Luciferase-expressing Leishmania infantum allows the monitoring of amastigote population size, in vivo, ex vivo and in vitro

Abstract

Here we engineered transgenic Leishmania infantum that express luciferase, the objectives being to more easily monitor in real time their establishment either in BALB/c mice--the liver and spleen being mainly studied-or in vitro. Whatever stationary phase L. infantum promastigotes population--wild type or engineered to express luciferase-the parasite burden was similar in the liver and the spleen at day 30 post the intravenous inoculation of BALB/c mice. Imaging of L. infantum hosting BALB/C mice provided sensitivity in the range of 20,000 to 40,000 amastigotes/mg tissue, two tissues-liver and spleen-being monitored. Once sampled and processed ex vivo for their luciferin-dependent bioluminescence the threshold sensitivity was shown to range from 1,000 to 6,000 amastigotes/mg tissue. This model further proved to be valuable for in vivo measurement of the efficiency of drugs such as miltefosine and may, therefore, additionally be used to evaluate vaccine-induced protection.

Figure 1. Infectivity of LUC-parasites and sensitivity of bioluminescence in vivo and ex vivo.

A. Infectivity of Luc-parasites as compared to L. infantum WT parasites. Both luciferase transgenic and wild type L. infantum stationary phase promastigotes establish themselves in BALB/c mice. Groups of 4 BALB/c mice were given either 0.12×10⁸ to 3×10⁸ stationary phase luciferase-transgenic or wild type L. infantum. One month later, mice were imaged, the bioluminescence being recorded before their sacrifice. L. infantum burdens were estimated by ELISA in the liver and the spleen. Burdens were calculated as parasite/mg organ x organ weight (in mg). Data representative of three experiments are presented as box whisker plots. For statistical analysis Man Whitney Wilcoxon test was performed and did not show any significant difference between parasite loads of mice inoculated with WT or luciferase parasites; ns =  not-significant. B. Bioimaging of BALB/c mice one month post the IV inoculation of luciferase transgenic L. infantum. Mice inoculated with LUC-parasite and with parasite loads depicted in figure 1A, were given luciferin via IP. The photon emission was recorded once the anesthetized mice were deposited in the imaging chamber of the Photon Imager. Red zones are ROIs that delineate the liver or spleen. C. Sensitivity of BLI. Luminescence (photon/s/cm²) was recorded in ROIs corresponding to liver and spleen of the 11 mice infected with LUC-parasites depicted in figure 1A. Luminescence recorded in ROIs was plotted versus parasite density measured by ELISA as described in figure 1A to generate regression curves. The threshold sensitively of BLI calculated as three times luminescence of naïve mice (indicated by the gray zone) was 20,000 to 40,000 parasites/mg for spleen and liver respectively. D. Ex-vivo quantification of parasite loads by bioluminescence. Luminescence of equal numbers of the LUC-parasite under exponentially growing promastigote form or liver and spleen amastigotes was analyzed using a luminometer. Equal numbers of liver and spleen amastigotes from mice infected with WT promastigotes were used as specificity control. Sensitivity of ex vivo analysis of parasite density by bioluminescence (1,000 to 6,000 amastigotes/mg for spleen and liver respectively) was calculated as parasite numbers corresponding to twice luminescence background values. E. Accuracy of ex vivo bioluminescence analysis for estimation of parasite density. Liver and spleen samples from 18 IV infected BALB/c mice were detergent extracted and assayed both by ELISA and bioluminescence. Values generated by both techniques were plotted to generate regression curves.

Figure 2. Monitoring of L. infantum infection using BLI and application for leishmanicidal drug screening.

A. Follow up of infection by BLI and drug screening in vivo. Groups of 2 BALB/c mice were IV infected with 1×10⁸ or 3×10⁸ of stationary phase LUC-parasites and animals were imaged at the indicated times. BLI of mice shows the typical liver infection followed by spleen parasite colonization. By day 40, 3 mice with different levels of spleen infection were treated with miltefosine during 5 days (+) and reimaged 2 days later. Miltefosine efficacy was illustrated by the loss of luminescence signal compared to a control non treated mouse. Efficacy was confirmed by parasite counts (number of parasites/mg organ) measured by ELISA following dissection (indicated below each mouse tested). The illustration is representative of three different experiments. B. In vitro drug screening. PMA-differentiated THP-1 cells in 96 well microplates were incubated for 3 h with stationary phase LUC-parasites at a parasite-to-THP-1 cell ratio of 10∶1. After 48 h incubation, miltefosine at concentrations ranging from 10⁻⁴ to 10⁻⁸ M were delivered. 48 h later, the plate was washed and wells were lysed with 50 µl of reporter lysis buffer. 20 µl of supernatant was assayed for luminescence activity and showed a clear negative correlation between the drug concentration and luminescence of parasites. IC 50 was measured at 1.5 µM. Cell viability measured by trypan blue exclusion was 83% and 96% for miltefosine concentration of 100 µM and 10 µM, respectively. The dose dependency of miltefosin efficacy on parasite killing is representative of 3 different experiments.

Figure 3. Localization of bioluminescent parasites in intra-abdominal mesenteric fat in BALB/c mice infected by IP route.

4 Mice were infected with 5×10⁸ of LUC-parasites by IP route. Infection was monitored by BLI. On day 40 post infection (Fig 3A), mice showed intraperitoneal parasites localisation by BLI. One representative mouse (Fig 3B) was dissected and the adipose tissue localization detected by bioluminescence was verified in situ (Fig 3C) and after removing the adipose tissue and reimaging (Fig 3D). The illustrations are representative of at least three different experiments.