Kurzbeschreibung
(Englisch)
|
The genomes of Theileria parva and T. annulata have been sequenced, but genetic tools to manipulate Theileria are not yet available. This project aims to establish transient and stable transfection technology for Theileria parasites and to create the tools that will allow us to study the complex biology of this organism, in particular the interaction with the cells it infects.
|
Partner und Internationale Organisationen
(Englisch)
|
AT, CH, CZ, DE, DK, ES, FR, Gr, IT, NL, NO, PT, SE, UK
|
Abstract
(Englisch)
|
Theileria is an obligate intracellular protozoan parasite of cattle belonging to the phylum Apicomplexa. The parasites have the ability to transform their host cells, a unique event among eukaryotes, and thereby cause a severe lymphoproliferative disease called tropical theileriosis. Molecular genetics approaches have been extremely useful to investigate host-parasite interactions in other Apicomplexan parasites as Plasmodium or Toxoplasma. Our aim was to establish experimental conditions that will allow transfection of the Theileria parasite to be performed on a routine basis. A wide range of vectors were constructed expressing reporter genes as chloramphenicol acetyl transferase (CAT) and different green fluorescent protein (GFP) variants under the control of various putative Theileria promoters, including a bidirectional promoter (Hsp90/110) which should simultaneously drive the expression of two genes (a reporter gene and a resistance gene) in transfected Theileria parasites. Different transfection methods such as electroporation, lipofection, microinjection, magnet-assisted transfection, laser assisted transfection, gold particle diffusion and gold particle bombardment have been applied to the schizont stage of the parasite. The fact that three membranes (host cell membrane, parasite membrane, parasite nuclear membrane) have to be crossed for successful transfection of Theileria parasites might be a major handicap and leading to a very low transfection efficiency with any of the applied methods. Only with gold particle bombardment could transfected parasites expressing GFP be observed. It cannot be excluded that transfection methods other than ballistic bombardment also resulted in transfected parasites, but in numbers so small that they could not be detected using normal methods. To overcome the problem of low transfection efficiency we screened for drugs that could be used for selection after transfection. Pyrimethamine, G418, chloramphenicol, tetracycline, puromycine, blasticidine, erythromycin and WR99210 were tested. For some of these drugs, we noticed that resistance developed in a short time in T. annulata infected macrophages. Other drugs did not have any effect on the cells, even when applied at high doses. Interestingly, some drugs induced differentiation of the schizont to the next life cycle stage of the parasite, merogony. Nevertheless, we were able to transfect Theileria parasites using gold particle bombardment, confirming that the promoter(s) we selected are indeed funtional. This will now allow us to optimise the procedures towards the development of a protocol that could be used on a daily basis in the lab. For that purpose, two collaboration have now been established with laboratories in Belgium and Kenya and we are hopeful that this technology will allow us to tackle a number of important biolgogical questions in the near future.
|
