3R-Project 60-97

Transgenic protozoa (Trypanosoma spp.) as an alternative to transgenic animals

Isabel Roditi
Institutes of General Microbiology and Parasitology, University of Bern, 3012 Bern, Switzerland
isabel.roditi@imb.unibe.ch

Keywords: parasites, ectoparasites: trypanosoma; cytokines, growth factors; transgenic models; cell cultures: parasites; cell cultures: transgenic; molecular biology: recombinant proteins; replacement; toxicity testing: pharmaceuticals

Duration: 2 years Project Completion: 2001

Background and Aim
Unicellular organisms (protozoa) can provide an attractive system for medium-scale production of biologically active recombinant proteins. The production of transgenic trypanosomes expressing foreign proteins has several advantages compared to transgenic animals as shown with a series of recently developed vectors in our laboratory. These include stable and precisely targeted integration into the genome by homologous recombination , a choice of integration into several defined sites, allowing expression of multi-subunit complexes, and easy maintenance of cells in a semi-defined medium and growth to high densities (>2 x 10⁷ ml^-1). The aim of this study is to exploit transgenic trypanosomes, firstly as a source of recombinant proteins that might otherwise be produced in animals, and secondly as a tool for drug-screening in culture, thereby replacing animals as a source of parasites.

Method and Results

1) Expression of Theileria parva sporozoite antigen p67
Background: More than half a million cattle a year die of East Coast Fever, a disease caused by tick-borne transmission of the protozoan parasite T. parva. The infection is initiated when sporozoite forms of the parasite invade host lymphocytes and cause them to proliferate in an uncontrolled manner. More than ten years after its discovery, the sporozoite surface antigen p67 is still the only vaccine candidate against East coast fever. It is not possible to obtain sporozoites from infected ticks without allowing them to feed on experimental animals (cattle and rabbits). The application of ticks to rabbit ears can cause severe irritation, especially when larger numbers are used, and is sometimes accompanied by inflammatory responses that affect large areas of the ear epidermis.
Recombinant p67 that has been produced in either bacteria or baculoviruses cannot replace Theileria p67, since it is not recognised by antibodies against the native protein. Up to now we have expressed three different forms of p67 in trypanosomes. The most promising is a hybrid protein that contains the signal sequence and GPI-anchor addition sequence of an endogenous trypanosome surface protein (procyclin). A further set of experiments will establish whether this form of p67 can substitute for the Theileria protein.

2) A new system for testing drugs in vitro
Background: Bloodstream forms of African trypanosomes survive and replicate in the blood of the mammalian host whereas procyclic forms (which normally first appear in the insect vector) lyse immediately upon contact with serum. Identifying compounds that can induce premature differentiation and the subsequent destruction of trypanosomes in the bloodstream, could be of therapeutic value. To this end we have produced transgenic bloodstream form trypanosomes that synthesise the bacterial enzyme beta-glucuronidase when they differentiate into procyclic forms. Enzyme activity can be detected by adding a substrate which results in a colour change. This assay is so sensitive that it can be performed with small numbers of trypanosomes that are easily obtainable in vitro (whereas larger numbers would require the infection of mice or rats). Thus drugs which induce premature differention can be detected with this approach which is suitable for rapid screening of chemicals (see also 3R-Info-Bulletin Nr. 14).

Conclusions and Relevance for 3R
The use of transgenic trypanosomes, firstly for the expression of recombinant proteins, and secondly as a readout for the detection of novel drugs with antitrypanosomal activities, provides alternatives to transgenic animals for the production of biologically active eukaryotic proteins and reduces the requirement for animals as a source of parasites in drug and vaccine development.

(see also 3R-INFO-BULLETIN Nr. 14)

References
1. Sbicego, S., Vassella, E., Kurath, U., Blum, B and Roditi, I. (1999). The use of transgenic Trypanosoma brucei to identify compounds inducing the differentiation of bloodstream to procyclic forms. Mol. Biochem. Parasitol., 104, 311-322.

2. Furger, A., Jungi, T.W., Salomone, J.Y., Weynants, V. and Roditi, I. (2001). Stable expression of biologically active recombinant bovine IL-4 in Trypanosoma brucei , submitted.