A panel of Trypanosoma brucei strains tagged with blue and red-shifted luciferases for bioluminescent imaging in murine infection models.

Nick Van Reet,Patient Pati Pyana,Anne Marie Van Der Linden,Philippe Büscher,Hélène Van De Vyver,Jayne Raper

doi:10.1371/journal.pntd.0003054

Abstract

BackgroundGenetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients.Methodology/Principal findingsWe transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT.Conclusions/SignificanceWe established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better kinetics of their substrate D-luciferin.

Highlights

African trypanosomes pose a threat to millions of humans and animals in sub-Saharan Africa
Out of four clones of T.b. brucei AnTaR 1 transfected with pHD PpyRE9 luciferase (P9) (AnTaR 1 P9), ten clones of T.b. brucei AnTaR 1 transfected with pHD click beetle red (CBR) (AnTaR 1 CBR), 3 clones of T.b. rhodesiense RUMPHI transfected with pHD CBR (RUMPHI CBR), 7 clones of T.b. gambiense LiTaR 1 transfected with pHD CBR (LiTaR 1 CBR) and 2 clones of T.b. gambiense 348 BT transfected with pHD CBR (348 BT CBR) that were simultaneously tested, we identified for each strain and luciferase reporter combination, the clone with the highest relative luciferase activity (Figure 2: A and Figure S1)
Post-hoc analysis revealed that the relative luciferase activity of clone 4 of P9modified T.b. brucei AnTaR 1 was significantly higher than the highest relative luciferase activity of the CBR-modified clones from T.b. brucei AnTaR 1, T.b. gambiense LiTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT (p,0.05)

Summary

Introduction

African trypanosomes pose a threat to millions of humans and animals in sub-Saharan Africa. Two species readily infect humans and both are subspecies of Trypanosoma brucei (T.b.). T.b. gambiense is responsible for the chronic form of human African trypanosomiasis (HAT) in West and Central Africa and accounts for more than 97% of the near 10,000 sleeping sickness patients who are diagnosed and treated annually [1]. T.b. rhodesiense causes a more acute form of HAT in South and East Africa, only differing from a third non human-infective subspecies, T.b. brucei, by a single gene, SRA, that confers resistance against human serum [2]. First stage gambiense and rhodesiense HAT are treated with pentamidine and suramin respectively. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients

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