%, 67·9–92·0) with sustained response at 12-month follow-up. After 1 week of treatment, mean quantitative PCR repeated measurements showed a significant reduction in parasite load in all treatment arms versus placebo. Parasite levels in the low-dose and short-dose E1224 groups gradually returned to placebo levels. Both treatments were well tolerated. Reversible, dose-dependent liver enzyme increases were seen with E1224 and benznidazole. 187 (81%) participants developed treatment-emergent adverse events and six (3%) developed treatment-emergent serious adverse events. Treatment-emergent adverse events were headaches, nausea, pruritus, peripheral neuropathy, and hypersensitivity. Interpretation: E1224 is the first new chemical entity developed for Chagas disease in decades. E1224 displayed a transient, suppressive effect on parasite clearance, whereas benznidazole showed early and sustained efficacy until 12 months of follow-up. Despite PCR limitations, our results support increased diagnosis and access to benznidazole standard regimen, and provide a development roadmap for novel benznidazole regimens in monotherapy and in combinations with E1224. Funding: Drugs for Neglected Diseases initiative.Fil: Torrico, Faustino. Universidad Mayor de San Simon Bolivia; Bolivia. Fundación Ceades; BoliviaFil: Gascon, Joaquim. Instituto de Salud Global de Barcelona; EspañaFil: Ortiz, Lourdes. Universidad Autónoma Juan Misael Saracho de Tarija; BoliviaFil: Alonso Vega, Cristina. Drugs For Neglected Diseases Initiative; SuizaFil: Pinazo, María-Jesús. Instituto de Salud Global de Barcelona; EspañaFil: Schijman, Alejandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Almeida, Igor C. University of Texas at El Paso; Estados UnidosFil: Alves, Fabiana. Drugs For Neglected Diseases Initiative; SuizaFil: Strub-Wourgaft, Nathalie. Drugs For Neglected Diseases Initiative; SuizaFil: Ribeiro, Isabela. Drugs For Neglected Diseases Initiative; SuizaFil: Santina, Glaucia. Drugs For Neglected Diseases Initiative; SuizaFil: Blum, Bethania. Drugs For Neglected Diseases Initiative; SuizaFil: Correia, Erika. Drugs For Neglected Diseases Initiative; SuizaFil: García Bournissen, Facundo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Vaillant, Michel. Competence Center in Methodology and Statistics; LuxemburgoFil: Ramos Morales, Jimena. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Pinto Rocha, Jimy Jose. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Rojas Delgadillo, Gimena. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Magne Anzoleaga, Helmut Ramon. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Mendoza, Nilce. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Quechover, Roxana Challapa. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Caballero, Maria Yurly Escobar. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Lozano Beltran, Daniel Franz. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Zalabar, Albert Mendoza. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Rojas Panozo, Lizeth. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Palacios Lopez, Alejandro. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Torrico Terceros, Dunia. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Fernandez Galvez, Violeta Alejandra. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Cardozo, Letty. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Cuellar, Gabriela. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Vasco Arenas, Rudy Nelson. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Gonzales, Isabel. Platform for Comprehensive Care of Patients with Chagas Disease; BoliviaFil: Hoyos Delfin, Carlos Florencio. Universidad Juan Misael Saracho; BoliviaFil: Garcia, Lineth. Universidad Mayor de San Simón; BoliviaFil: Parrado, Rudy. Universidad Mayor de San Simón; BoliviaFil: de la Barra, Anabelle. Universidad Mayor de San Simón; BoliviaFil: Montaño, Nair. Universidad Mayor de San Simón; BoliviaFil: Villarroel, Sandro. Universidad Mayor de San Simón; BoliviaFil: Duffy, Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bisio, Margarita María Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Ramirez Gomez, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Duncanson, Fred. Eisai; JapónFil: Everson, Michael. Eisai; JapónFil: Daniels, Antonia. Eisai; JapónFil: Asada, Makoto. Eisai; JapónFil: Cox, Eugene. Quantitative Solutions; Países BajosFil: Wesche, David. Quantitative Solutions; Países BajosFil: Diderichsen, Paul Matthias. Quantitative Solutions; Países BajosFil: Marques, Alexandre F. Universidade Federal de Minas Gerais; BrasilFil: Izquierdo, Luis. ISGlobal; EspañaFil: Sender, Silvia Sanz. ISGlobal; EspañaFil: Reverter, Joan Carlos. Hospital Clinic Barcelona; EspañaFil: Morales, Manuel. Hospital Clinic Barcelona; EspañaFil: Jimenez, Wladimiro. Hospital Clinic Barcelona; Españ