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

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.

Pharmacokinetics and pharmacodynamics in the treatment of cutaneous leishmaniasis – challenges and opportunities

“…95 The usage of the elegant biosensor Leishmania model that has a GFP reporter gene integrated within the 18S rDNA allows monitoring of the expression of 18S rRNA to test drug efficacy within quiescent populations. 96 Digital spatial profiling (DSP). DSP is a recently developed technique that allows multiplex and spatial detection and quantification of proteins or RNAs on formalin-fixed, paraffin-embedded samples.…”

Leishmania: Responding to environmental signals and challenges without regulated transcription

“…Axenically induced amastigote differentiation coincided with reduced overall translation and phosphorylation of translation factor eIF2α [45] . Reduction of protein synthesis in amastigotes may reflect the adaptation to the reduced proliferation rate observed for the intracellular stage [46] . Furthermore, it is known for eukaryotes that stress causes a switch from cap-dependent to cap-independent translation.…”

Trypanosomatid-Caused Conditions: State of the Art of Therapeutics and Potential Applications of Lipid-Based Nanocarriers

“…Therefore, novel therapeutic options are urgently required to improve both accessibility and therapeutic output and control potential resistance issues. Interestingly, the existence of quiescent and dormant stages (characterized by low or no replication and diminished metabolic activity) has been described, respectively, in Leishmania and Trypanosoma cruzi (Berg et al, 2013;Kloehn et al, 2015;Jara et al, 2017Jara et al, , 2019Sánchez-Valdez et al, 2018). Depending on the case, such forms may emerge spontaneously (Sánchez-Valdez et al, 2018) or as an adaptive response to stress conditions (e.g., drug pressure) (Berg et al, 2013;Jara et al, 2019), contributing to treatment failure.…”

“…Interestingly, the existence of quiescent and dormant stages (characterized by low or no replication and diminished metabolic activity) has been described, respectively, in Leishmania and Trypanosoma cruzi (Berg et al, 2013;Kloehn et al, 2015;Jara et al, 2017Jara et al, , 2019Sánchez-Valdez et al, 2018). Depending on the case, such forms may emerge spontaneously (Sánchez-Valdez et al, 2018) or as an adaptive response to stress conditions (e.g., drug pressure) (Berg et al, 2013;Jara et al, 2019), contributing to treatment failure. It has been suggested that these new resistance mechanisms, which add to classical genetic-based drug resistance, may be treated using current drugs in extended therapeutic regimens that extend beyond the dormancy potential (Sánchez-Valdez et al, 2018).…”