Laboratorios de Investigación y Desarrollo de la Facultad de Ciencias y Filosofía & Instituto de Medicina Tropical "Alexander von Humboldt", Universidad Peruana Cayetano Heredia, Lima, Peru.
Institute of Tropical Medicine Antwerp, Molecular Parasitology Unit, Antwerp, Belgium.
Sci Rep. 2019 Dec 12;9(1):18951. doi: 10.1038/s41598-019-55486-z.
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.
在应激条件下，一些微生物会进入休眠状态，其特征是可逆的非增殖或缓慢增殖状态，使其能够存活。这种适应现象最近才在利什曼原虫中被发现。我们开发了一种体外模型和生物传感器来跟踪群体和单细胞水平的休眠状态。该生物传感器是一个整合在 18S rDNA 基因座内的 GFP 报告基因，可监测 18S rRNA（rGFP 表达）的表达。我们发现，在从细胞外前鞭毛体向细胞内无鞭毛体的转变过程中，rGFP 表达显著且迅速下降，并且在体外与 BrdU 掺入测量的复制减少相关。rGFP 表达可用于跟踪活细胞中休眠的可逆性，并首次显示了在潜伏感染的动物模型中，无鞭毛体群体中生理阶段的异质性，其中可能同时存在浅休眠和深休眠阶段。我们还验证了 rGFP 表达作为生物传感器在潜伏感染动物模型中的用途。我们的模型和生物传感器应该能够进一步在代谢和分子水平上对休眠进行特征描述。
