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.

Evaluating Aziridinyl Nitrobenzamide Compounds as Leishmanicidal Prodrugs

“…4). In all cases, the ANBs screened had no growth-inhibitory effect against this mammalian line at concentrations of up to 100 M. Comparison of the mammalian cell IC 50 with the equivalent value obtained against amastigote parasites revealed that CB1964 and NH11 displayed Ͼ2,000-and Ͼ1,667-fold selective toxicity toward the intracellular pathogen, respectively. This clearly demonstrates that the antiparasitic activity displayed by these two compounds was specifically due to growth inhibition of L. major and not due to induction of host cell toxicity.…”

“…This screening demonstrated that cells expressing lower levels of the oxidoreductase were between 2-and 4-fold more resistant to the ANB than controls; wild-type L. major had IC 50 …”

“…Of these, the luciferase-based systems have proven to be sensitive, rapid, reproducible, and versatile, with tagged parasites now being used to follow the fate of the pathogen during the course of an animal model infection (49)(50)(51)(52). Here, we constructed a luciferase-expressing L. major line making use of an existing T. cruzi integration vector, modifying the expression construct such that it would integrate into the leishmanial ribosomal array (39).…”

“…The luminescence was proportional to the number of live cells. The IC 50 for each compound was then established.…”

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“…This 'bioluminescent approach' highlighted the well-characterized liverto-spleen parasite colonization. Furthermore, this model was used to study the effect of miltefosine, showing that 5 days after treatment both parasite load and the luminescence signal were undetectable in spleen or lymph nodes [29].…”