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...

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.

A major factor in the resilience of Listeria monocytogenes is the alternative sigma factor B (σB). Type II Toxin/Antitoxin (TA) systems are also known to have a role in the bacterial stress response upon activation via the ClpP or Lon proteases. Directly upstream of the σB operon in L. monocytogenes is the TA system mazEF, which can cleave mRNA at UACMU sites. In this study, we showed that the mazEF TA locus does not affect the level of persister formation during treatment with antibiotics in lethal doses, but exerts different effects according to the sub-inhibitory stress added. Growth of a ΔmazEF mutant was enhanced relative to the wildtype in the presence of sub-inhibitory norfloxacin and at 42 °C, but was decreased when challenged with ampicillin and gentamicin. In contrast to studies in Staphylococcus aureus, we found that the mazEF locus did not affect transcription of genes within the σB operon, but MazEF effected the expression of the σB-dependent genes opuCA and lmo0880, with a 0.22 and 0.05 fold change, respectively, compared to the wildtype under sub-inhibitory norfloxacin conditions. How exactly this system operates remains an open question, however, our data indicates it is not analogous to the system of S. aureus, suggesting a novel mode of action for MazEF in L. monocytogenes.

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