Establishment of an In Bacterio Assay for the Assessment of Carbon Storage Regulator A (CsrA) Inhibitors.

Autor: Wu Y; Department of Antiviral & Antivirulence Drugs (AVID), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, 66123, Saarbrücken, Germany.; Cluster of Excellence RESIST (EXC 2155), Hanover Medical School, Carl-Neuberg-Straße 1, 30625, Hanover, Germany., Zoller BGE; Department of Antiviral & Antivirulence Drugs (AVID), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, 66123, Saarbrücken, Germany., Kamal MAM; Department of Drug Delivery (DDEL), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, 66123, Saarbrücken, Germany.; Department of Pharmacy, Saarland University, 66123, Saarbrücken, Germany., Hotop SK; Department of Chemical Biology, Helmholtz Centre for Infection Research, German Center for Infection Research (DZIF), 38124, Braunschweig, Germany., Lehr CM; Department of Drug Delivery (DDEL), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, 66123, Saarbrücken, Germany.; Department of Pharmacy, Saarland University, 66123, Saarbrücken, Germany., Brönstrup M; Department of Chemical Biology, Helmholtz Centre for Infection Research, German Center for Infection Research (DZIF), 38124, Braunschweig, Germany., Dersch P; Institute of Infectiology, Center for Molecular Biology of Inflammation (ZMBE), University of Münster, 48149, Münster, Germany., Empting M; Department of Antiviral & Antivirulence Drugs (AVID), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, 66123, Saarbrücken, Germany.; Department of Pharmacy, Saarland University, 66123, Saarbrücken, Germany.; Cluster of Excellence RESIST (EXC 2155), Hanover Medical School, Carl-Neuberg-Straße 1, 30625, Hanover, Germany.
Jazyk: angličtina
Zdroj: Chembiochem : a European journal of chemical biology [Chembiochem] 2023 Aug 15; Vol. 24 (16), pp. e202300369. Date of Electronic Publication: 2023 Jul 25.
DOI: 10.1002/cbic.202300369
Abstrakt: 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.
(© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)
Databáze: MEDLINE
Externí odkaz: