Towards modulation of polymicrobial communities in chronic lung diseases through targeting the Carbon Storage regulator A (CsrA)

Multidrug resistance is an ongoing problem in infectious diseases; especially considering difficult-to-treat Gram-negative pathogens. Additional complexity is provided by the polymicrobial nature of our respiratory and gastro-intestinal systems involving the beneficial microbiome, but also associated polymicrobial infections. Acute and chronic diseases are caused by a variety of pathogenic Gram-negative microorganisms like e.g. Escherichia Coli, Pseudomonas Aeruginosa or Yersinia Pseudotuberculosis. Investigating new modalities to achieve anti-virulence effects in this polymicrobial scenario is of high interest.

Our aim is to modulate microbial communities through addressing the post-translational regulator system called carbon storage regulator A (CsrA) in E. Coli and Y. Pseudotuberculosis. In P. aeruginosa, it is called repressor of secondary metabolites (RsmA). CsrA/RsmA is an astonishingly widespread bacterial RNA binding protein mediating global effects on translation by binding to GGA-motifs in mRNAs. We identified novel, easily accessible CsrA binding scaffolds through biophysical screening (Fluorescence Polarization competition assay) and a macrocyclic CsrA binding peptide via phage display technology. In order to find new binders, a commercial structurally diverse compound library was employed for a two-step screening procedure using pharmacophore-guided docking as an in silico prescreening filter and fluorescence polarization as a functional in vitro assay. By this means, we identified four new classes of CsrA binding scaffolds for medicinal chemistry optimization. However, only two classes were confirmed to be effective CsrA inhibitors via cell assays.

We aim to assess cellular effects of optimized inhibitors in a bacterial test system using qRT-PCR gene expression and Luciferase reporter gene assay methodolgies in E. Coli. These assays are still in development and further optimizations are needed. We also included transport studies to evaluate the accumulation of the potential scaffolds in the cells.