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

Abstract

Initial attachment of bacteriophage P22 to the Salmonella host cell is known to be mediated by interactions between lipopolysaccharide (LPS) and the phage tailspike proteins (TSP), but the events that subsequently lead to DNA injection into the bacterium are unknown. We used the binding of a fluorescent dye and DNA accessibility to DNase and restriction enzymes to analyze DNA ejection from phage particles in vitro. Ejection was specifically triggered by aggregates of purified Salmonella LPS but not by LPS with different O-antigen structure, by lipid A, phospholipids, or soluble O-antigen polysaccharide. This suggests that P22 does not use a secondary receptor at the bacterial outer membrane surface. Using phage particles reconstituted with purified mutant TSP in vitro, we found that the endorhamnosidase activity of TSP degrading the O-antigen polysaccharide was required prior to DNA ejection in vitro and DNA replication in vivo. If, however, LPS was pre-digested with soluble TSP, it was no longer able to trigger DNA ejection, even though it still contained five O-antigen oligosaccharide repeats. Together with known data on the structure of LPS and phage P22, our results suggest a molecular model. In this model, tailspikes position the phage particles on the outer membrane surface for DNA ejection. They force gp26, the central needle and plug protein of the phage tail machine, through the core oligosaccharide layer and into the hydrophobic portion of the outer membrane, leading to refolding of the gp26 lazo-domain, release of the plug, and ejection of DNA and pilot proteins.

Conformational Changes Leading to T7 DNA Delivery upon Interaction with the Bacterial Receptor

“…Similar findings have been reported previously for P22 (37). In our experiments with T7 bacteriophage, no proteins were observed inside the capsid after ejection (Fig.…”

Atomic Structure of Bacteriophage Sf6 Tail Needle Knob

“…4). This is surprising as a protein receptor is neither known to be required for P22 infec- tion in vivo nor necessary for P22 genome ejection in vitro (22). Second, a molecule of L-Glu is tightly bound at each subunit interface of the Sf6 tail needle knob.…”

“…Furthermore, recent in vitro work has demonstrated that P22 tail needle can be ejected from virions by treatment with lipopolysaccharide (22), suggesting that gp26 does not interact with a proteinaceous receptor and is mechanically ejected into the cell during infection. Enzymatic studies using either isolated tailspike or mature P22 virions have determined that O antigen cleavage by tailspike stops about five repeat units from the core LPS (22). The tail needle was suggested to function as a simple "pressure sensor" that triggers genome ejection after contacting the LPS inner core or lipid A.…”

“…Although the molecular details of subsequent events are largely unknown, the tail needle is released from the virion to open a passage for the ejection proteins and DNA (10, 13). Andres et al (22) recently developed an in vitro assay to study P22 genome ejection, which is specifically triggered by aggregates of purified Salmonella LPS. This suggests that the signal for tail needle ejection and genome release lays in the tailspike, an idea supported by observations that LPS with different O antigen structure, lipid A, phospholipids, or soluble O antigen polysaccharide fail to trigger P22 gp26 ejection and genome release (22).…”

“…Andres et al (22) recently developed an in vitro assay to study P22 genome ejection, which is specifically triggered by aggregates of purified Salmonella LPS. This suggests that the signal for tail needle ejection and genome release lays in the tailspike, an idea supported by observations that LPS with different O antigen structure, lipid A, phospholipids, or soluble O antigen polysaccharide fail to trigger P22 gp26 ejection and genome release (22). Thus, P22 and likely all P22-like phages do not appear to use a secondary protein receptor located in the bacterial outer membrane.…”