Engineered phage with antibacterial CRISPR-Cas selectively reduce E. coli burden in mice.

Antibiotic treatments have detrimental effects on the microbiome and lead to antibiotic resistance. To develop a phage therapy against a diverse range of clinically relevant Escherichia coli, we screened a library of 162 wild-type (WT) phages, identifying eight phages with broad coverage of E. coli, complementary binding to bacterial surface receptors, and the capability to stably carry inserted cargo. Selected phages were engineered with tail fibers and CRISPR-Cas machinery to specifically target E. coli. We show that engineered phages target bacteria in biofilms, reduce the emergence of phage-tolerant E. coli and out-compete their ancestral WT phages in coculture experiments. A combination of the four most complementary bacteriophages, called SNIPR001, is well tolerated in both mouse models and minipigs and reduces E. coli load in the mouse gut better than its constituent components separately. SNIPR001 is in clinical development to selectively kill E. coli, which may cause fatal infections in hematological cancer patients.

Structure and Function of the Branched Receptor-Binding Complex of Bacteriophage CBA120.

Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120-TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1-TSP2-TSP3-TSP4 branched complex in CBA120 and its related ViI-like phages.