Question: Staphylococcus aureus can cause life-threatening infections that are often resistant to many different antibiotics. One way to combat these antibiotic resistant infections are bacteriophages. The host range of S. aureus phages is determined by the species-specific structure of wall teichoic acids (WTA), the only known S. aureus phage receptor. Nearly all S. aureus strains possess WTA consisting of ribitol-phosphate repeats. This study aims to investigate the binding capabilities of S. aureus ribitol-phosphate binding phages to their host cells through the identification of receptor-binding proteins (RBPs). Through this, we want to understand the underlying mechanism leading to adsorption and thus infection of the phage, which might enable us to predict the host range of S. aureus phages.

Methods: We used bioinformatic analysis to identify putative RBPs necessary for phage adsorption. Over 350 S. aureus phage genomes were analyzed to identify S. aureus ribitol-phosphate binding RBPs, which were then classified based on amino acid homology. Protein fusion constructs were created by addition of a fluorescent N-terminus to the phage RBPs, and the binding specificity of these proteins to different S. aureus WTA-mutants was investigated via flow cytometry and fluorescence microscopy.

Results: We found various RBPs necessary for phage adsorption and identified several different clusters of S. aureus ribitol-phosphate binding phages based on their predicted RBPs. Analysis of the binding specificity revealed distinct binding patterns for each RBP cluster. The created phage clusters allow prediction of phage adsorption to different WTA glycosylation types during the initial stage of phage infection.

Conclusion: This study provides insights into the host range of both known and novel phages that are useful for the development of phage-based therapeutics against S. aureus. The findings suggest that S. aureus ribitol-phosphate binding phages can be classified into different groups based on their RBPs, which can be used to predict their binding capabilities and success in phage adsorption to different WTA glycosylation types.