We set out to develop scalable assays to measure bacterial adhesion to mammalian extracellular matrix proteins, with the aim to perform high-throughput screening for inhibitors. Our model system is the trimeric autotransporter adhesin YadA from Yersinia enterocolitica that binds to collagen. Using bacterial cells expressing GFP under an inducible promotor, and co-expressing the adhesin of choice, we were able to establish a 384-well plate-based assay that allowed us to screen 28,000 compounds in 8 days (3520 compounds per day). We have collected all parameters that were essential in assay development, and describe how they can be tuned for improved performance. Out of 28,000 compounds, 5 compounds showed significant inhibitory activity, measured as loss of fluorescence compared to control wells. Our assay is easy to scale up, and can be adopted to different ECM component/Adhesin combinations. Alternatively, bacterial pathogens (harboring deletion mutants of adhesins compared to wildtype) could be used directly in the same assay if they express GFP as a reporter at high levels.
Keywords: Adhesin; Assay development; Bacterial adhesion; HTS; TAA; YadA.
© 2019 The Authors.