Abstract：
Polymicrobial infections involving various combinations of microorganisms, such as Escherichia, Pseudomonas, or Yersinia, can lead to acute and chronic diseases in for example the gastrointestinal and respiratory tracts. Our aim is to modulate microbial communities by targeting the posttranscriptional regulator system called carbon storage regulator A (CsrA) (or also repressor of secondary metabolites (RsmA)). In previous studies, we identified easily accessible CsrA binding scaffolds and macrocyclic CsrA binding peptides through biophysical screening and phage display technology. However, due to the lack of an appropriate in bacterio assay to evaluate the cellular effects of these inhibitor hits, the focus of the present study is to establish an in bacterio assay capable of probing and quantifying the impact on CsrA-regulated cellular mechanisms. We have successfully developed an assay based on a luciferase reporter gene assay, which in combination with a qPCR expression gene assay, allows for the monitoring of expression levels of different downstream targets of CsrA. The chaperone protein CesT was used as a suitable positive control for the assay, and in time-dependent experiments, we observed a CesT-mediated increase in bioluminescence over time. By this means, the cellular on-target effects of non-bactericidal/non-bacteriostatic virulence modulating compounds targeting CsrA/RsmA can be evaluated.
摘要：
涉及各种微生物组合的多微生物感染，如埃希氏菌，假单胞菌,或者耶尔森氏菌,可导致例如胃肠道和呼吸道的急性和慢性疾病。我们的目标是通过靶向称为碳储存调节因子A（CsrA）（或次级代谢产物的阻遏物（RsmA））的转录后调节系统来调节微生物群落。在以往的研究中,我们通过生物物理筛选和噬菌体展示技术鉴定了易于获得的CsrA结合支架和大环CsrA结合肽。然而,由于缺乏适当的细菌测定法来评估这些抑制剂的细胞作用，本研究的重点是建立一种能够探测和量化对CsrA调节的细胞机制的影响的细菌学试验。我们已经成功开发了一种基于荧光素酶报告基因检测的检测方法，结合qPCR表达基因测定，允许监测CsrA的不同下游靶标的表达水平。伴侣蛋白CesT用作测定的合适阳性对照，在依赖于时间的实验中，我们观察到CesT介导的生物发光随时间增加。通过这种方式，可以评估靶向CsrA/RsmA的非杀菌/非抑菌毒力调节化合物的细胞中靶效应。
