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An essential role for the baseplate protein Gp45 in phage adsorption to Staphylococcus aureus.

ABSTRACT: Despite the importance of phages in driving horizontal gene transfer (HGT) among pathogenic bacteria, the underlying molecular mechanisms mediating phage adsorption to S. aureus are still unclear. Phage ?11 is a siphovirus with a high transducing efficiency. Here, we show that the tail protein Gp45 localized within the ?11 baseplate. Phage ?11 was efficiently neutralized by anti-Gp45 serum, and its adsorption to host cells was inhibited by recombinant Gp45 in a dose-dependent manner. Flow cytometry analysis demonstrated that biotin-labelled Gp45 efficiently stained the wild-type S. aureus cell but not the double knockout mutant ?tarM/S, which lacks both ?- and ?-O-GlcNAc residues on its wall teichoic acids (WTAs). Additionally, adsorption assays indicate that GlcNAc residues on WTAs and O-acetyl groups at the 6-position of muramic acid residues in peptidoglycan are essential components of the ?11 receptor. The elucidation of Gp45-involved molecular interactions not only broadens our understanding of siphovirus-mediated HGT, but also lays the groundwork for the development of sensitive affinity-based diagnostics and therapeutics for S. aureus infection.

SUBMITTER: Li X

PROVIDER: S-EPMC4876445 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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An essential role for the baseplate protein Gp45 in phage adsorption to Staphylococcus aureus.

Li Xuehua X Koç Cengiz C Kühner Petra P Stierhof York-Dieter YD Krismer Bernhard B Enright Mark C MC Penadés José R JR Wolz Christiane C Stehle Thilo T Cambillau Christian C Peschel Andreas A Xia Guoqing G

Scientific reports 20160523

Despite the importance of phages in driving horizontal gene transfer (HGT) among pathogenic bacteria, the underlying molecular mechanisms mediating phage adsorption to S. aureus are still unclear. Phage ϕ11 is a siphovirus with a high transducing efficiency. Here, we show that the tail protein Gp45 localized within the ϕ11 baseplate. Phage ϕ11 was efficiently neutralized by anti-Gp45 serum, and its adsorption to host cells was inhibited by recombinant Gp45 in a dose-dependent manner. Flow cytome ...[more]

PMID: 27212064

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Project description:BackgroundSpontaneous Bacillus anthracis mutants resistant to infection by phage AP50c (AP50R) exhibit a mucoid colony phenotype and secrete an extracellular matrix.MethodsHere we utilized a Roche/454-based whole genome sequencing approach to identify mutations that are candidates for conferring AP50c phage resistance, followed by genetic deletion and complementation studies to validate the whole genome sequence data and demonstrate that the implicated gene is necessary for AP50c phage infection.ResultsUsing whole genome sequence data, we mapped the relevant mutations in six AP50R strains to csaB. Eleven additional spontaneous mutants, isolated in two different genetic backgrounds, were screened by PCR followed by Sanger sequencing of the csaB gene. In each spontaneous mutant, we found either a non-synonymous substitution, a nonsense mutation, or a frame-shift mutation caused by single nucleotide polymorphisms or a 5 base pair insertion in csaB. All together, 5 and 12 of the 17 spontaneous mutations are predicted to yield altered full length and truncated CsaB proteins respectively. As expected from these results, a targeted deletion or frame-shift mutations introduced into csaB in a different genetic background, in a strain not exposed to AP50c, resulted in a phage resistant phenotype. Also, substitution of a highly conserved histidine residue with an alanine residue (H270A) in CsaB resulted in phage resistance, suggesting that a functional CsaB is necessary for phage sensitivity. Conversely, introduction of the wild type allele of csaB in cis into the csaB deletion mutant by homologous recombination or supplying the wild type CsaB protein in trans from a plasmid restored phage sensitivity. The csaB mutants accumulated cell wall material and appeared to have a defective S-layer, whereas these phenotypes were reverted in the complemented strains.ConclusionsTaken together, these data suggest an essential role for csaB in AP50c phage infection, most likely in phage adsorption. (The whole genome sequences generated from this study have been submitted to GenBank under SRA project ID: SRA023659.1 and sample IDs: AP50 R1: SRS113675.1, AP50 R2: SRS113676.1, AP50 R3: SRS113728.1, AP50 R4: SRS113733.1, AP50 R6: SRS113734.1, JB220 Parent: SRS150209.1, JB220 Mutant: SRS150211.1).

Project description:Phages and bacteria have a long history of co-evolution. However, these dynamics of phage-host interactions are still largely unknown; identification of phage inhibitors that remodel host metabolism will provide valuable information for target development for antimicrobials. Here, we perform a comprehensive screen for early-gene products of ΦNM1 that inhibit cell growth in Staphylococcus aureus. A small membrane protein, Gp11, with inhibitory effects on S. aureus cell division was identified. A bacterial two-hybrid library containing 345 essential S. aureus genes was constructed to screen for targets of Gp11, and Gp11 was found to interact with MurG and DivIC. Defects in cell growth and division caused by Gp11 were dependent on MurG and DivIC, which was further confirmed using CRISPRi hypersensitivity assay. Gp11 interacts with MurG, the protein essential for cell wall formation, by inhibiting the production of lipid II to regulate peptidoglycan (PG) biosynthesis on the cell membrane. Gp11 also interacts with cell division protein DivIC, an essential part of the division machinery necessary for septal cell wall assembly, to disrupt the recruitment of division protein FtsW. Mutations in Gp11 result in loss of its ability to cause growth defects, whereas infection with phage in which the gp11 gene has been deleted showed a significant increase in lipid II production in S. aureus. Together, our findings reveal that a phage early-gene product interacts with essential host proteins to disrupt PG biosynthesis and block S. aureus cell division, suggesting a potential pathway for the development of therapeutic approaches to treat pathogenic bacterial infections.ImportanceUnderstanding the interplay between phages and their hosts is important for the development of novel therapies against pathogenic bacteria. Although phages have been used to control methicillin-resistant Staphylococcus aureus infections, our knowledge related to the processes in the early stages of phage infection is still limited. Owing to the fact that most of the phage early proteins have been classified as hypothetical proteins with uncertain functions, we screened phage early-gene products that inhibit cell growth in S. aureus, and one protein, Gp11, selectively targets essential host genes to block the synthesis of the peptidoglycan component lipid II, ultimately leading to cell growth arrest in S. aureus. Our study provides a novel insight into the strategy by which Gp11 blocks essential host cellular metabolism to influence phage-host interaction. Importantly, dissecting the interactions between phages and host cells will contribute to the development of new and effective therapies to treat bacterial infections.

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An essential role for the baseplate protein Gp45 in phage adsorption to Staphylococcus aureus.

Publications

An essential role for the baseplate protein Gp45 in phage adsorption to Staphylococcus aureus.

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