Bioluminescent Imaging of Trypanosoma brucei Shows Preferential Testis Dissemination Which May Hamper Drug Efficacy in Sleeping Sickness

Abstract

Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs.

The in vivo dynamics of antigenic variation in Trypanosoma brucei

“…To measure VSG expression within populations of T. brucei , we infected four mice with ~5 EATRO1125 parasites, originally expressing VSG AnTat1.1 (14, 15) but now heterogeneous and each expressing a distinct VSG , and tracked VSG expression dynamics for 30 days. A few variants made up the majority of the population at each time, but, surprisingly, each sample also contained many rare variants that would have been undetectable using previous approaches (Fig.…”

In Vitro and In Vivo Activities of 2-Aminopyrazines and 2-Aminopyridines in Experimental Models of Human African Trypanosomiasis

“…gambiense (MBA), and stable Renilla luciferase-expressing recombinant parasite T. brucei subsp. brucei (Rluc AnTat1.1E) (11,12), kindly provided by P. Büscher (Institute of Tropical Medicine, Antwerp, Belgium), were cultured at 37°C and 5% CO 2 in Iscove's modified Dulbecco's medium (IMDM; HyClone) containing 10% heat-inactivated calf serum (Invitrogen, Carlsbad, CA), 28 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.14% glucose, 1.5% sodium bicarbonate, 2 mM L-glutamine, 0.14 mg/ml gentamicin, 0.3 mM dithiothreitol (DTT), 1.4 mM sodium pyruvate, 0.7 mM L-cysteine, 28 M adenosine, and 14 M guanosine (13). T. brucei forms were harvested by centrifugation at 2,500 rpm for 6 min followed by resuspension in fresh medium.…”

“…luciferase (Rluc AnTat1.1E) was used to determine the killing effect of the compound on concentrated parasite cultures (1.25 ϫ 10 6 /ml) at different time points after compound exposure. The recombinant Renilla enzyme is constitutively expressed, and the luminescence emitted efficiently monitors cell viability as opposed to proliferation (13).…”

Trypanosomatids see the light: recent advances in bioimaging research

“…Furthermore, in vivo imaging was useful to explain the subcurative drug efficacy of a series of adenosine analogs in a murine model of African trypanosomiasis. Authors found preferential testis tropism of T. brucei parasites, which might be protected from the drug effect by the blood-testis barrier [36].…”