Malaria is the deadliest parasitosis worldwide, causing 216 milion cases and 445.000 casualities in 2016. Among the malaria causative agents, Plasmodium falciparum is the main cause of death in humans. The identification of new antimalarial drug targets is a fundamental challenge, given the continuous emergence of drug-resistant strains of P. falciparum in endemic areas. The human GTPase Rac1 is known to be involved in infection of several intracellular pathogens, including bacteria (e.g.: Salmonella enterica, Shigella flexneri, Staphylococcus aureus) and parasites (e.g.: Toxoplasma gondii, wich belongs to the same phylum as Plasmodium falciparum. Rac1 was also identified in a proteomic analysis of erythrocyte membrane microdomains, subcellular organelles shown to be essential for P. falciparum invasion of the host cell. In order to investigate whether Rac1 plays a role in P. falciparum infection, we tested 16 Rac1 chemical inhibitors both commercial and newly designed on asynchronous parasite cultures, and showed that 13 compounds have anti-malarial activity. Four inhibitors showed IC50 below 1 uM. In particular we wanted to assess the role of Rac1 in P. falciparum invasion of host erythrocytes. To do this, we performed invasion assays and measured invasion rates by FACS analysis. Three different Rac1 inhibitors were able to reduce P. falciparum invasion efficiency, in a dose-dependent way. We also investigated Rac1 subcellular localization during P. falciparum invasion by immuno-fluorescence assay and showed that the GTPase is recruited to the site of parasite entrance, colocalizing with the moving junction, and is then gradually depleted from the eyrthocyte membrane, being relocated to the parasitophorous vacuole membrane (PVM). Furthermore, the GTPase is activated by the parasite both during invasion and parasite intracellular growth. These results indicate that malaria parasites exploit the host cell transduction machinery, activating Rac1 in order to invade human RBCs. Future experiments will include co-immunoprecipitation of Rac1 and its partners in order to estabilish wich interactions the GTPase estabilishes during the invasion process and erythrocyte transfection of a Rac1 dominant-negative protein in order to confirm the functional analysis with a different approach.
Role of human GTPase Rac1 in Plasmodium falciparum infection / Paone, S.; Celani, F.; Tirelli, V.; D’Alessandro, S.; Parapini, S.; Contini, A.; Modiano, D.; Ponzi, M.; Olivieri, A.. - (2018). (Intervento presentato al convegno IX congresso del Corso di Dottorato in Malattie Infettive, Microbiologia e Sanità Pubblica tenutosi a Roma, Italia).
Role of human GTPase Rac1 in Plasmodium falciparum infection
V. Tirelli;D. Modiano;
2018
Abstract
Malaria is the deadliest parasitosis worldwide, causing 216 milion cases and 445.000 casualities in 2016. Among the malaria causative agents, Plasmodium falciparum is the main cause of death in humans. The identification of new antimalarial drug targets is a fundamental challenge, given the continuous emergence of drug-resistant strains of P. falciparum in endemic areas. The human GTPase Rac1 is known to be involved in infection of several intracellular pathogens, including bacteria (e.g.: Salmonella enterica, Shigella flexneri, Staphylococcus aureus) and parasites (e.g.: Toxoplasma gondii, wich belongs to the same phylum as Plasmodium falciparum. Rac1 was also identified in a proteomic analysis of erythrocyte membrane microdomains, subcellular organelles shown to be essential for P. falciparum invasion of the host cell. In order to investigate whether Rac1 plays a role in P. falciparum infection, we tested 16 Rac1 chemical inhibitors both commercial and newly designed on asynchronous parasite cultures, and showed that 13 compounds have anti-malarial activity. Four inhibitors showed IC50 below 1 uM. In particular we wanted to assess the role of Rac1 in P. falciparum invasion of host erythrocytes. To do this, we performed invasion assays and measured invasion rates by FACS analysis. Three different Rac1 inhibitors were able to reduce P. falciparum invasion efficiency, in a dose-dependent way. We also investigated Rac1 subcellular localization during P. falciparum invasion by immuno-fluorescence assay and showed that the GTPase is recruited to the site of parasite entrance, colocalizing with the moving junction, and is then gradually depleted from the eyrthocyte membrane, being relocated to the parasitophorous vacuole membrane (PVM). Furthermore, the GTPase is activated by the parasite both during invasion and parasite intracellular growth. These results indicate that malaria parasites exploit the host cell transduction machinery, activating Rac1 in order to invade human RBCs. Future experiments will include co-immunoprecipitation of Rac1 and its partners in order to estabilish wich interactions the GTPase estabilishes during the invasion process and erythrocyte transfection of a Rac1 dominant-negative protein in order to confirm the functional analysis with a different approach.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
