Journal: Journal of Virology

Article Title: Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

doi: 10.1128/jvi.00810-25

Figure Lengend Snippet: Identification of phage-targeted receptor types. ( A ) Adsorption of phage GSP004 to S . Enteritidis SE006 and E. coli ATCC 35150 treated with periodate (IO 4 - ). ( B ) Adsorption of phage GSP004 to S . Enteritidis SE006 and E. coli ATCC 35150 treated with proteinase K. ( C ) Competitive inhibition of phage adsorption by LPS. Phage GSP004 was mixed with LPS (0–10 µg/mL) extracted from S . Enteritidis SE006 or E. coli ATCC 35150, and then its adsorption capacity to the host was assessed. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: To further validate LPS as a receptor for phage GSP004, we performed adsorption assays and plaque formation tests using SE006 and ATCC 35150 strains harboring deletions in the LPS biosynthesis genes rfaL and rfaC .

Techniques: Adsorption, Inhibition

Journal: Journal of Virology

Article Title: Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

doi: 10.1128/jvi.00810-25

Figure Lengend Snippet: LPS O-antigen is the target receptor for phage GSP004. ( A, B ) Adsorption capacity of phage GSP004 to Δ rfaL (LPS O-antigen-deficient) and Δ rfaC (LPS core-deficient) mutants of S . Enteritidis SE006 ( A ) or E. coli ATCC 35150 ( B ). ( C, D ) Lysis efficiency of phage GSP004 against Δ rfaL and Δ rfaC mutants of S . Enteritidis SE006 ( C ) or E. coli ATCC 35150 ( D ) on solid agar medium. ( E, F ) Lysis kinetics of phage GSP004 against S . Enteritidis SE006 ( E ) and E. coli ATCC 35150 ( F ) mutants (Δ rfaL , Δ rfaC ) in liquid culture. ( G ) Schematic diagram showing the structure of LPS and the various truncated mutants, modified from references ( , ). *** P < 0.001; ns, not significant.

Article Snippet: To further validate LPS as a receptor for phage GSP004, we performed adsorption assays and plaque formation tests using SE006 and ATCC 35150 strains harboring deletions in the LPS biosynthesis genes rfaL and rfaC .

Techniques: Adsorption, Lysis, Modification

Journal: Journal of Virology

Article Title: Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

doi: 10.1128/jvi.00810-25

Figure Lengend Snippet: In vitro DNA ejection from phage GSP004 particles. ( A, B ) Silver staining analysis of LPS. Lane 1, LPS extracted from wild-type strains SE006 ( A ) or ATCC 35150 ( B ). Lane 2, LPS from Δ rfaL mutants of SE006 ( A ) or ATCC 35150 ( B ). The positions of the fully synthesized LPS, containing the attached O-antigen and the lipid A-core oligosaccharide (OS) precursor, are indicated. ( C, D ) In vitro monitoring of LPS-induced DNA ejection. Phage GSP004 was incubated with LPS from S . Enteritidis SE006 ( C ) or E. coli ATCC 35150 ( D ), and DNA release was quantified using a fluorescent DNA-binding dye. Increased fluorescence intensity after LPS treatment and decreased after the addition of DNase I, confirming that DNA was released from phage particles. The standard deviations (SDs) from three independent experiments were less than 1% of total fluorescence for every experiment.

Article Snippet: To further validate LPS as a receptor for phage GSP004, we performed adsorption assays and plaque formation tests using SE006 and ATCC 35150 strains harboring deletions in the LPS biosynthesis genes rfaL and rfaC .

Techniques: In Vitro, Silver Staining, Synthesized, Incubation, Binding Assay, Fluorescence

Journal: Journal of Virology

Article Title: Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

doi: 10.1128/jvi.00810-25

Figure Lengend Snippet: Prediction and identification of the RBP from phage GSP004 . ( A ) Purification of recombinant ORF208 protein analyzed by SDS-PAGE with Coomassie blue staining. Lane M, Precision Plus Protein Dual Color Protein Marker (Bio-Rad). Lane 1, unpurified recombinant ORF208 protein. Lane 2, Ni-NTA affinity chromatography purified recombinant ORF208 protein. ( B ) Competitive binding assay of ORF208 protein to host bacteria. Pre-incubation of S. Enteritidis SE006 or E. coli ATCC 35150 with ORF208 reduced phage adsorption. Conversely, the addition of LPS restored adsorption by competing with ORF208 binding. ( C ) Antibody blocking assay. Pre-incubation of phage GSP004 with anti-ORF208 antibody significantly inhibited bacterial infectivity. ( D ) Fluorescence microscopy analysis of EGFP-tagged ORF208 binding to wild-type, Δ rfaL mutants, and Δ rfaL -complemented strains. EGFP-tagged ORF208 binds to wild-type and complemented strains, but not to Δ rfaL mutant. * P < 0.05, *** P < 0.001.

