Luminescent multiplex viability assay for Trypanosoma brucei gambiense
© Van Reet et al.; licensee BioMed Central Ltd. 2013
Received: 23 April 2013
Accepted: 9 July 2013
Published: 15 July 2013
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.
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.
In a 96-well format, the RLuc transfected strain showed a detection limit of 2 × 104 cells ml-1 for the Renilla luciferase measurement and 5 × 103 cells ml-1 for the ATP measurement. Both assays of the LMVA showed linearity up to 106 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.
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.
KeywordsTrypanosoma brucei gambiense Democratic Republic of the Congo Viability Multiplex Luminescence Renilla luciferase
Human African trypanosomiasis (HAT), or sleeping sickness, is caused by two subspecies of Trypanosoma brucei (T.b.) and is transmitted through tsetse flies (Glossina spp). T.b. rhodesiense causes an acute form of sleeping sickness in East Africa. T.b. gambiense is responsible for the chronic form in West and Central Africa and accounts for more than 95% of the near 10,000 sleeping sickness patients that are diagnosed and treated annually . In both forms, the disease evolves from a first stage with peripheral tissue invasion, towards a second stage with invasion of the central nervous system. The drugs for treating sleeping sickness are subspecies specific due to their different metabolisation, and are disease stage specific depending on their ability to cross the blood–brain-barrier . T.b. gambiense HAT is treated with pentamidine (first stage) and nifurtimox-eflornithine combination therapy or melarsoprol (second stage). T.b. rhodesiense HAT is treated with suramin (first stage) or melarsoprol (second stage). All these drugs are toxic and require intramuscular or intravenous injection except for nifurtimox which is an oral drug . Research into new drugs for HAT aims at drugs that are safe, that are active against both subspecies and both disease stages, that can be given orally and that need only one administration . 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 strain 427[5–14]. Less often a hit is confirmed in vitro and in vivo on a collection of Trypanozoon strains, including T.b. rhodesiense and T.b. gambiense[15–20]. To be relevant for the current situation in the field, T.b. rhodesiense and T.b. gambiense strains that are recently isolated from patients with known treatment outcomes and that underwent few in vivo and in vitro passages, should be included in these drug discovery validation panels [21–23]. It would be even better to include T.b. gambiense strains already in the initial HTS screening, because, despite the high sequence similarity between the genomes of T.b. brucei and T.b. gambiense, the latter is often found to be more susceptible to drugs than other T. brucei subspecies, as is the case for eflornithine and pentamidine [15, 16, 25–27]. Several factors hamper inclusion of T.b. gambiense as a primary target organism in HTS. T.b. gambiense is particularly difficult to isolate from patients and to adapt to mice and to in vitro culture [23, 28]. Often, T.b. gambiense leads to silent or chronic infections in mice with hardly detectable parasites . Generally, bloodstream form parasites from in vivo or in vitro cultures are exposed to compounds for up to 72 hours, whereafter the remaining viability of the cells is assessed using either radioactive, colorimetric, fluorimetric, or luminescent detection [5, 9, 26, 30, 31]. The fluorimetric resazurin viability assay is very cost-effective, but performance is limited with T.b. gambiense strains due to lengthy incubation times with resazurin before detection of resorufin yields high enough signal to background for detection. The reason for low conversion of resazurin to resorufin is unknown, but long term incubation times with resazurin with live or lysed trypanosomes may affect the IC50 value of a drug [26, 31, 32]. Because of their easy, sensitive and fast readout, viability assays based on ATP detection (such as the luminescent CellTiter-Glo assay) have been proposed and used as an alternative viability assay for HTS in T.b. brucei strain 427 [5, 7]. Currently, there is no reporter gene based in vitro assay employed for HTS compound screening, either for T.b. gambiense or for T.b. brucei and T.b. rhodesiense, unlike for other protozoan parasites such as Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi[33–39]. Renilla luciferase tagged parasites have previously been generated for T.b. brucei and T.b. gambiense and have proved effective for in vivo parasite tracking in murine models of experimental trypanosomiasis [29, 40] and for preclinical in vivo drug efficacy testing . Yet up to now, no in vitro application of Renilla luciferase parasites has been described. Recently, it has been shown that the EnduRen assay, for measurement of vital in vitro Renilla luciferase activity, can be combined with the CellTiter-Glo assay as an efficient luminescent multiplex viability assay for HTS compound screening against Dengue . Assays that measure multiple fitness parameters within the same wells, such as multiplex assays, may decrease false-positive rates and increase confidence for hit selection in HTS . In the present study, a T.b. gambiense strain that was recently isolated in the Democratic Republic of the Congo was made bioluminescent by expression of Renilla luciferase. With this strain, the feasibility of the Enduren/CellTiter-Glo luminescent multiplex viability assay, abbreviated as LMVA, was compared to the fluorimetric resazurin assay for drug sensitivity testing of the main drugs to treat T.b. gambiense sleeping sickness: pentamidine, eflornithine nifurtimox and melarsoprol.
