Superinfection exclusion strategy of siphophage T5: analysis of the FhuA:Llp complex.

Abstract

Superinfection exclusion is a widespread viral mechanism that protects the infected cell from overinfection by other identical or closely related viruses. Shortly after infection by bacteriophage T5, the bacterium Escherichia coli produces Llp, encoded by the newly injected viral genome. Llp is a small lipoprotein targeted to the inner leaflet of the host outer membrane. It binds to FhuA, an outer-membrane iron-ferrichrome transporter, which is the T5 receptor at the E. coli cell surface. The interaction between Llp and FhuA prevents any further bacteriophage binding and infection. Here, we determined the NMR structure of Llp and analyzed the formation of the FhuA:Llp complex using a wide range of techniques and mutants, both in vivo and in vitro. Interaction of Llp with FhuA is governed by a two-step equilibrium with a strong contribution of induced fit: Llp binding requires remodeling of FhuA periplasmic plug surface, enabling the subsequent large conformational reorganization of the extracellular loops. Analysis of FhuA mutants shows the importance of the intertwined interactions between the extracellular loops and the plug in the communication between the periplasmic and the extracellular faces of FhuA.IMPORTANCEBacteriophages (phages) are viruses that infect bacteria. As their enemies, phages play an important role in the dynamics, diversity, evolution, and pathogenicity of bacterial populations, but also represent an alternative to antibiotics in our fight against the rise of multi-resistant pathogenic bacterial strains. Infection is triggered by phage recognition of a receptor at the host surface, inducing phage DNA injection into the bacterial cytoplasm. The host cell is then transformed into a viral factory. To secure this viral factory, the infecting phage develops superinfection exclusion strategies, preventing other phages from interfering. Understanding how phages achieve the "locking" of the host will allow us to optimize phage use in phage therapy. Here, we analyze the structure of the receptor-exclusion protein complex using integrated in vivo, biochemical, and biophysical approaches. Our analysis reveals an unusually slow association process, consistent with an induced-fit mechanism, ultimately transmitting information from the bacterial periplasm to its surface.