My PhD project started within the framework of a European FP7-Project called NEUROTRYP. In this project I was responsible to genetically modify the different subspecies of Trypanosoma brucei with a bioluminescent reporter gene. In order to render trypanosomes luminescent, we first required in vitro culture adapted trypanosomes. Although methods were described to culture T.b. brucei and T.b rhodesiense in vitro using an axenic culture medium, the in vitro isolation of T.b. gambiense was largely based on the use of a feeder cell layer system that interferes with downstream applications, such as transfection or drug resistance analysis. We successfully tested an axenic medium containing methylcellulose to culture T.b. gambiense strains from our own collection of strains at ITM and later also T.b. gambiense strains from treatment refractory patients from Mbuji-Mayi (DRC) that were isolated by Dr Pati Pyana, with whom I would closely collaborate over the next years.
Within our NEUROTRYP project we succeeded in generating Renilla luciferase modified T.b. brucei trypanosomes, thanks to collaboration with Dr. Cross (Rockefeller University, New York, US), who donated his trypanosomal expression vector, pHD309. The Renilla luciferase confers to trypanosomes the ability to express blue light upon oxidation of its substrate coelenterazine. One of the most interesting findings in rodent models infected with Renilla modified T.b. brucei trypanosomes was that the testes are a hideout for trypanosomes. On the other hand, Renilla luciferase modified T.b. gambiense allowed us to develop very different murine disease models that range from silent to chronic and acute infections. Later it has been shown that the Renilla luciferase trypanosomes could be used in the preclinical in vivo assessment of drugs where they could be used to monitor the outcome of treatment. The Renilla luciferase trypanosomes have been distributed to researchers in the USA, France, Italy, Portugal and Cuba; including a compound screening facility (OTRADI, USA).
Whole cell in vitro high-throughput screenings (HTS) are now in use to discover novel trypanotoxic compounds. However, these HTS assays are almost exclusively performed with one particular non human pathogenic strain: T.b. brucei 427. With one of our most drug resistant Renilla luciferase modified strains, T.b. gambiense 348 BT, I was able to deliver the proof-of-principle for a HTS screening with T.b. gambiense for which I developed a multiplexed luminescent viability assay that can be easily implemented in HTS research.
Despite the high activity of Renilla luciferase in trypanosomes, the blue light is not transmitted very well because of absorption and scattering in tissue. Often firefly luciferases are chosen to create BLI models that in contrast to Renilla luciferase use D-luciferin as substrate and emit light of yellow or red color. Under a Material Transfer Agreement with Dr. Bruce Branchini, I received the PpyRE9 red luciferase for use with trypanosomes. Our latest results show that this luciferase is several tenfold more sensitive than Renilla luciferase in vivo. At the same time, we explored to use of fluorescent trypanosomes for in vivo fluorescence imaging. We have expressed green, red, far-red and near infrared fluorescent genes in our T. brucei strains. Although they do not appear to be sensitive enough for whole body in vivo fluorescence imaging, they are particularly fit for use in multiphoton intravital microscopy. Ongoing experiments are performed in collaboration with the University of Verona (Italy) and recently also with Prof Meiqing Shi (University of Maryland, USA) but also in ITM (Dr Caljon, Unit of Veterinary Protozoology). Over the years, I gained a lot of experience in transfection and generation of new trypanosomal expression vectors. Starting with the initial pHD309 vector we constructed alternative versions that allow us to fuse a reporter gene to eGFP either N or C terminally or to express two reporter genes independently from this vector by using b-tubulin intergenic regions.
Thus, during my previous PhD work, I generated a unique collection of biophotonic T. brucei, T.b. gambiense and T.b. rhodesiense that deserves valorization in new research lines.
I have been involved in the study of mechanisms of treatment failure for several years now in collaboration with Dr. Vet. Pati Pyana who has isolated a collection of T.b. gambiense strains from relapsed and cured infection that have been characterized for melarsoprol in vivo.