Iscove’s modified Dulbecco’s medium powder (IMDM) and fetal calf serum (FCS; heat-inactivated; EU approved South American origin) were purchased from Invitrogen. Methylcellulose (5140 mPa.s) was purchased from Fluka. All other culture media ingredients were from Sigma–Aldrich. An HMI-9 based stock solution  was adapted to prepare two culture media for use with in vitro culture of bloodstream form T.b. gambiense. Briefly, HMI-human serum (HH) contains HMI-9 with 15% v/v FCS and 5% v/v heat-inactivated human serum. HMI – human serum – methylcellulose (HHM) is HH containing a final concentration of 1.1% w/v methylcellulose. For fluorescent and luminescent activity assays, HH was prepared from IMDM without phenol red (Invitrogen).
T. b. gambiense MHOM/CD/INRB/2006/23A
T. b. gambiense strain MHOM/CD/INRB/2006/23A, alias 348 BT, was isolated in Mbuyi-Mayi in the Democratic Republic of the Congo in 2006, from the blood of a second stage patient who was cured after a 10 day melarsoprol treatment . The isolation of the bloodstream form in vivo in rodents and the adaptation in vitro to HHM and the confirmation of its gambiense type I genotype have been described previously [23, 28].
In vitro T.b. gambiense culture
An HHM adapted population of 348 BT, was inoculated in 500 μl of HHM in a 48-well plate at densities between 103 – 105 cells ml-1 and maintained in logarithmic phase growth by sub passages at appropriate dilutions after 24 to 72 hours of incubation at 37°C and 5% CO2. Cultures were monitored by phase contrast inverted microscopy. For cell counting, 20 μl were withdrawn and dispensed in a disposable Uriglass counting chamber (Menarini). Cultures were stepwise scaled up to 40 ml, by addition of four volumes of fresh medium in 25 cm2 flasks once the parasites reached a density of 5 × 105 cells ml-1. For long term storage, cells were cryostabilised in liquid nitrogen as tenfold concentrated log phase cultures in 90% HH with 10% glycerol.
In vivo T.b. gambiense culture
All animal experiments were approved by the Animal Ethics Committee of the Institute of Tropical Medicine Antwerp, under licence PAR019. Female OF-1 mice (40 – 50 g) (Charles River, Belgium), either immune suppressed with 200 mg kg-1 cyclophosphamide (Endoxan, Baxter) 48 h before infection or not, were infected intraperitoneally with 2 × 105 parasites in 200 μl, obtained from rodent blood or in vitro culture, and diluted at least 1:1 in phosphate buffered saline + 1% glucose pH 8.0 (PSG) . The matching method was used to monitor parasitemia in tail-blood  for 4 weeks after which the animals were sacrificed.