Article Snippet: To further validate LPS as a receptor for phage GSP004, we performed adsorption assays and plaque formation tests using SE006 and ATCC 35150 strains harboring deletions in the LPS biosynthesis genes rfaL and rfaC .

Techniques: Purification, Recombinant, SDS Page, Staining, Marker, Affinity Chromatography, Competitive Binding Assay, Bacteria, Incubation, Adsorption, Binding Assay, Antibody Blocking Assay, Infection, Fluorescence, Microscopy, Mutagenesis

Journal: Journal of Virology

Article Title: Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

doi: 10.1128/jvi.00810-25

Figure Lengend Snippet: Analysis and identification of the interaction mechanism of ORF208 protein with LPS receptor . ( A ) Domain architecture of ORF208. N-terminal domain of the phage G7C tail spike protein (TSP) is located at 90–155 aa and C-terminal domain of the phage P22 TSP is located at 161–697 aa. ( B ) Sequence alignment of ORF208 with tail spike proteins of phage P22 and 9NA. ( C ) The model of the ORF208 trimer was constructed using AlphaFold 2.0. Three monomers are each labeled with a different color. ( D, E ) LPS digestion assay. Lane 1, LPS from S . Enteritidis SE006 ( D ) or E. coli ATCC 35150 ( E ) incubated with heat-inactivated ORF208. Lane 2: LPS treated with active ORF208. The positions of the fully synthesized LPS with attached O-antigen and lipid A-core oligosaccharide (OS) precursor are indicated.

Article Snippet: To further validate LPS as a receptor for phage GSP004, we performed adsorption assays and plaque formation tests using SE006 and ATCC 35150 strains harboring deletions in the LPS biosynthesis genes rfaL and rfaC .

Techniques: Sequencing, Construct, Labeling, Incubation, Synthesized

Journal: Journal of Virology

Article Title: Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

doi: 10.1128/jvi.00810-25

Figure Lengend Snippet: Identification of phage-targeted receptor types. ( A ) Adsorption of phage GSP004 to S . Enteritidis SE006 and E. coli ATCC 35150 treated with periodate (IO 4 - ). ( B ) Adsorption of phage GSP004 to S . Enteritidis SE006 and E. coli ATCC 35150 treated with proteinase K. ( C ) Competitive inhibition of phage adsorption by LPS. Phage GSP004 was mixed with LPS (0–10 µg/mL) extracted from S . Enteritidis SE006 or E. coli ATCC 35150, and then its adsorption capacity to the host was assessed. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant.

Article Snippet: LPS was extracted from S . Enteritidis SE006 or E. coli ATCC 35150 using the LPS Extraction Kit (Bestbio, Shanghai, China) for subsequent experiments.

Techniques: Adsorption, Inhibition

Journal: Journal of Virology

Article Title: Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

doi: 10.1128/jvi.00810-25

Figure Lengend Snippet: LPS O-antigen is the target receptor for phage GSP004. ( A, B ) Adsorption capacity of phage GSP004 to Δ rfaL (LPS O-antigen-deficient) and Δ rfaC (LPS core-deficient) mutants of S . Enteritidis SE006 ( A ) or E. coli ATCC 35150 ( B ). ( C, D ) Lysis efficiency of phage GSP004 against Δ rfaL and Δ rfaC mutants of S . Enteritidis SE006 ( C ) or E. coli ATCC 35150 ( D ) on solid agar medium. ( E, F ) Lysis kinetics of phage GSP004 against S . Enteritidis SE006 ( E ) and E. coli ATCC 35150 ( F ) mutants (Δ rfaL , Δ rfaC ) in liquid culture. ( G ) Schematic diagram showing the structure of LPS and the various truncated mutants, modified from references ( , ). *** P < 0.001; ns, not significant.

Article Snippet: LPS was extracted from S . Enteritidis SE006 or E. coli ATCC 35150 using the LPS Extraction Kit (Bestbio, Shanghai, China) for subsequent experiments.

