Interactions of phage P22 tails with their cellular receptor, Salmonella O-antigen polysaccharide

Abstract

Bacteriophage P22 binds to its cell surface receptor, the repetitive O-antigen structure in Salmonella lipopolysaccharide, by its six homotrimeric tailspikes. Receptor binding by soluble tailspikes and the receptor-inactivating endorhamnosidase activity of the tailspike protein were studied using octa- and dodecasaccharides comprising two and three O-antigen repeats of Salmonella enteritidis and Salmonella typhimurium lipopolysaccharides. Wild-type tailspike protein and three mutants (D392N, D395N, and E359Q) with defective endorhamnosidase activity were used. Oligosaccharide binding to all three subunits, measured by a tryptophan fluorescence quench or by fluorescence depolarization of a coumarin label attached to the reducing end of the dodecasaccharide, occurs independently. At 10 degrees C, the binding affinities of all four proteins to oligosaccharides from both bacterial strains are identical within experimental error, and the binding constants for octa- and dodecasaccharides are 1 x 10(6) M(-1) and 2 x 10(6) M(-1), proving that two O-antigen repeats are sufficient for lipopolysaccharide recognition by the tailspike. Equilibration with the oligosaccharides occurs rapidly, but the endorhamnosidase produces only one cleavage every 100 s at 10 degrees C or about 2 min(-1) at the bacterial growth temperature. Thus, movement of virions in the lipopolysaccharide layer before DNA injection may involve the release and rebinding of individual tailspikes rather than hydrolysis of the O-antigen.

Tailspike Interactions with Lipopolysaccharide Effect DNA Ejection from Phage P22 Particles in Vitro

“…Whereas oligosaccharide binding is unaffected by the mutations at Asp-392 or Asp-395 (5,8), no binding of O-antigen octasaccharide to TSP T307K was detected by isothermal titration calorimetry at saccharide concentrations up to 0.5 mM. Despite the strongly reduced binding affinity to the short oligosaccharide, TSP T307K displayed high endorhamnosidase activity toward soluble polysaccharide substrate.…”

“…titrations were performed and data fitted to an independent binding site model as described previously (8,26). Hydrolytic activity of 0.09 M TSP with 12 mg/ml O-antigen polysaccharide from S. typhimurium was measured at 37°C in 50 mM phosphate buffer, pH 7.…”

“…Three subunits with parallel right-handed ␤-helix fold make up the native trimer of 215 kDa (6,7). A long surface-exposed groove on each subunit specifically recognizes the O-antigen portion of the Salmonella host LPS and harbors an endorhamnosidase activity, cleaving the ␣-(133) glycosidic linkages between rhamnose and galactose and producing dimers of 2 O-antigen repeat units (RU) as the main product (8,9).…”

Effects of Lactobacillus Probiotic, P22 Bacteriophage and Salmonella Typhimurium on the Heterophilic Burst Activity of Broiler Chickens

“…This is explained by the fact that P22 does not interact with eukaryotic cells, binding only to specific receptors of the cell wall of the host, which is Salmonella in this case (Baxa et al, 1996;Matsuzaki et al, 2005).…”

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Bacteriophage SPN3UB

“…To develop phages as a biocontrol agent for controlling this pathogen in foods, understanding of the host infection mechanism of Salmonella phages is important (2,3,10). While the O antigen of lipopolysaccharides (LPS) is a common host receptor for infection in the Myoviridae (FelixO1) (11) and Podoviridae (P22, 34) families (4,13,14), phages in the Siphoviridae family that use this host receptor, such as SETP3, are rare (8). The S. Typhimurium-targeting SPN3UB phage, which belongs to the Siphoviridae family, could not infect the rfaL (Oantigen ligase)-deficient mutant strain of S. Typhimurium SL1344 (data not shown), suggesting that it infects the host via the O antigen of LPS (12).…”