Transgenic Plasmodium chabaudi for analysis of host/parasite interactions
Final Report Abstract
The project originally had two different aims, first the creation of transgenic Plasmodium chabaudi and secondly the analysis of the role of CIR proteins in antigenic variation using these transgenic parasites as tools. The success obtained regarding the first aim was only very limited. While a transgenic line was isolated, it turned out to contain the transgene in a non-stable episomal form and not integrated into the genome. This resulted in loss of the transgene unless strong selection with pyrimethamine was applied for three days. In immunocompetent mice, this resulted in only minimal peak parasitemia. Passage was only possible in immunodeficient SCID mice. Moreover, expression of the GFP/lacZ reporter genes encoded by the plasmid was very low. GFP expression was only detectable by RT-PCR but not by fluorescence microscopy or Western blotting. Luciferase activity was clearly detectable in the blood of mice infected with transgenic lines. There were no significant differences in transgene expression between early trophozoites and late trophozoites/early schizonts. Despite the fact that no transgenic parasites were available, analysis of cir mRNA was performed in the course of P. chabaudi infection. Phylogenetic analysis identified two major subfamilies of cir genes, encoding relatively uniform proteins with hypervariable regions and a third very heterogeneous group including several genes that strongly differ in their size from the two conventional subfamilies. Expression patterns for subfamily 1 and 2 were analysed by sequencing of cloned RT-PCR products as well as by RT-PCR followed by restriction fragment length polymorphism (RFLP) analysis. Both methods revealed tissue specific differences in cir gene expression. While marked differences for subfamily 1 could be found, only small differences were seen for subfamily 2. Finally a small set of experiments was conducted to examine the suitability of reverse transcription loopmediated isothermal amplification (RT-LAMP) for quantification of cir gene expression directly from small blood samples without RNA purification. While detection of parasite specific nucleic acids was possible without any problems, it turned out to be impossible to design primer sets that did not also amplify genomic DNA. The small number and size of introns in cir genes and the very high AT content of P. chabaudi limiting the number of primer sets useful for LAMP are probably the main reason for this failure.
Publications
- (2009). Charakterisierung der cir Multigenfamilie und ihre Bedeutung für Sequestrierung bei Plasmodium chabaudi Malaria-Infektionen in der Maus. LBH: Proceedings, Tagung der DVG-Fachgruppe „Parasitologie und parasitäre Krankheiten“
Ebbinghaus P., von Samson Himmelstjerna G., Krücken J.
- (2010). The Plasmodium chabaudi cir multigene family: Antigenic Variation and its role in sequestration. Dusseldorf university press, Abstracts of the Joint Meeting of the German Societies of Parasitologiy and Protozoology at Düsseldorf University
Ebbinghaus P., von Samson Himmelstjerna G., Krücken J.
- (2011). Antigenic variation of the Plasmodium chabaudi cir multigene family and its implication in host tissue localization. Proceedings 23rd International Conference of the World Association for the Advancement of Veterinary Parasitology (WAAVP) Buenos Aires, Argentinia, ISBN978-978-27164-0-0
Ebbinghaus P., von Samson Himmelstjerna G., Krücken J.
- Characterization and tissue-specific expression patterns of the Plasmodium chabaudi cir multigene family. Malaria J. 10: 272, 2011
Ebbinghaus P., Krücken J.