Techniques: Adsorption, Lysis, Modification

Journal: Journal of Virology

Article Title: Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

doi: 10.1128/jvi.00810-25

Figure Lengend Snippet: In vitro DNA ejection from phage GSP004 particles. ( A, B ) Silver staining analysis of LPS. Lane 1, LPS extracted from wild-type strains SE006 ( A ) or ATCC 35150 ( B ). Lane 2, LPS from Δ rfaL mutants of SE006 ( A ) or ATCC 35150 ( B ). The positions of the fully synthesized LPS, containing the attached O-antigen and the lipid A-core oligosaccharide (OS) precursor, are indicated. ( C, D ) In vitro monitoring of LPS-induced DNA ejection. Phage GSP004 was incubated with LPS from S . Enteritidis SE006 ( C ) or E. coli ATCC 35150 ( D ), and DNA release was quantified using a fluorescent DNA-binding dye. Increased fluorescence intensity after LPS treatment and decreased after the addition of DNase I, confirming that DNA was released from phage particles. The standard deviations (SDs) from three independent experiments were less than 1% of total fluorescence for every experiment.

Article Snippet: LPS was extracted from S . Enteritidis SE006 or E. coli ATCC 35150 using the LPS Extraction Kit (Bestbio, Shanghai, China) for subsequent experiments.

Techniques: In Vitro, Silver Staining, Synthesized, Incubation, Binding Assay, Fluorescence

Journal: Journal of Virology

Article Title: Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

doi: 10.1128/jvi.00810-25

Figure Lengend Snippet: Prediction and identification of the RBP from phage GSP004 . ( A ) Purification of recombinant ORF208 protein analyzed by SDS-PAGE with Coomassie blue staining. Lane M, Precision Plus Protein Dual Color Protein Marker (Bio-Rad). Lane 1, unpurified recombinant ORF208 protein. Lane 2, Ni-NTA affinity chromatography purified recombinant ORF208 protein. ( B ) Competitive binding assay of ORF208 protein to host bacteria. Pre-incubation of S. Enteritidis SE006 or E. coli ATCC 35150 with ORF208 reduced phage adsorption. Conversely, the addition of LPS restored adsorption by competing with ORF208 binding. ( C ) Antibody blocking assay. Pre-incubation of phage GSP004 with anti-ORF208 antibody significantly inhibited bacterial infectivity. ( D ) Fluorescence microscopy analysis of EGFP-tagged ORF208 binding to wild-type, Δ rfaL mutants, and Δ rfaL -complemented strains. EGFP-tagged ORF208 binds to wild-type and complemented strains, but not to Δ rfaL mutant. * P < 0.05, *** P < 0.001.

Article Snippet: LPS was extracted from S . Enteritidis SE006 or E. coli ATCC 35150 using the LPS Extraction Kit (Bestbio, Shanghai, China) for subsequent experiments.

Techniques: Purification, Recombinant, SDS Page, Staining, Marker, Affinity Chromatography, Competitive Binding Assay, Bacteria, Incubation, Adsorption, Binding Assay, Antibody Blocking Assay, Infection, Fluorescence, Microscopy, Mutagenesis

Journal: Journal of Virology

Article Title: Polyvalent phage GSP004 recognizes O-antigen polysaccharide receptors in Salmonella and Escherichia coli through tail fiber protein ORF208

doi: 10.1128/jvi.00810-25

Figure Lengend Snippet: Analysis and identification of the interaction mechanism of ORF208 protein with LPS receptor . ( A ) Domain architecture of ORF208. N-terminal domain of the phage G7C tail spike protein (TSP) is located at 90–155 aa and C-terminal domain of the phage P22 TSP is located at 161–697 aa. ( B ) Sequence alignment of ORF208 with tail spike proteins of phage P22 and 9NA. ( C ) The model of the ORF208 trimer was constructed using AlphaFold 2.0. Three monomers are each labeled with a different color. ( D, E ) LPS digestion assay. Lane 1, LPS from S . Enteritidis SE006 ( D ) or E. coli ATCC 35150 ( E ) incubated with heat-inactivated ORF208. Lane 2: LPS treated with active ORF208. The positions of the fully synthesized LPS with attached O-antigen and lipid A-core oligosaccharide (OS) precursor are indicated.

Article Snippet: LPS was extracted from S . Enteritidis SE006 or E. coli ATCC 35150 using the LPS Extraction Kit (Bestbio, Shanghai, China) for subsequent experiments.

Techniques: Sequencing, Construct, Labeling, Incubation, Synthesized