Abstract
Under stressful conditions some microorganisms adopt a quiescent stage characterized by a reversible non or slow proliferative condition that allows their survival. This adaptation was only recently discovered in Leishmania. We developed an in vitro model and a biosensor to track quiescence at population and single cell levels. The biosensor is a GFP reporter gene integrated within the 18S rDNA locus, which allows monitoring the expression of 18S rRNA (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during the transition from extracellular promastigotes to intracellular amastigotes and that it was coupled in vitro with a decrease in replication as measured by BrdU incorporation. rGFP expression was useful to track the reversibility of quiescence in live cells and showed for the first time the heterogeneity of physiological stages among the population of amastigotes in which shallow and deep quiescent stages may coexist. We also validated the use of rGFP expression as a biosensor in animal models of latent infection. Our models and biosensor should allow further characterization of quiescence at metabolic and molecular level.
Highlights
Leishmania is a digenetic parasite involving two life stages
We postulated that if the GFP gene was integrated within the rDNA locus, the subsequent measurement of its expression would serve as a quiescence biosensor and would allow to discriminate quiescent populations among alive parasites
In L. braziliensis, the analysis at whole population level showed that rGFP expression in alive cells was the highest in the highly proliferative ProLog, with an average value of 1995.0 ± 5.7
Summary
Leishmania is a digenetic parasite involving two life stages. The motile promastigotes reside in the invertebrate sand fly and the non-motile amastigotes reside within the parasitophorous vacuole of the macrophage and other phagocytic cells[1–3]. The authors found that in persistent infections of the self-healing C57BL/6J mice model, there are two populations among amastigotes: one in a slow proliferative stage (generation time of 60 hours, semi quiescent) and another with no evidence of proliferation (quiescent). We used here GFP as a reporter gene and demonstrated in L. braziliensis and L. mexicana reference strains that it can be used as a biosensor of the proliferative stage of parasites and as a tool to distinguish quiescent stages among amastigotes at a single cell level Using this biosensor, we have characterized in vitro the heterogeneity of physiologically downregulated stages among Leishmania amastigotes and the kinetic of the entrance and exit of these stages. The results of experimental work in vivo and the evaluation of quiescence in a panel of clinical isolates are shown
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