Renilla luciferase T.b. gambiense
The pHD309 RLuc expression vector, containing RLuc cDNA from pGL4 vector (Promega) was used for transfection . Parasites from flask cultures were harvested at 5 × 105 cells ml-1 and washed twice in cytomix (2 mM EGTA, 120 mM KCl, 0.15 mM CaCl2, 10 mM potassium phosphate pH 7.6, 25 mM Hepes, 1 mM hypoxanthine, 5 mM MgCl2, 5 g l-1 glucose, 100 mg l-1 BSA). Next, they were concentrated to 5 × 107 cells ml-1 and 400 μl of this suspension was transferred into a 4 mm cuvette (BioRad), whereafter 50 μl containing 10 μg of Not I (New England Biolabs) linearised pHD309 RLuc DNA was added. Subsequently, this mixture was pulsed once in a Gene Pulse Xcell electroporator (BioRad; 1250 V, 25 Ω, 50 μF), transferred to 12 ml of HH, plated in a 48-well plate in 250 μl volumes and incubated at 37°C for 24 h. Next, 250 μl of HH containing 2 μg ml-1 hygromycin was added. Populations were maintained in 1 μg ml-1 hygromycin for four weeks before cryostabilisation. The Renilla luciferase assay system (RLAS, Promega) was used to measure the RLuc activity from lysed cells. Forty μl of trypanosome suspension was mixed with 10 μl of 5 x Renilla lysis buffer and 20 μl of this solution was mixed with 100 μl of RLAS assay reagent (via dispenser) in a white opaque 96-well plate (Perkin Elmer). Each time an aliquot was dispensed into a well, the plate was shaken for 2 seconds and after a further 2 seconds delay, the number of photons per second (CPS) was measured for 10 seconds with a VictorX3 multimodal plate reader (Perkin Elmer).
Luminescent multiplex viability assay
The luminescent multiplex viability assay (LMVA) measures first the RLuc activity in live cells with EnduRen (Promega) and next measures the cell ATP content with the luminescent CellTiter-Glo reagent (Promega) within the same assay wells. Luminescence in CPS was measured with a VictorX3 multimodal plate reader (Perkin Elmer). The EnduRen reagent was used according to the manufacturer’s instructions (Promega). Forty-five μl of trypanosome suspension was transferred to a half area white opaque 96 well plate (Perkin Elmer) to which 5 μl of 60 μM EnduRen in HH (or 5 μl of HH) was added. After 2 hours incubation at 37° C and a 10 minutes equilibration at 25°C, the luminescence was measured by integrating the number of photons per 1 second. Then, an equal volume (50 μl) of reconstituted CellTiter-Glo reagent (Promega) was added to this parasite suspension, after 2 minutes of shaking, and a 10 minutes delay, the number of photons per second was integrated.
To assess the lower detection limit of wild type and recombinant trypanosome cells in HH, log phase trypanosomes at 105 cells ml-1 were harvested, concentrated and a tenfold dilution series was made in triplicate from 106 down to 102 cells ml-1 in 100 μl. This series was tested with the LMVA (using a 45 μl trypanosome suspension, as described above) and the RLAS (using 40 μl, as described above). The luminescent values (in CPS) of the cell containing samples were divided by the value of the blanks without cells (signal to background) and this relative luminescence value was plotted against the number of trypanosomes for each assay. Linear fits were used to find the lower detection limit of the number of trypanosome cells in each assay (at a signal to background ratio of 3 to 1). To measure the performance of the LMVA during the whole growth period, a trypanosome suspension of 2 × 104 cells ml-1 in 100 μl was inoculated in 15 wells and every day, for four days, triplicate wells were sampled for cell counting (using 20 μl cell suspension, as described above) and for the LMVA (using 45 μl cell suspension, as described above). Doubling times were calculated using non-linear regression in Prism (Graphpad).
Drug sensitivity testing
Eflornithine (Sanofi Aventis) and hygromycin B (Sigma) were prepared as 10 mg ml-1 stock solutions in distilled water. Melarsoprol (Arsobal, Sanofi Aventis), pentamidine isethionate (Sanofi Aventis) and nifurtimox (Sigma) were stored as 10 mg ml-1 stock solutions in DMSO. A method to measure the IC50 values of compounds in 96-well plates was performed as described elsewhere . Threefold drug dilutions in triplicate were made in HH from 100 to 0.14 μg ml-1 for eflornithine and hygromycin, from 50 to 0.07 μg ml-1 for nifurtimox and from 500 to 0.7 ng ml-1 for pentamidine and melarsoprol. Each drug concentration was inoculated with either 2 × 104 cells ml-1 or 5 × 103 cells ml-1 in a final volume of 100 μl. Next the plate was incubated for 72 hours. For detection of hygromycin sensitivity in the fluorimetric resazurin assay, 10 μl of 12 mg resazurin in 100 ml PBS were added to 100 μl trypanosome suspension in a 96 well transparent plate (Nunc). Alternatively, for comparison of IC50 values between the LMVA and resazurin assay, the 100 μl trypanosome suspension was split: 45 μl were used for LMVA as described above and 45 μl were transferred to a half area black opaque plate (Perkin Elmer) containing 5 μl of resazurin. After 24 h at 37°C, fluorescence was measured (excitation λ = 560 nm; emission λ = 590 nm) with a VictorX3 multimodal plate reader using top reading (Perkin Elmer) . The results were expressed as the percent reduction in parasite viability compared to parasite viability in control wells without drugs, and a 50% inhibitory concentration (IC50) was calculated using non-linear regression in Prism (GraphPad).
