Thanks to their wide host range and virulence, staphylococcal bacteriophages (phages) belonging to the genus Twortlikevirus
(staphylococcal Twort-like phages) are regarded as ideal candidates for clinical application for Staphylococcus aureus infections
due to the emergence of antibiotic-resistant bacteria of this species. To increase the usability of these phages, it is necessary to
understand the mechanism underlying host recognition, especially the receptor-binding proteins (RBPs) that determine host
range. In this study, we found that the staphylococcal Twort-like phageSA012 possesses at least two RBPs. Genomic analysis of
five mutant phages ofSA012 revealed point mutations in orf103, in a region unique to staphylococcal Twort-like phages.
Phages harboring mutated ORF103 could not infect S. aureus strains in which wall teichoic acids (WTAs) are glycosylated with
-N-acetylglucosamine (-GlcNAc). A polyclonal antibody against ORF103 also inhibited infection bySA012 in the presence
of -GlcNAc, suggesting that ORF103 binds to -GlcNAc. In contrast, a polyclonal antibody against ORF105, a short tail fiber
component previously shown to be an RBP, inhibited phage infection irrespective of the presence of -GlcNAc. Immunoelectron
microscopy indicated that ORF103 is a tail fiber component localized at the bottom of the baseplate. From these results, we conclude
that ORF103 binds -GlcNAc in WTAs, whereas ORF105, the primary RBP, is likely to bind the WTA backbone. These
findings provide insight into the infection mechanism of staphylococcal Twort-like phages.