The tailspike protein (TSP) of Salmonella typhimurium phage P22 is a part of the apparatus by which the phage attaches to the bacterial host and hydrolyzes the O antigen. It has served as a model system for genetic and biochemical analysis of protein folding. The x-ray structure of a shortened TSP (residues 109 to 666) was determined to a 2.0 angstrom resolution. Each subunit of the homotrimer contains a large parallel beta helix. The interdigitation of the polypeptide chains at the carboxyl termini is important to protrimer formation in the folding pathway and to thermostability of the mature protein.

The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans).

The tailspike protein of Salmonella phage P22 is a viral adhesion protein with both receptor binding and destroying activities. It recognises the O-antigenic repeating units of cell surface lipopolysaccharide of serogroup A, B and D1 as receptor, but also inactivates its receptor by endoglycosidase (endorhamnosidase) activity. In the final step of bacteriophage P22 assembly six homotrimeric tailspike molecules are non-covalently attached to the DNA injection apparatus, mediated by their N-terminal, head-binding domains. We report the crystal structure of the head-binding domain of P22 tailspike protein at 2.3 A resolution, solved with a recombinant telluromethionine derivative and non-crystallographic symmetry averaging. The trimeric dome-like structure is formed by two perpendicular beta-sheets of five and three strands, respectively in each subunit and caps a three-helix bundle observed in the structure of the C-terminal receptor binding and cleaving fragment, reported here after full refinement at 1.56 A resolution. In the central part of the receptor binding fragment, three parallel beta-helices of 13 complete turns are associated side-by-side, while the three polypeptide strands merge into a single domain towards their C termini, with close interdigitation at the junction to the beta-helix part. Complex structures with receptor fragments from S. typhimurium, S. enteritidis and S. typhi253Ty determined at 1.8 A resolution are described in detail. Insertions into the beta-helix form the O-antigen binding groove, which also harbours the active site residues Asp392, Asp395 and Glu359. In the intact structure of the tailspike protein, head-binding and receptor-binding parts are probably linked by a flexible hinge whose function may be either to deal with shearing forces on the exposed, 150 A long tailspikes or to allow them to bend during the infection process.

e Artilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophageencoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and kill Pseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (ϳ5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect against P. aeruginosa persisters (up to >4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections.

The O-antigenic repeating units of lipopolysaccharides from Salmonella serogroups A, B, and DI serve as receptors for the phage P22 tailspike protein, which also has receptor destroying endoglycosidase (endorhamnosidase) activity, integrating the functions of both hemagglutinin and neuraminidase in influenza virus. Crystal structures of the tailspike protein in complex with oligosaccharides, comprising two 0-antigenic repeating units from Sabnonella typhiumurium, SalmoneUa enteritidis, and Salmonella typhi 253Ty were determined at 1.8 A resolution. The active-site topology with Asp-392, Asp-395, and Glu-359 as catalytic residues was identified. Kinetics of binding and cleavage suggest a role of the receptor destroying endorhamnosidase activity primarily for detachment of newly assembled phages.The infection of Salmonella by phage P22 starts with the recognition of the 0-antigenic repeating units of the cell surface lipopolysaccharide by the homotrimeric tailspike protein (TSP), which is present in six copies on the attachment apparatus (1, 2). Phages that use lipopolysaccharide as receptors are faced with the chemical diversity of the 0-antigenic repeats, which differ in carbohydrate composition and stereochemistry of the 0-glycosidic linkage. In addition, microheterogeneity conceming the number of repeating units and additional modifications as acetylation or glucosylation known as form variation is an important feature of lipopolysaccharide (3-5). P22 has adapted to Salmonella serotypes A, B, and Dl ( Fig. 1), sharing a common trisaccharide repeating unit a-Dmannose-(1,4)-a-L-rhamnose-(1,3)-a-D-galactose for the 0-antigen but differing in their branching carbohydrate moieties, a 3,6-dideoxyhexose a-(1,3)-linked to D-mannose. Dideoxyhexoses are paratose (serogroup A), abequose (serotype B), or tyvelose (serotype D1) and reflect the correlation of serotype classification and chemical structure of the 0-antigenic repeats of Salmonella (6, 7). In addition, P22 tolerates the 0-antigen 122, which shows an a-(1,4)-linked D-glucose at D-galactose as in Salmonella typhi 253Ty (8). In the phage-host interaction, the dideoxyhexose can be viewed as a wobble position that allows for some flexibility in a specific interaction.Receptor destroying enzymatic activities are well known for viruses that use carbohydrates as receptors. Influenza A and B virus and paramyxovirus have neuraminidases releasing terminal N-acetylmuraminic acid from glycoproteins and glycolipids (9), whereas influenza virus C and some coronaviruses that recognize an 0-acetylated sialic acid epitope have a sialate 9-0-acetylesterase, removing an acetyl group (10, 11). Endoglycosidase or acetylesterase activities have also been demonstrated for a large number of phages acting on encapsulated Gram-negative bacteria like Escherichia coli, Salmonella, or Klebsiella (12). TSP possesses receptor destroying endorhamnosidase activity cleaving the a(1,3)-O-glycosidic bond between rhamnose and galactose of the 0-antigenic repeats (13,14). To elucidate the s...