Prudêncio, MiguelMeireles, Patrícia dos Santos, 1987-2017-09-062018-05-0920162016http://hdl.handle.net/10451/28899Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2016Malaria is an acute febrile illness and one of the most prevalent infectious diseases in the world. It is caused by protozoan parasites of the genus Plasmodium that are transmitted to their mammalian hosts through the bite of an infected female Anopheles mosquito [1, 2]. In 2015, there were an estimated 214 million cases of malaria, which resulted in 438 000 deaths, most of them occurring in sub-Saharan Africa [1]. Overall, 3.2 billion people are estimated to be at risk of being infected by Plasmodium and developing the disease [1]. Upon the bite of an infected mosquito, Plasmodium parasites, termed sporozoites at this stage of the lifecycle, are deposited under the skin of their mammalian host, and are carried by the circulatory system to the liver, where they traverse several hepatocytes before productively invading a final cell [3]. Inside hepatocytes, each sporozoite develops into an exoerythrocytic form (EEF), containing thousands of newly formed merozoites that are eventually released into the bloodstream where they invade red blood cells, initiating the symptomatic, erythrocytic stage of the disease [4]. During the asymptomatic liver stage of Plasmodium infection, the parasite undergoes a remarkably rapid growth, with 104- to 105-fold replication of its genome [5, 6]. To support their rapid multiplication and supply their metabolic pathways, parasites scavenge nutrients from the host hepatocyte [7-10]. It was previously shown that glucose plays a crucial role during the liver stage of P. berghei, establishing the importance of the import of this nutrient through the parasite’s membrane hexose transporter (HT) for its replication inside liver cells [8]. On the other hand, microarray analyses of P. berghei-infected hepatoma cells and P. yoelii-infected mouse hepatocytes revealed that Plasmodium modulates the host cell’s transcriptome towards biosynthetic pathways and identified 63 transport proteins or regulatory subunits whose expression is altered in infected cells [11]. We employed RNA interference (RNAi) to down-modulate the expression of a selected group of genes, in order to identify host cell membrane transporters that play a role during Plasmodium infection of hepatic cells. This approach led to the identification of glucose transporter 1 (GLUT1) and cationic amino acid transporter 2 (CAT2) as major players in this process and warranted further investigation of the host-parasite interaction in which they are involved. GLUT1 is one of the 14 members of the family of integral membrane glucose transporter (GLUT) molecules. It is expressed in liver cells and is overexpressed in various tumors [12, 13]. Glucose is transported by GLUT1 from the plasma into the cytosol of Plasmodium-infected erythrocytes, and is then taken up by the parasite via a parasite-encoded facilitative hexose transporter (HT) [14-16]. This transporter is also expressed during the liver and transmission phases of Plasmodium parasites, suggesting that glucose might be essential also for these stages [8, 17]. In this study, we employed rodent P. berghei parasites, a well-established model of malaria infection, to investigate the uptake and utilization of glucose by Plasmodium liver stages. We established the importance of glucose availability for the intra-hepatic development of the malaria parasite. We observed that glucose uptake increases specifically in infected cells from 30 hours post-infection onwards and identified GLUT1 as the major host transporter involved in glucose uptake by those cells. The importance of this molecule during hepatic infection was further validated by employing a specific GLUT1 inhibitor, both in vitro and in vivo. Finally, we demonstrated that P. berghei infection leads to a small depletion of intracellular ATP and enhances the translocation of GLUT1 to the cell membrane of infected hepatoma cells in a phosphoinositide 3-kinase (PI3K)-independent manner, contributing to the significantly higher uptake of glucose by infected cells, when compared with their non-infected counterparts. This increase in glucose uptake was not observed when hepatic cells were subjected to a variety of other stimuli, including temperature and oxidative stress, and viral infection, suggesting that it constitutes a specific response to Plasmodium infection. Overall, these data suggest that the extensive replication of intra-hepatic Plasmodium parasites leads to a decrease of intracellular glucose availability, consequently decreasing the host cells’ ATP levels. GLUT1 translocation to the plasma membrane of Plasmodium-infected cells compensates for this decrease by enhancing glucose uptake and preventing further ATP depletion that could lead to cell death. CAT2, encoded by SLC7A2, is part of the CAT family of Na+-independent y+ transporters, which constitute the main mechanism of cellular uptake of cationic amino acids, such as arginine, lysine and ornithine [18-20]. Arginine is involved in many metabolic pathways, including the synthesis of nitric oxide (NO), polyamines, agmatine, creatine, proline, glutamate, and urea [21]. Arginine and the arginase pathway have been implicated in several infections, including Trypanosoma spp., Leishmania spp., Toxoplasma gondii, Shistosoma mansoni, Candida albicans, and Helicobacter pylori (reviewed in [22]). Plasmodium parasites were also shown to express arginase, the first enzyme of the metabolism of arginine into polyamines, and to be dependent on polyamines for survival in the blood stage of infection [23-28]. Our results showed that the normal developmental process of liver stage P. berghei parasites is also dependent on arginine availability and that this arginine is acquired by the infected host cell mostly via the CAT2A and CAT2B transporters, the two splice variants encoded by the SLC7A2 gene [29]. We also showed that the arginine taken up by the infected cell is mainly employed as a precursor of polyamine synthesis, to promote parasite growth. Because the inhibition of polyamine production by the host cell alone has no impact on P. berghei development in Huh7 cells, we hypothesize that the parasite relies mostly on its own polyamine synthesis pathway to fulfill its developmental needs. The bimodal hepatic infectivity behavior displayed by the arginase-KO P. berghei parasite partially supports this hypothesis but also suggests that the parasite may have compensatory mechanisms to guarantee its survival when this pathway is compromised. Finally, we investigated whether Plasmodium infection could be impaired by appropriate amino acid supplementation. Lysine, ornithine and valine have been described as arginase inhibitors and could therefore be employed to inhibit polyamine synthesis [30, 31]. Under these conditions, increasing the amount of arginine available might in turn enhance the production of NO, which was previously shown to kill malaria parasites in vitro [32-39]. For these reasons, we investigated the effect of these 4 amino acids, individually and in combination, on liver stage Plasmodium infection. Our data showed that P. berghei hepatic infection can be markedly inhibited in vitro and ex vivo through supplementation with lysine and valine, either alone or in combination. Importantly, we further showed that the simultaneous administration of lysine, valine and arginine in the drinking water of mouse models of P. berghei infection resulted in a striking decrease of in vivo liver parasite load. We showed that this decrease results from an impairment of parasite growth, as well as a significant reduction in the number of P. berghei-infected hepatocytes, suggesting that this combination of amino acids effectively inhibits parasite growth and contributes to its elimination. Our data strongly suggest that the lysine and valine employed in the supplementation lead to arginase inhibition and consequent impairment of the synthesis of polyamines, which are important for parasite development. Concurrently, the extra arginine provided in the supplementation stimulates NO production by inducible NO synthase (iNOS), leading to parasite death. Overall, this dissertation highlights the relevance of glucose and arginine uptake and metabolism for parasite survival and intra-hepatic development in Plasmodium-infected cells. This work elucidates hitherto unknown host-parasite interactions and identifies a novel, drugless and non-toxic approach to modulate liver-stage of Plasmodium infection.engPlasmodiumHepatócitosMaláriaNutrientesMetabolismoTeses de doutoramento - 2016doctoral thesis101386931