Studies on the function of Rhoptry Neck Protein 11 (RON11) in Plasmodium falciparum merozoites and sporozoites
Ribeiro Dinis, Luciana
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Malaria is a life-threatening disease that mainly affects pregnant women and children under 5, with a mortality rate of 12 deaths per 1,000 individuals per year. Over 94% of P.falciparum cases occur in Sub-Saharan Africa. Eradication campaigns have been implemented that consist of vector control, chemotherapy and chemoprophylaxis. However, effective implementation of these strategies remains a challenge. The emergence of resistance to front-line antimalarials exacerbates this problem. RTS,S (Mosquirix) the first malaria vaccine shown to protect children against malaria is currently being implemented. However, vaccine efficacy wanes from ~40% in the first year to less than 30% in subsequent years. Importantly, this vaccine does not target the disease-causing forms of the parasite. Understanding the molecular mechanisms that regulate parasite invasion of host cells can provide new antimalarial targets and lead to development of novel approaches to prevent disease. Second messenger calcium (Ca2+) signaling plays an important role during merozoite egress and invasion. How the parasite senses changes in ionic concentrations such as Ca2+ is an active area of interest. The goal of this study is to characterize the role of PfRON11, an EF-hand domain containing protein, in P. falciparum merozoites and sporozoites. Our recent mass spectrometry analysis of invasive merozoites identified RON11 to be in higher abundance than in non-invasive merozoites. Interestingly, a high throughput genetic screen in the rodent malaria parasite P. berghei identified RON11 to be essential during parasite asexual growth and subsequently also shown to be required for sporozoite invasion of hepatocytes. In this study our aims were, 1) examine the expression and localization of RON11 in P. falciparum merozoite and sporozoite, 2) evaluate activity of antibodies targeting RON11 in neutralizing P. falciparum merozoites, and 3) test the Ca2+ binding potential of the EF hand domain of RON11. We expressed recombinant protein corresponding to the N-terminus of RON11 in E.coli, generated antibodies in rats and use these antibodies to study protein expression and localization in merozoites and sporozoites. We further demonstrate that anti-RON11 antibodies can neutralize merozoites. Furthermore, we optimized expression of the putative calcium binding RON11 EF hand domain in HEK293T cells to study its ability to bind Ca2+. Lastly, we generated a dual conditional knock out/conditional knock down plasmid to replace the endogenous RON11 gene using Crispr/Cas9 - mediated homologous recombination in P. falciparum and attempted parasite transfections.