Analysis of phage resistance in Staphylococcus aureus SA003 reveals different binding mechanisms for the closely related Twort-like phages ɸSA012 and ɸSA039

Abstract

We have previously generated strains of Staphylococcus aureus SA003 resistant to its specific phage ɸSA012 through a long-term coevolution experiment. However, the DNA mutations responsible for the phenotypic change of phage resistance are unknown. Whole-genome analysis revealed eight genes that acquired mutations: six point mutations (five missense mutations and one nonsense mutation) and two deletions. Complementation of the phage-resistant strains by the wild-type alleles showed that five genes were linked to phage adsorption of ɸSA012, and two mutated host genes were linked to the inhibition of post-adsorption. Unlike ɸSA012, infection by ɸSA039, a close relative of ɸSA012, onto early coevolved phage-resistant SA003 (SA003R2) was impaired drastically. Here, we identified that ɸSA012 and ɸSA039 adsorb to the cell surface S. aureus SA003 through a different mechanism. ɸSA012 requires the backbone of wall teichoic acids (WTA), while ɸSA039 requires both backbone and the β-GlcNAc residue. In silico analysis of the ɸSA039 genome revealed that several proteins in the tail and baseplate region were different from ɸSA012. The difference in tail and baseplate proteins might be the factor for specificity difference between ɸSA012 and ɸSA039.

Analysis host-recognition mechanism of staphylococcal kayvirus ɸSA039 reveals a novel strategy that protects Staphylococcus aureus against infection by Staphylococcus pseudintermedius Siphoviridae phages

“…ɸSA039 requires β-GlcNAc glycosylation of WTA by TarS (Azam et al 2018). In this study, we found that ɸSA039 could generate spontaneous mutants capable of infecting TarS-null S. aureus .…”

“…The gene was deleted by pCasSA plasmid with clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system (Chen et al 2017). Plasmid construction was performed as previously described (Chen et al 2017; Azam et al 2018). Spacers were manually selected by searching the protospacer adjacent motif (PAM) region.…”

“…Most phages belonging to this group use the backbone of wall teichoic acid (WTA), the most abundant molecule in the cell wall of Staphylococcus , as their receptor (Xia et al 2011). However, previous work showed that kayvirus ɸSA039 uses the β-GlcNAc moiety in WTA of S. aureus SA003, a unique feature within the group (Azam et al 2018).…”

“…In addition, a previous study showed that kayviruses such as ɸSA012 and ɸK have at least two RBPs (RBP1 and RBP2), which are responsible for these phages’ broad host range (O’Flaherty et al 2004; Takeuchi et al 2016). Mutant ɸSA012 has a modified RBP2 (ORF103) that enables the phage to infect mutant S. aureus SA003R38, which has an altered WTA and overproduces capsular polysaccharide (Takeuchi et al 2016; Osada et al 2017; Azam et al 2018), indicating that mutated RBP2 may allow the phage to counter-adapt to resistant hosts by using an alternative component as a novel receptor (Takeuchi et al 2016). Like ɸSA012, ɸSA039 also has two RBPs, RBP1 (ORF100) and RBP2 (ORF102) (Azam et al 2018).…”

“…Mutant ɸSA012 has a modified RBP2 (ORF103) that enables the phage to infect mutant S. aureus SA003R38, which has an altered WTA and overproduces capsular polysaccharide (Takeuchi et al 2016; Osada et al 2017; Azam et al 2018), indicating that mutated RBP2 may allow the phage to counter-adapt to resistant hosts by using an alternative component as a novel receptor (Takeuchi et al 2016). Like ɸSA012, ɸSA039 also has two RBPs, RBP1 (ORF100) and RBP2 (ORF102) (Azam et al 2018). Hence, in light of the unique infection strategy of ɸSA039, we also investigated the ability of the phage to counter-adapt to phage-resistant host and analyzed the underlying mechanism.…”