In vitro adaptation in HH
Transfection with pHD309 RLuc
Sensitivity to hygromycin and lower detection limits of RLAS, EnduRen and CellTiter-Glo with the T.b. gambiense 348BT wild type and three RLuc transfected strains
218 ± 14
7.5 ± 1.1
207 ± 22
991 ± 131
212 ± 9
2.4 ± 0.7
810 ± 80
4804 ± 511
4.2 ± 0.9
488 ± 50
3225 ± 423
215 ± 22
Activity of RLuc
To select the most luminescent population, activity of the expressed luciferase in the wild type and recombinant populations was quantified with two assays that measure RLuc activity either in lysed cells (RLAS, Promega) or in live cells (EnduRen, Promega). With both RLuc activity assays, a linear fit between the number of log phase recombinant trypanosomes and relative luminescence (signal to background) data was obtained until up to 106 cells ml-1, the most dense trypanosome suspension tested. The lower detection limit generated from these linear fits was different for each of the recombinant populations and was also different between both RLuc assays (ANOVA, p < 0.05) (Table 1). Due to its lowest detection limit in both RLuc assays, population #2.1 was identified as the most luminescent population.
LMVA performed on cells in the logarithmic growth phase
LMVA performance during growth profile
In vitro doubling time of the T.b. gambiense 348BT wild type and the recombinant RLuc #2.1 strain assessed in triplicate with EnduRen, CellTiter-Glo and by microscopy
12.7 ± 0.8
12.6 ± 0.9
12.9 ± 0.7
12.4 ± 0.7
12.6 ± 0.5
LMVA performance in drug sensitivity screening
IC 50 values for eflornithine, melarsoprol, pentamidine and nifurtimox obtained with the T.b. gambiense 348BT wild type and the recombinant RLuc #2.1 strain assessed with EnduRen, CellTiter-Glo and resazurin
RLuc # 2.1
RLuc # 2.1
RLuc # 2.1
5 x 103
1.0 ± 0.4
1.1 ± 0.4
1.0 ± 0.5
1.1 ± 0.6
1.4 ± 0.4
5.5 ± 2.1
4.5 ± 2.2
5.0 ± 1.9
6.3 ± 3.0
6.5 ± 2.3
40.1 ± 11.1
43.6 ± 10.3
41.1 ± 7.5
67.5 ± 11.1
64.1 ± 13.6
334 ± 47
274 ± 93
380 ± 84
437 ± 139
410 ± 132
2 x 104
2.8 ± 0.5
3.0 ± 0.7
2.6 ± 1.0
2.6 ± 0.9
2.9 ± 0.7
11.0 ± 3.2
12.0 ± 2.8
11.5 ± 2.6
8.9 ± 2.2
11.9 ± 2.6
47.5 ± 8.1
43.7 ± 10.8
48.9 ± 9.1
74.7 ± 12.3
72.5 ± 6.3
700 ± 63
670 ± 78
720 ± 75
751 ± 174
768 ± 125
In vivo infections of wild type and recombinant 348BT
Differences in infectivity, as determined by the number of days until the first parasite is detected (prepatent period), and virulence, as determined by the number of days of survival of the rodents, between the original cell line (adapted in vivo but not in vitro), the HHM and HH (adapted in vitro) cell lines, and the resulting RLuc cell line, were compared by expanding each population in 4 mice treated or not with endoxan. All mice were found parasitemic after 3 to 7 days of infection. The mean prepatent period was not significantly different between mice treated or not with endoxan and between the different trypanosome cell lines (data not shown). During the next 3 weeks of follow up, we could sporadically detect waves of parasitemia in all mice of all groups. No mice died from the infection during the experiment and all mice were sacrificed at day 30.
