The invasion of erythrocytes by Plasmodium falciparum parasites produces an infection that causes major changes in the structure, composition, antigenicity and function of the host cell membrane. During infection, the parasite actively imports obligate nutrients from the serum and exports lipids, proteins and membranes to the erythrocyte cytoplasm and the erythrocyte membrane. The regulation of these pathways must be under parasite control since mature erythrocytes have limited lipid synthetic or metabolic capabilities and do not have the machinery to synthesize and process proteins. Elucidation of trafficking pathways is complex in Plasmodia-infected erythrocytes compared to other eukaryotic cells, since the parasite is separated from the serum by its plasma membrane, the parasitophorous vacuolar membrane (PVM) and the erythrocyte membrane. There remains a long-felt need to identify the cellular components of these trafficking pathways to gain a better understanding of transport mechanisms in malaria-infected erythrocytes. To explore the pathway of uptake of serum nutrients, the origin of the parasitophorous duct will be determined. We ask whether the duct is a remnant of merozoite invasion or forms at a latter stage in the life cycle. The duct will be visualized by serial sectioning of infected erythrocytes by confocal fluorescence imaging microscopy (CFIM) and transmission electron microscopy. The internal diameter of the duct will be determined using sized latex spheres. To determine whether the duct is selective, the internalization of macromolecules having different surface charge will be examined. It will be determined if the duct is a transient structure. Investigations will be implemented to separate endocytosis by the erythrocyte from parasite endocytosis. To explore the pathways of parasite protein export to the erythrocyte membrane, the cellular distribution of a variety of parasite proteins at different stages of parasite development will be examined. Double label CFIM will be employed to determine if mobile vesicles observed by CFIM in the erythrocyte cytoplasm bear parasite proteins. The timecourse for the appearance of lipid vesicles and their morphology will be compared to that obtained for the various proteins. Double label CFIM will be used to determine whether proteins are trafficked constitutively or are segregated into discrete vesicles. The origin of the membranes/vesicles (i.e parasite plasma membrane, PVM) identified in the erythrocyte cytoplasm will be investigated. Information gleaned from these investigations may contribute to the design of new chemotherapeutic strategies and agents and development of a blood-stage malaria vaccine.
