The Presence of Two Receptor-Binding Proteins Contributes to the Wide Host Range of Staphylococcal Twort-Like Phages

Abstract

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 phage ΦSA012 possesses at least two RBPs. Genomic analysis of five mutant phages of ΦSA012 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 α-N-acetylglucosamine (α-GlcNAc). A polyclonal antibody against ORF103 also inhibited infection by ΦSA012 in the presence of α-GlcNAc, suggesting that ORF103 binds to α-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 α-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 α-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.

Importance: Staphylococcus phages belonging to the genus Twortlikevirus (called staphylococcal Twort-like phages) are considered promising agents for control of Staphylococcus aureus due to their wide host range and highly lytic capabilities. Although staphylococcal Twort-like phages have been studied widely for therapeutic purposes, the host recognition process of staphylococcal Twort-like phages remains unclear. This work provides new findings about the mechanisms of host recognition of the staphylococcal Twort-like phage ΦSA012. The details of the host recognition mechanism of ΦSA012 will allow us to analyze the mechanisms of infection and expand the utility of staphylococcal Twort-like phages for the control of S. aureus.

FIG 1

Comparisons of main structural proteins among Twortlikevirus and related phages. Host bacteria for each phage are as follows: A511, Listeria monocytogenes; ΦSA012, K, and ISP, Staphylococcus aureus; SPO1, Bacillus subtilis; and ΦEF24C, Enterococcus faecalis. Asterisks (*) represent the locations of mutations. Gp108 in A511 is an RBP (25). The figure was generated using GenomeMatcher (59).

FIG 2

Spot tests of phages on S. aureus strains. Two microliters of concentrated phage lysate (10⁷ to 10¹⁰ PFU/ml) was dropped onto an LB plate overlaid with S. aureus cells. (A) SA003 (ΔtarM); (B) RN4220; (C) RN4220 ΔtarM; (D) RN4220 ΔtarM::pLIP3_tarM. w, wild type.

FIG 3

Adsorption efficiencies with 10⁸ CFU/ml of S. aureus strains in 60 min. Error bars indicate standard deviations (SD). Three biological replicates were conducted. **, P < 0.01; ns, not significant.

FIG 4

EOP of ΦSA012 with anti-ORF103 and anti-ORF105 antibodies. Preimmune serum (pre-serum) was used in controls. The number of plaques observed in the presence of preimmune serum was set as 100%. Plaques were not detected (ND) in the presence of anti-ORF105 serum. Three biological replicates were conducted. Error bars indicate SD. *, P < 0.05; **, P < 0.01; ns, not significant.

FIG 5

Localization of ORF103 in ΦSA012 stained with a gold-conjugated secondary antibody. Bars, 100 nm (top) and 50 nm (bottom). The size of the gold particles is 12 nm. Black spots in the images represent gold particles conjugated to the secondary antibody.

FIG 6

Effects of mutations in ORF103. (A) Spot tests and adsorption assays with ΦSA012-resistant strains. Upper and lower panels show the results of adsorption assays (10⁹ CFU/ml of cells in 270 min) and spot tests, respectively, with SA003R11 (a) and SA003R20 (b). Three or five biological replicates were conducted. Error bars indicate SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) EOP of ΦSA012M20 with anti-ORF013 and anti-ORF105. Preimmune serum was used in controls. The number of plaques formed in the presence of preimmune serum was defined as 100%. ND, not detected. Three biological replicates were conducted. Error bars indicate SD. ns, not significant.

FIG 7

Scheme of putative adsorption mechanism of ΦSA012. In most S. aureus strains, the WTA polymer consists of 11 to 40 ribitol phosphate (RboP) repeats substituted with d-alanine and α- and β-GlcNAc, covalently attached to a peptidoglycan with a WTA linkage unit. ΦSA012 recognizes the WTA backbone. α-GlcNAc masks the WTA backbone, which is the binding site of the primary RBP ORF105, and ORF103 helps the phage to bind the WTA backbone by binding α-GlcNAc. Hence, a phage in which ORF103 is disabled by point mutations or antibodies cannot infect S. aureus strains in the presence of α-GlcNAc, whereas the loss of ORF103 function does not affect phage infection in the absence of α-GlcNAc.