A Targeted Genome-wide Approach to Elucidate and Control Bacterial Adhesion to Physicochemically Diverse Biomaterials
Escherichia coli is the leading cause of catheter-associated urinary tract infections, caused by biofilm formation on implanted biomaterial surfaces. Understanding the genes that cause cellular adhesion to diverse biomaterial surfaces may aid in the design of targeting anti-biofouling chemicals to prevent these biofilm infections, but our current knowledge on such surfaces is limited. Here, we incorporate a platform of six biomaterials of varying hydrophilicities and stiffnesses and a comprehensive genome-wide CRISPR interference (CRISPRi) library to elucidate genotype-phenotype relationships for cellular adhesion to physicochemically varied biomaterial surfaces in E. coli MG1655. After characterization of the biomaterials and CRISPRi tool, we designed a CRISPRi library of 34,315 unique designs targeting 99.0% of the genome in MG1655 (up to 8 designs per gene). We then performed pooled selections for adhesion to each biomaterial surface, elucidating over 400 novel gene hits to each biomaterial surface. Data analysis revealed greater correlations between biomaterials of the same hydrophilicity rather than stiffness, in addition to more gene hits associated with decreased adhesion across all six surfaces than increased adhesion. Monoclonal verification of select designs from the library exhibited strong correlations between results from the pooled selections and individual measurements for adhesion to each gel for most designs. The results from this study provide comprehensive gene sets for cellular adhesion to physicochemically diverse biomaterial surfaces that may be potential gene targets for the design of targeting anti-biofouling agents and offer insight that could be used for the design of “smart” biomaterials, both with potential to prevent biofilm infections. Overall design: Competitive CRISPR interference (CRISPRi) selections for cellular adhesion of an E. coli MG1655 pooled cell library to a biomaterial surface were performed in duplicate for polydimethylsiloxane (PDMS) and polyethylene glycol (PEG) biomaterials of three different stiffness regimes each (Soft, Medium, or Stiff). Selections were also performed for planktonic growth in stationary media above each biomaterial chemistry. The CRISPRi library contained 34,315 unique gRNA designs targeting 99.0% of all annotated genes in the genome of MG1655, with both the Sp-dCas9 protein and gRNA expressed from a plasmid with a p15a replicon. The change in distribution of each gRNA design in the library before and after selection was used to determine gRNA fitness for cellular adhesion to these physicochemically diverse biomaterials