Abstract
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.
Highlights
Antimicrobial resistance in bacterial pathogens is a critical problem, and in recent years, hardly any novel antibiotics to overcome this issue have been brought to market
Wild type/commercial grade Wild type/commercial grade Wild type/commercial grade Top10 arsB::superfolder GFP (sfGFP) carried by J23100 constitutive promoter AS43 + pASK-IBA2 AS43 + pASK-IBA2-YadAwt Top10 glmS::sfGFP carried by J23100 constitutive promoter AS61 + pASK-IBA2 AS61 + pASK-IBA2-YadAwt AS61 + pASK-IBA2-YadAwt Top10 glmS::sfGFP carried by arabinose inducible promoter AS75 + pASK-IBA2-YadAwt BL21DE3 glmS::sfGFP carried by J23100 constitutive promoter AS89 + pASK-IBA2-YadAwt
When we investigated the impact of different sources of collagen type I (Fig. 3A), we observed that all collagen type I sources display similar binding of Yersinia enterocolitica Adhesin A (YadA)-expressing cells based on statistical significance analysis
Summary
Antimicrobial resistance in bacterial pathogens is a critical problem, and in recent years, hardly any novel antibiotics to overcome this issue have been brought to market. The future of antimicrobial therapy might lie in more targeted substances that inhibit only a subset of pathogenic bacteria, combined with better (pre-treatment) diagnostics (Spaulding et al, 2018). One family of adhesins exclusive to Gram-negative bacteria are the trimeric autotransporter adhesins (TAAs). They are essential for the pathogenesis of a number of Enterobacteriaceae, including Yersinia enterocolitica and Yersinia pseudotuberculosis (Mikula et al, 2013; Sabina et al, 2011), and appear to be important in various stages of pathogenesis in entero- and uropathogenic E. coli (Totsika et al, 2012), Salmonella (Raghunathan et al, 2011), and Proteus (Alamuri et al., 2010). Experimental approaches as well as sequence and structure comparisons show that the domain mainly responsible for binding to extracellular matrix molecules, and to collagen, is the socalled head domain of TAAs (Leo et al, 2012; Linke et al, 2006)
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