Identification and characterization of bacterial repeat-in-toxin adhesins using long-read genome analysis

Gram-negative bacteria attach to host surfaces using ligand-binding domains at the distal tips of fibrillar Repeats-in-ToXin adhesins. Blocking these initial interactions could prevent colonization, biofilm formation, and infection. To achieve this, the adhesins must be identified and, in species encoding multiple adhesins, the predominant type determined. These adhesins are often the largest proteins encoded by a genome (1,500-15,000 aa) and are frequently misannotated as incomplete or pseudogene products because their repetitive sequences complicate short-read genome assemblies. Our bioinformatic pipeline collects predicted proteins from long-read assemblies and clusters them according to similarity in their C-terminal regions, where ligand-binding domains are typically located. Adhesins are identified by their size and domain architecture and modelled using AlphaFold3. Analysis of multiple strains from seven species identified 35 adhesin isoforms distributed across 16 loci, exhibiting diverse combinations of putative binding domains such as carbohydrate-binding modules and von Willebrand factor A-like domains. Similar adhesins were sometimes shared among species through common ancestry or horizontal gene transfer. Three species encoded an adhesin of unknown function that lacked an obvious ligand-binding domain.