1. Luciferase-expressing Leishmania infantum allows the monitoring of amastigote population size, in vivo, ex vivo and in vitro
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Emmanuel Lemichez, Pierre Marty, Madhavi P. Maddugoda, Patrick Munro, Grégory Michel, Christelle Pomares, Thierry Lang, Bernard Ferrua, Centre méditérannéen de médecine moléculaire (C3M), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunophysiologie et Parasitisme Intracellulaire, Institut Pasteur [Paris] (IP), Laboratoire de Parasitologie-Mycologie, Centre Hospitalier Universitaire de Nice (CHU Nice)-Hôpital l'Archet, This work was supported by the Foundation Infectiopole Sud, the Groupe d'Action Contre la Leishmaniose (GACL), the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Université de Nice Sophia-Antipolis, the Conseil Régional Provence-Alpes-Cote d'Azur, the Conseil Général des Alpes Maritimes for Photon Imager acquisition., Université Nice Sophia Antipolis (... - 2019) (UNS), Institut Pasteur [Paris], and Autard, Delphine
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Quantitative Parasitology ,MESH: Spleen ,Drug Evaluation, Preclinical ,Protozoan Proteins ,MESH: Spectrum Analysis ,Parasite Load ,Mice ,[SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,Whole Body Imaging ,MESH: Animals ,Leishmania infantum ,Luciferases ,MESH: Protozoan Proteins ,Mice, Inbred BALB C ,0303 health sciences ,MESH: Statistics, Nonparametric ,lcsh:Public aspects of medicine ,MESH: Leishmania infantum ,3. Good health ,MESH: Reproducibility of Results ,Infectious Diseases ,medicine.anatomical_structure ,Liver ,MESH: Phosphorylcholine ,[SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,Leishmaniasis, Visceral ,MESH: Drug Evaluation, Preclinical ,Female ,Research Article ,medicine.drug ,lcsh:Arctic medicine. Tropical medicine ,MESH: Organisms, Genetically Modified ,lcsh:RC955-962 ,Phosphorylcholine ,Antiprotozoal Agents ,MESH: Mice, Inbred BALB C ,Spleen ,Biology ,Microbiology ,Sensitivity and Specificity ,Statistics, Nonparametric ,03 medical and health sciences ,MESH: Whole Body Imaging ,In vivo ,medicine ,Animals ,Luciferase ,MESH: Antiprotozoal Agents ,Amastigote ,MESH: Mice ,MESH: Life Cycle Stages ,030304 developmental biology ,Life Cycle Stages ,Miltefosine ,Organisms, Genetically Modified ,030306 microbiology ,Spectrum Analysis ,Public Health, Environmental and Occupational Health ,Reproducibility of Results ,lcsh:RA1-1270 ,biology.organism_classification ,Virology ,Molecular biology ,In vitro ,MESH: Sensitivity and Specificity ,MESH: Parasite Load ,MESH: Luminescent Measurements ,MESH: Leishmaniasis, Visceral ,Luminescent Measurements ,Parasitology ,MESH: Luciferases ,MESH: Female ,Ex vivo ,MESH: Liver - 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., Author Summary Leishmania infantum/L. chagasi parasites are inoculated in the skin of mammals by sand flies. Though most often these L. infantum-mammal interactions are asymptomatic, they can proceed, in some individuals, to a systemic disease known as visceral leishmaniasis. If left untreated this disease is fatal. The lack of protective or curative vaccines and the limited number of parasite-targeting drugs were incentive to set up experimental conditions that could allow easy monitoring of the fluctuation of the population size of parasites in living laboratory animals. Thus, in the present report, we depict two distinct readout assays that rely on a population of L. infantum we genetically engineered for stably expressing the firefly luciferase gene. These transgenic parasites were either inoculated to BALB/c mice or added to a culture of monocytic cells. Post intravenous inoculation, BALB/c mice were imaged over time, with special attention being given to the liver and the spleen. The sensitivity of this technique ranged from 20,000 to 40,000 parasites/mg of tissue and from 1,000 to 6,000 parasites/mg tissue, for in vivo and ex vivo measurements, respectively. Though preliminary, the data, relying on monocytic cells, are promising for further in vitro screening of small compound libraries.
- Published
- 2011
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