The ins and outs of macromolecular transport in malaria-infected erythrocytes
The investigations reported in this thesis have uncovered several pathways by which intra-erythrocytic P. falciparum, the protozoan parasite which causes human malaria, obtains nutrients and exports molecules to extracellular locations. Chapter One investigated 2 parasitic pathways for the procurement of metabolic precursors; cytostomal uptake of erythrocyte cytosolic contents as well as fluid phase endocytic uptake of soluble serum components. The cytostomal uptake pathway was found to begin shortly after invasion, continuing throughout the erythrocytic life cycle and culminating in the accumulation of erythrocytic contents within the food vacuole. Fluid-phase internalization of fluorescently- and digoxigenin-labeled dextrans from the external medium seemed to be compartmentally distinct from cytostomal uptake in that it was localized in the parasite cytosol and within the lumen of intra-erythrocytic membranes.
The results in Chapter Two established, through the use of polymerization inhibitors (e.g. cytochalasin B) and stabilizers (Taxol, Epothilone A (EpA) and phalloidin), that the cytoskeleton of intra-erythrocytic P. falciparum has several features common to higher eukaryotic cells. Treatment of asexual P. falciparum with Taxol and EpA established that microtubules are central to chromosomal segregation during merozoite formation, but seemed to have no role in the morphogenesis or migration of secretory organelles such as the rhoptries and micronemes or in the cytostome-mediated feeding process. Cytochalasin B- and phalloidin-treatment of erythrocytic P. falciparum indicated that cytostomal activity seems to rely upon the polymerization of actin, which was found to be associated with structures involved in hemoglobin internalization.
Chapter Three reported the first ultrastructural evidence for a eukaryotic-like secretory apparatus in P. falciparum-infected erythrocytes. Intra-erythrocytic P. falciparum were found not only to contain coated ER- and Golgi-based vesicles, but these secretory structures were also found to have distinct morphology at different parasite developmental stages. Smooth tubo-vesicular membrane elements were observed in sub-membranous regions of both trophozoite- and schizont stage limiting membranes, which were associated with vesicles which appear to be morphologically similar to secretory vesicles identified in higher eukaryotes.
Results reported in Chapter Four establish the basis of a transfection system for the investigation of protein trafficking pathways within asexual P. falciparum. Through the use of several different expression vectors, it was found that transient transfection of intra-erythrocytic parasites with plasmids which encode chimeric fusion proteins resulted in levels of expression of those proteins which were undetectable by immunofluorescence, immunogold labeling and Western analysis. In light of these results, construction of stable transfection vectors and the identification of more transcriptionally active promoter elements seemed to be the only plausible alternative for the detectable expression of chimeric transgenes in transfected asexual P. falciparum. To this end, this investigation not only established that the KAHRP 5$\sp\prime$ UTR contained a viable promoter region for transient and stable gene expression, it also identified a 1.4 kb fragment of that UTR (e.g. pHRPP2) which contained over 3 times the promoter activity than the parent molecule, as indicated by CAT expression. (Abstract shortened by UMI.)
0307: Molecular biology