MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF SECRETORY ORGANELLES OF THE TRANSMISSION STAGES OF THE MALARIA PARASITE PLASMODIUM FALCIPARUM.

Plasmodium falciparum is responsible for the immense majority of malaria deaths. Plasmodium sexual stages, the gametocytes, are the only parasite forms capable of becoming gametes and propagate in mosquitoes, which in turn infect a new human host, completing the malaria life cycle. Osmiophilic bodies (OBs) are membranous secretory organelles found only in Plasmodium gametocytes. They have been shown to play a role in gamete egress from the host erythrocyte and to be important for mosquito infectivity. Gametocytes defective for Pfg377, the only known protein localized to OBs of P. falciparum, are depleted from these organelles. This unique feature of the Pfg377 defective parasites was successfully exploited in this project to perform a comparative proteomic analysis between WT and stably disrupted pfg377KO gametocytes to identify candidate proteins as OB candidate resident proteins (OBCs). Together with Pfg377 co-IP experiments and previous knowledge of protein components of OBs in related species, five OBCs were identified. Localization of OBCs was performed either by generating antibodies to recombinant fragments of individual OBCs or by producing transgenic P. falciparum parasite lines where the gene of interest was C-terminally fused with the gene of the Green Fluorescent Protein. Immunofluorescence assay (IFA) analysis exploring co-localization of the OBC proteins with antibodies against Pfg377, in some instances complemented by immunoelectron microscopy observation, led to the identification of four OBCs, including two proteases, as components of the OBs of P. falciparum. In order to functionally characterize the OBC encoding genes and to investigate a possible role in gamete egress, parasite lines were obtained where the respective coding sequence were disrupted. In order to improve current tools for the analysis of P. falciparum gametogenesis, a protocol to measure gamete egress was developed, based on the staining of the host erythrocyte membrane. Functional analysis of the mutant parasites using the new protocol revealed that the pfg377-disrupted lines and one other OBC-depleted line produced gametocytes with no obvious defect in egress, with the only exception of one KO line with a mild egress phenotype. The data generated in this thesis revealed novel proteins localized in these organelles. The functional analysis showing no obvious gamete egress phenotype in gametocytes depleted of these organelles or of some of the OB resident proteins revised current hypotheses on the role of these organelles in the release of gametes from the surrounding erythrocyte membrane, opening the question of a different role of these organelles in later mosquito stages of the parasite