TY  - GEN
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/23530/
N2  - Plasmodium species are unicellular eukaryotic parasites that are the causative agents of malaria. This disease places a high burden on most tropical countries due to the severity of its symptoms and high prevalence. The absence of sterile immunity under natural transmission conditions, rapidly evolving drug resistance and the lack of a protective vaccine intensify the problem. Plasmodium is a highly adapted intracellular parasite. Plasmodium sporozoites develop within oocysts in the mosquito midgut, enter the salivary glands and are injected into the skin of their mammalian host. Sporozoites reach the liver, develop intracellular within hepatocytes into merozoites and enter red blood cells. Multiplication of parasites in the blood causes the disease. If a mosquito takes a blood meal at this time, the parasites enter the midgut, causing an infection.
It has been shown in the past that parasites lacking genes required for liver stage development can be used as an experimental live-attenuated vaccine. Their arrest in the liver allows the host to launch a protective immune response, resulting in sterile protection from sporozoite challenge. Here I investigated if it is possible to exploit parts of the particular biology of Plasmodium, its haploid genome and the limitation of DNA repair
to homologous recombination to generate a new generation of experimental vaccines. I generated parasites that express zinc finger nucleases causing a single well-timed double strand break within their own genome using stage specific promoters. This resulted in delayed death of the parasites as they failed to repair their split chromosome. I show that parasites arresting within the liver stage using this method can cause sterile protection from lethal sporozoite challenge. Additionally, rare breakthrough events resulting
in blood stage infection were investigated. In these parasites I observed the genetic signature of DNA repair events by microhomology-mediated end joining with as little as four base pairs of homology.
The circumsporozoite protein (CSP) is the major surface protein of sporozoites, essential for their formation in the oocyst and despited extensive research its function at this stage is not understood. I used interdomain tagging with green fuorescent protein to study the function of the circumsporozoite protein (CSP). Using live cell microscopy of complete mosquito midguts, electron microscopy of midgut sections and analysis of
protein extracts via western blotting I investigated the role of CSP during sporozoite formation. I could show that CSP undergoes previously unrecognized proteolytic processing at the C-terminus within the oocyst. Additionally my results suggest that extensive invagination of the plasma membrane has to occur prior to sporozoite formation and that CSP plays a role in orchestration of these events. This work also highlighted the importance of a previously unappreciated membranous network, the labyrinthine structure, for sporozoite formation. In conclusion this part of my work focuses on sporozoite biology, on their formation in the oocyst and on how to block their development in the liver.
Y1  - 2017///
TI  - Timed genome editing and sporozoite formation in Plasmodium berghei
AV  - public
ID  - heidok23530
A1  - Singer, Mirko
ER  -