This study was undertaken to develop a Renilla luciferase based luminescent multiplex viability assay (LMVA) for in vitro compound screening on bloodstream form T.b. gambiense. To obtain the RLuc transfected T.b. gambiens e, we started with a recently isolated strain that underwent few in vivo passages in rodents and that was adapted to in vitro HMI-9 based medium with human serum (HH). Although nucleofection has been described to be very efficient for transient and for stable transfection of African trypanosomes [29, 49, 50], our study confirms that transfection with pHD309 RLuc is also successful by electroporation . Due to the presence of hygromycin phosphotransferase as resistance selection marker for stable genomic integration of Renilla luciferase, cross resistance against trypanotoxic hygromycin analogues may pose a problem, as has been described for pyrimethamine resistance of a transgenic firefly luciferase Plasmodium strain . To select the most RLuc transfected trypanosome population, two RLuc activity assays were used. The RLAS system is very sensitive, but is not compatible with CellTiter-Glo and requires more manipulations than the EnduRen assay. In the EnduRen assay, only the most hygromycin resistant population allowed fast detection of low numbers of cells (± 2 × 104 cells ml-1). In contrast to the EnduRen assay, the CellTiter-Glo assay does not require genetic manipulation of the trypanosome strain, the assay is performed faster and has a lower detection limit (± 5 × 103 cells ml-1). Combining the EnduRen assay with CellTiter-Glo as two independent assays measuring respectively the RLuc activity and the ATP content of the cells, we were able to establish a multiplex viability assay for which the luminescence signals correlate well with the cell density during the proliferation of the long slender bloodstream form trypanosomes. The multiplex luminescent format has several advantages over the fluorimetric resazurin assay: first, viability assessment requires less incubation time with substrate before detection takes place (2 hours vs 16 – 24 hours) and second, two independent viability parameters are assayed in the same experiment (RLuc activity and ATP content). Disadvantages of this LMVA are its higher cost compared to the resazurin assay and the need for an RLuc transfected trypanosome strain. On the other hand, transfecting a trypanosome strain with a luminescence reporter gene makes it possible to first select trypanocidal compounds in vitro and subsequently test their activity in vivo with the same trypanosome strain by bioluminescence imaging of infected mice. Here we confirm that even after transfection with pHD 309 RLuc, the in vitro and in vivo growth characteristics of the recombinant T.b. gambiense strain are not different from the wild type strain. We used several drugs that are in use against T.b. gambiense sleeping sickness to investigate whether the pHD 309 RLuc integration would have altered the drug sensitivity profile compared to the wild type. No such influence could be observed. Furthermore, the IC50 values for these drugs fall within range of other T.b. gambiense field strains isolated in Mbuji-Mayi, Democratic Republic of the Congo, that have been used recently for validation of fexinidazole [20, 26]. Before the present LMVA can be adopted as HTS assay, further evaluation and optimisation is necessary. Also, the assay should be tested for its applicability on other RLuc transfected Trypanozoon strains that have become available recently, including T.b. brucei, T.b. rhodesiense and T. evansi strains.
In conclusion, we showed that a luminescent multiplex viability assay with an RLuc transfected T.b. gambiense strain can be used as an alternative to the resazurin viability assay in drug discovery. Both the LMVA assay and the trypanosome strain represent valuable assets in the fight against sleeping sickness, complementing the available tools for HTS compound screening, particularly where it comes to confirm in vivo trypanocidal activity of molecules that have been selected in vitro.
The pHD 309 vector was a kind gift of Dr George Cross. Authors like to thank Maarten Van Den Boogaerde, Jeroen Swiers en Nicolas Bebronne for technical assistance. This work was supported by the Fonds voor Wetenschappelijk onderzoek (FWO) [grant 1.5.147.09, G.0229.10 N]. The funding agency had no role in the collection, analysis, interpretation of data, writing of the manuscript and in the decision to submit the manuscript for publication.
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