Luminescent multiplex viability assay for Trypanosoma brucei gambiense

Abstract

Background: New compounds for the treatment of human African trypanosomiasis (HAT) are urgently required. Trypanosoma brucei (T.b.) gambiense is the leading cause of HAT, yet T.b. gambiense is often not the prime target organism in drug discovery. This may be attributed to the difficulties in handling this subspecies and the lack of an efficient viability assay to monitor drug efficacy.

Methods: In this study, a T.b. gambiense strain, recently isolated in the D.R. Congo, was made bioluminescent by transfection with Renilla luciferase (RLuc) without altering its in vitro and in vivo growth characteristics. A luminescent multiplex viability assay (LMVA), based on measurement of the Renilla luciferase activity and the ATP content of the cells within the same experiment, was investigated as an alternative to the standard fluorimetric resazurin viability assay for drug sensitivity testing of T.b. gambiense.

Results: In a 96-well format, the RLuc transfected strain showed a detection limit of 2 × 10(4) cells ml(-1) for the Renilla luciferase measurement and 5 × 10(3) cells ml(-1) for the ATP measurement. Both assays of the LMVA showed linearity up to 10(6) cells ml(-1) and correlated well with the cell density during exponential growth of the long slender bloodstream forms. The LMVA was compared to the fluorimetric resazurin viability assay for drug sensitivity testing of pentamidine, eflornithine, nifurtimox and melarsoprol with both the wild type and the RLuc transfected population. For each drug, the IC50 value of the RLuc population was similar to that of the wild type when determined with either the fluorimetric resazurin method or the LMVA. For eflornithine, nifurtimox and melarsoprol we found no difference between the IC50 values in both viability assays. In contrast, the IC50 value of pentamidine was higher when determined with the fluorimetric resazurin method than in both assays of the LMVA.

Conclusions: LMVA has some advantages for viability measurement of T.b. gambiense: it requires less incubation time for viability detection than the fluorimetric resazurin assay and in LMVA, two sensitive and independent viability assays are performed in the same experiment.

Figure 1

Growth profiles of an HHM line, and from the nine subpassages (HH1 – HH9) that resulted in the HH adapted line of T.b. gambiense 348 BT. Vertical dotted lines mark the start of each new subpassage made using 90 to 50% new medium.

Figure 2

Relative luminescence of LMVA in function of cell density tested with population RLuc # 2.1. Horizontal dotted line represents a signal to background ratio of 3 to 1.Vertical dotted lines mark the cell density necessary for detection at this signal to background ratio (5 × 10³ cells ml^-1 for CellTiter-Glo and 2 × 10⁴ cells ml^-1 for EnduRen).

Figure 3

Growth curve of RLuc# 2.1 in function of time, assessed by LMVA (in relative luminescence, left Y-axis) and by microscopy (cell density, right Y-axis).