Tracking of quiescence in Leishmania by quantifying the expression of GFP in the ribosomal DNA locus

Abstract

Under stressful conditions some microorganisms adopt a reversible non or slow proliferative quiescent stage that allows their survival. Although quiescence has been described broadly in bacteria, this phenotype has been only recently discovered in Leishmania. In the present work we developed a biosensor of quiescence that allows to monitor the physiological stage of the parasite at population and single cell levels. We inserted a GFP gene into the ribosomal DNA locus and followed the expression of this reporter gene, driven by the ribosomal promotor (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during the in vitro transition from extracellular promastigotes to intracellular amastigotes of L. mexicana an L. braziliensis and that the decrease in rGFP expression was coupled in vitro with a decrease in replication as measured by BrdU incorporation. Quiescence was not only observed in reference laboratory strains, but also among clinical isolates. We found that quiescence was reversible as the parasites could rapidly resume their metabolically active and proliferative stage when they were put back in an optimal environment for growth. We demonstrated for the first time in live cells that amastigotes are a heterogeneous population in which shallow and deep quiescent stages may coexist. Finally, we showed that rGFP expression could be monitored in vivo and that quiescent amastigotes could reside in tissues of animals with latent infections of L. braziliensis or L. mexicana. We propose rGFP expression as a simple parameter to define quiescent cells and further characterize them.\n\nIMPORTANCEQuiescence is a physiological diversification that allows pathogens to overcome chemotherapy without the development of drug resistance and to be invisible to the immune system of their host. Quiescent pathogens can cause latent infections and (re-) emerge in an unpredictable time during the lifetime of the individual. The phenomenon was recently described in Leishmania in which it could explain several clinical and sub-clinical features, like therapeutic failure, reactivation of the disease and asymptomatic infections. However, a simple biosensor of quiescence for Leishmania is not yet available. We show for the first time that the integration of GFP within the rDNA locus and the subsequent quantification of its expression can be used as a biosensor to distinguish quiescent subpopulations among live amastigotes. Moreover, we show quiescence is quickly reversible both in vivo and in vitro. We offer a tool that will allow the further molecular characterization of quiescent parasites.