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

Abstract

Background: Genetic 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 findings: We 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/significance: We 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.

Figure 1. Definition of ROI.

BLI data were analysed in function of 3 ROIs; (A) abdomen, (B) thorax and (C) head.

Figure 2. Characteristics of wild-type and luminescent strains.

Comparison of (A) relative luciferase activity (values are the mean ± SD from 6 to 11 cultures) (B) doubling time (values are the mean ± SD from 3 cultures) and (C) hygromycin resistance IC₅₀ (values are the mean ± SD from 2 to 7 cultures).

Figure 3. Drug sensitivity profiles of wild-type and luminescent strains.

IC₅₀ (mean ± SD) of wild-type and luminescent strains against (A) eflornithine (values are the mean ± SD from 4 to 9 cultures), (B) nifurtimox (values are the mean ± SD from 4 to 9 cultures), (C) diminazene diaceturate (values are the mean ± SD from 2 cultures), (D) melarsoprol (values are the mean ± SD from 4 to 9 cultures), (E) suramin (values are the mean ± SD from 4 to 9 cultures) and (F) pentamidine isethionate (values are the mean ± SD from 2 cultures).

Figure 4. Quantification of BLI data of OF-1 mice infected with luminescent T.b. brucei, T.b. rhodesiense and T.b. gambiense.

OF-1 mice (n = 3) were infected with T.b. brucei AnTaR 1 wild-type, T.b. brucei AnTaR 1 RLuc, T.b. brucei AnTaR 1 CBR, T.b. brucei AnTaR 1 P9, T.b. rhodesiense RUMPHI CBR, T.b. gambiense LiTaR 1 CBR and their luminescence was measured at 1, 4, 7, 18 and 26 post-infection in BLI using their respective substrates. The BLI data were divided in 3 ROIs; (A) abdomen, (B) thorax and (C) head and expressed as ph s⁻¹ cm⁻² sr⁻¹.

Figure 5. Visualisation of BLI data of OF-1 mice infected with T.b. brucei, T.b. rhodesiense and T.b. gambiense.

OF-1 mice (n = 3) were infected with T.b. brucei AnTaR 1 RLuc (A–D), T.b. brucei AnTaR 1 CBR (E–H), T.b. brucei AnTaR 1 P9 (I–L), T.b. rhodesiense RUMPHI CBR (M–P), T.b. gambiense LiTaR 1 CBR (Q–R) and their luminescence was measured in BLI using their respective substrates. Rows represent measurements at 1, 4, 18 and 26 days post-infection.

Figure 6. Quantification of BLI data in untreated and CPA-treated mice infected with T.b. gambiense.

OF-1 mice (n = 3) untreated or CPA-treated were infected with T.b. gambiense 348 BT RLuc and T.b. gambiense 348 BT CBR and their luminescence was measured at 1, 3, 7, 11, 43 (for RLuc) and 60 days post-infection (for CBR) using their respective substrates. The BLI data were divided in 3 ROIs; (A) abdomen, (B) thorax and (C) head and expressed as ph s⁻¹ cm⁻² sr⁻¹.

Figure 7. Visualisation of BLI data in untreated and CPA-treated OF-1 mice infected with T.b. gambiense.

OF-1 mice (n = 3) were infected with T.b. gambiense 348 BT RLuc without (A–E) or with CPA treatment (F–J) and their luminescence was measured with ViviRen or were infected with T.b. gambiense 348 BT CBR without (K–P) or with (Q–V) CPA treatment and their luminescence was measured with D-luciferin. Rows represent measurements at 1, 3, 7, 11, 43 (for RLuc) and 60 days post-infection (only CBR). In square P one mouse woke up from anesthesia just before the end of acquisition.

Figure 8. Visualisation of ex vivo brain BLI data obtained from mice infected with different luminescent strains.

At 26 days post-infection for T.b. brucei AnTaR 1 and T.b. rhodesiense RUMPHI and at 43 and 60 days post-infection for T.b. gambiense 348 BT brains were extracted and immersed in PBSG and substrate. (A–B) T.b. gambiense 348 BT CBR, CPA-treated, with D-luciferin; (C) T.b. gambiense 348 BT CBR, untreated, with D-luciferin ;(D) uninfected with D-luciferin; (E) T.b. gambiense 348 BT RLuc, CPA-treated with ViviRen; (F–H) T.b. rhodesiense RUMPHI CBR with D-luciferin; (I) T.b. brucei AnTaR 1 CBR with D-luciferin; (J) T.b. brucei AnTaR 1 P9 with D-luciferin.