Transcriptional Shift and Metabolic Adaptations during Leishmania Quiescence Using Stationary Phase and Drug Pressure as Models

Abstract

Microorganisms can adopt a quiescent physiological condition which acts as a survival strategy under unfavorable conditions. Quiescent cells are characterized by slow or non-proliferation and a deep downregulation of processes related to biosynthesis. Although quiescence has been described mostly in bacteria, this survival skill is widespread, including in eukaryotic microorganisms. In Leishmania, a digenetic parasitic protozoan that causes a major infectious disease, quiescence has been demonstrated, but the molecular and metabolic features enabling its maintenance are unknown. Here, we quantified the transcriptome and metabolome of Leishmania promastigotes and amastigotes where quiescence was induced in vitro either, through drug pressure or by stationary phase. Quiescent cells have a global and coordinated reduction in overall transcription, with levels dropping to as low as 0.4% of those in proliferating cells. However, a subset of transcripts did not follow this trend and were relatively upregulated in quiescent populations, including those encoding membrane components, such as amastins and GP63, or processes like autophagy. The metabolome followed a similar trend of overall downregulation albeit to a lesser magnitude than the transcriptome. It is noteworthy that among the commonly upregulated metabolites were those involved in carbon sources as an alternative to glucose. This first integrated two omics layers afford novel insight into cell regulation and show commonly modulated features across stimuli and stages.

Unveiling drug tolerant and persister-like cells inLeishmania braziliensislines derived from patients with cutaneous leishmaniasis

“…Secondly, in vitro, we showed that a strain of L. lainsoni entered in quiescence under stationary phase or antimony pressure. Several transcriptional signatures were shared by the parasites surviving to both environmental insults: among these, a series of transcripts were present in higher abundance, in a general background of transcriptional shift (Jara et al, 2022). These molecular similarities validated the quiescent character of Leishmania under antimony pressure.…”

“…Nine of them were selected for present study, according to the following inclusion criteria: (i) first episode of leishmaniasis, (ii) cutaneous form of the disease, (iii) no previous treatment before attending to the clinic, (iv) complete therapy with antimonial post-diagnosis, (v) no concomitant disease and (vi) infecting species typed as Leishmania braziliensis. One strain of Leishmania lainsoni was included in the study as control: this is a line highly susceptible to antimonials, for which the existence of quiescence after PAT exposure was demonstrated (Jara et al, 2022). For each isolate, enhanced green fluorescent protein (EGFP) was integrated within the 18S ribosomal DNA locus, further called rEGFP with the use of the pLEXSY-hyg system (Jena Bioscience) as previously reported elsewhere (Bolhassani et al, 2011;Jara et al, 2022).…”

“…One strain of Leishmania lainsoni was included in the study as control: this is a line highly susceptible to antimonials, for which the existence of quiescence after PAT exposure was demonstrated (Jara et al, 2022). For each isolate, enhanced green fluorescent protein (EGFP) was integrated within the 18S ribosomal DNA locus, further called rEGFP with the use of the pLEXSY-hyg system (Jena Bioscience) as previously reported elsewhere (Bolhassani et al, 2011;Jara et al, 2022). After the generation of the transgenic rEGFP parasites, clones were obtained with the 'micro-drop' method, as described elsewhere (Van Meirvenne et al, 1975;Jara et al, 2022).…”

“…For each isolate, enhanced green fluorescent protein (EGFP) was integrated within the 18S ribosomal DNA locus, further called rEGFP with the use of the pLEXSY-hyg system (Jena Bioscience) as previously reported elsewhere (Bolhassani et al, 2011;Jara et al, 2022). After the generation of the transgenic rEGFP parasites, clones were obtained with the 'micro-drop' method, as described elsewhere (Van Meirvenne et al, 1975;Jara et al, 2022). All the work presented here was made with these clones, further called rEGFP strains.…”

“…Exponentially proliferating cells (promastigotes and amastigotes) were expose to PAT at concentration ranging from 1 to 83 µg/ mL and both the cell viability and the rEGFP expression were measured by flow cytometry at three time points: before drug exposure and after drug pressure at 24 and 48 hrs. The rEGFP expression is a negative marker of quiescence: highly expressed in proliferative cells and downregulated during quiescence (Jara et al, 2019(Jara et al, , 2022. The cell viability was evaluated by preincubating cells with the NucRed dead 647 (Thermo fisher scientific) for the staining of dead cells and Vybrant Dye Cycle violet (Thermo fisher scientific) for the staining of DNA in all cells.…”