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 ⌽SA012 possesses at least two RBPs. Genomic analysis of five mutant phages of ⌽SA012 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 by ⌽SA012 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. IMPORTANCE Staphylococcus phages belonging to the genus Staphylococcus aureus, a Gram-positive coccus, is a commensal and pathogenic bacterium that causes opportunistic infections in humans and animals. Currently, antibiotic resistance in this species poses a threat to public health. Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections around the world (1). The emergence of such antibiotic-resistant bacteria requires development of alternatives to antibiotic-based therapies. One promising alternative is phage therapy, in which bacteriophages (phages) are used to treat bacterial infections (2, 3). In preclinical trials performed in mice, S. aureus infections (including MRSA infections) were successfully treated by use of phages (4). Therefore, phage therapy has attracted great interest as an alternative to antibiotics.Previously, we isolated the virulent staphylococcal phage ⌽SA012, which exerts lytic effects on a wide range of S. aureus isolates from bovine mastitis cases (5). Genomic analysis of ⌽SA012 revealed that it belongs to the genus Twortlikevirus, which contains the Staphylococcus phages Twort, K, and G, whereas the genus SPO1likevirus contains the Bacillus phage SPO1 as well as Listeria phages P100 and A511. Lactobacillus phage LP65 and Enterococcus phage ⌽EF24C were classified as orphans within the subfamily (6). Representatives of the two genera share the following features: they (i) hav...

Analysis of phage-resistant mechanisms inStaphylococcus aureusSA003 reveals a different binding mechanism for the closely related Twort-like phages ϕSA012 and ϕSA039

“…As we described in a previous study, the mutant phage ɸSA012M20 carries a point mutation in 533 orf99, and subsequently accumulates two more point mutations to become the mutant phage 534 ɸSA012M38 (Takeuchi et al 2016), which may indicate the importance of orf99 in phage-host 535 interaction. Therefore, the different in orf96 of ɸSA039 may contribute to the specificity 536 different.…”

“…Only large-genome size phage (termed Jumbo phage), with genome 456 larger than 200 kbp, have been reported to have more than one paralogous gene for DNA 457 polymerase and RNA polymerase (RNAP) (Yuan and Gao 2017). Our phage in this study has a 458 genome size around 142 kbp and the complete set paralogous of RNA polymerase subunit was 459 not found (Takeuchi et al 2016). Therefore this phage may use host RNA polymerase as 460 machinery for their DNA transcription.…”

“…The phenotype as a result of coevolutionary adaptation of host and phage has previously 80 been observed and explained in our previous report (Takeuchi et al 2016;Osada et al 2017). In 81 this study, we focused on genetic analysis of the host cell by whole genome sequencing (WGS).…”

“…EOP with antibodies specific for ϕSA012 ORF103 (anti-ORF103) and ORF105 (anti-212 ORF105) was conducted according to a previous study (Takeuchi et al 2016). 213…”

“…The mutation in SA003R2 was followed by a decrease in total phosphate from 401 extracted WTA, which may indicate the alteration of WTA production. However, ɸSA012 has 402 been shown to have at least two RBPs (ORF103 and ORF105) (Takeuchi et al 2016). Therefore, 403 the phage may alter one of them to enhance the binding ability to its host.…”