Induction of apoptosis in the malarial parasite
Over the last few years it has become possible to demonstrate stress-induced apoptotic and apoptosis-related processes in unicellular organisms, including parasites such as Plasmodium and Leishmania. Plasmodium falciparum as a causative agent of tropical malaria is responsible for the death of 800,000 people annually and represents a most interesting target of modern apoptosis research. It has been hypothesized that the parasite regulates its cell density in human blood by apoptotic processes. Thus our detailed understanding of apoptotic signal transduction pathways in the parasite and their induction by external stimuli is a promising approach to the directed induction of cell death. The proposed studies will furthermore enhance our knowledge of parasite-host interactions and evolutionary aspects of apoptotic cascades.
Scientifically our group focuses on redox and energy metabolism of malarial parasites and their host cells, employing methods of molecular biology, protein biochemistry, and (structure-based) drug development. Over the last few years we have studied apoptotic marks of P. falciparum blood stages in vitro. Since Plasmodium is an intracellular parasite, this approach is methodologically challenging. We identified H2O2, human apoptosis-inducing agents, and various antimalarial agents as external inducers. In parallel we identified potential apoptosis-related genes, including a metacaspase, of P. falciparum in silico. These genes have been partially cloned and overexpressed. Apoptosis in malarial parasites has hardly been characterized but differs significantly from the respective processes in the human host. In order to enhance our knowledge of induced cell death in unicellular organisms and to estimate its potential as a target for pharmacological interventions, we would like to develop this project further.
Programmed cell death (PCD) is thought to have evolved not only to regulate growth and development in multicellular organisms but also to have a functional role in the biology of unicellular organisms. In protozoan parasites some features of PCD similar to those in multicellular organisms have been reported. They have, however, not yet been sufficiently studied to understand the respective PCD pathways and to define the internal and external factors that control PCD. This understanding could delineate the evolutionary origin of this pathway and provide information on the pathogenesis of the respective diseases and potential targeted drug development.
Apoptosis research in Plasmodium is a young discipline. Malarial parasites are transmitted from host to host by mosquitoes. Interactions between parasite and vector occur at all stages of the establishment and development of the parasite, and some of these result in the death of host and parasite cells by apoptosis. Apoptosis caused by neutrophil secretory products has been shown to play a major role in endothelial cell damage in malaria and thus in the pathogenesis of cerebral malaria. The antioxidants ascorbic acid and tocopherol and the protease inhibitor ulinastatin were found to reduce endothelial PCD in vitro. Active inhibition of apoptosis has been described in human host liver cells during infection by Plasmodium. It is conferred by hepatocyte growth factor/MET signalling and is required for a successful infection. Activation of cation channels in malaria-infected erythrocytes is required for intracellular growth of the pathogen; however, it also stimulates the exposure of phosphatidylserine at the red cell surface, resulting in phagocytosis. This erythrocyte "apoptosis" may represent a host defense mechanism that serves to eliminate infected erythrocytes.
Despite the absence of genes homologous to classical caspases in the genome of the parasite, caspase-like activity has been detected in the cytoplasm of the malarial ookinetes, and nitric oxide is likely to play a prominent role in the induction of PCD. Apoptosis could be demonstrated in the mosquito stages of Plasmodium berghei as a selection criterion; since apoptotic markers, chromatin condensation, phosphatidylserine externalization, and DNA fragmentation as reactions to the stress induced in the midgut of Anopheles were detected. Furthermore, it was possible to inhibit apoptosis via human caspase inhibitors. Interestingly, in a chloroquine-sensitive strain of P. falciparum, a stronger DNA fragmentation as a reaction to treatment with chloroquine was observed than for a resistant strain. This was a first hint that there might be a connection between apoptosis and resistance mechanisms.
A good model for successful apoptosis research on unicellular parasites was conferred by recent work on Leishmania. In Leishmania, apoptosis can be induced as a reaction to oxidative stress in the form of H2O2 or nitrogen monoxide. Furthermore – as in higher eukaryotes – a permeabilization of the mitochondrial membrane as well as an activation of cysteine proteases could be demonstrated. The signal transduction pathways involved in apoptotic processes of unicellular parasites differ significantly from human apoptotic cascades and are thus most promising as potential targets for antiparasitic interventions.