Bacteriophage phi92 is a large, lytic myovirus isolated in 1983 from pathogenic Escherichia coli strains that carry a polysialic acid capsule. Here we report the genome organization of phi92, the cryoelectron microscopy reconstruction of its virion, and the reinvestigation of its host specificity. The genome consists of a linear, double-stranded 148,612-bp DNA sequence containing 248 potential open reading frames and 11 putative tRNA genes. Orthologs were found for 130 of the predicted proteins. Most of the virion proteins showed significant sequence similarities to proteins of myoviruses rv5 and PVP-SE1, indicating that phi92 is a new member of the novel genus of rv5-like phages. Reinvestigation of phi92 host specificity showed that the host range is not limited to polysialic acid-encapsulated Escherichia coli but includes most laboratory strains of Escherichia coli and many Salmonella strains. Structure analysis of the phi92 virion demonstrated the presence of four different types of tail fibers and/or tailspikes, which enable the phage to use attachment sites on encapsulated and nonencapsulated bacteria. With this report, we provide the first detailed description of a multivalent, multispecies phage armed with a host cell adsorption apparatus resembling a nanosized Swiss army knife. The genome, structure, and, in particular, the organization of the baseplate of phi92 demonstrate how a bacteriophage can evolve into a multi-pathogen-killing agent.

Proteins fold into their functional three-dimensional structure based on the information encoded in the residue sequence 1 . In all domains of life, various strategies evolved to assist folding processes in order to prevent immediate misfolding and aggregation of the nascent polypeptide chain. In the crowded cellular environment, protein folding is often aided by molecular chaperones. Molecular chaperones differ in size, function and energy dependence; however, all have in common to bind to the unfolded state of the protein in order to facilitate or mediate assembly of the correct three-dimensional structure 2,3 . In contrast to molecular chaperones, the intramolecular chaperones (IMCs) constitute a different class of chaperones. As part of the polypeptide chain, the IMC is typically cleaved off the target protein after the folding process is completed. Two classes of IMCs can be distinguished: class I IMCs assist the protein to fold into the correct tertiary structure, whereas class II IMCs are involved in quaternary structure assembly 4 . In contrast to many molecular chaperones, no evidence for an ATP-driven cleavage reaction could be found in IMCs. An example of a class II intramolecular chaperone has been identified in viral tailspike and fiber proteins. These proteins are functionally unrelated but share a highly conserved chaperone domain at their C terminus (C-terminal intramolecular chaperone domain, CIMCD), which is cleaved at a conserved position in an autoproteolytic reaction 5 . It was shown that the covalent linkage between the CIMCD and N-terminal pre-protein is necessary for correct folding, indicating that an in trans function of the chaperone is impossible 5 . Furthermore, it could be shown that some of the chaperone domains are exchangeable between the different preproteins 5,6 . While the three-dimensional structures of the CIMCDs are as yet unknown, the crystal structure of N-terminally truncated mature endoNF shows that the homotrimeric enzyme comprises a triple β-helix involved in substrate recognition 7 . This triple β-helix and the related triple-β-spiral motif have been identified in various proteins, which often play a role as virulence factors 8 . In triple β-helices, three polypeptide chains wind around a common threefold symmetry axis, conferring an extraordinary stability to the protein. The rigid elongated shape allows triple β-helix-comprising proteins to protrude from a pathogen's surface in order to interact with flexible host cell receptors, like lipopolysaccharides 9 . However, proper assembly of triple-β-helical folds poses to be difficult in the absence of a trimerization domain 10 . Hence, most triple β-helices depend on a C-terminal extension for trimerization and correct assembly 11 .Here we present the crystal structures of two representatives of a large group of systematically, functionally and structurally similar intramolecular chaperones: the Escherichia coli phage K1F endosilidase CIMCD and the Bacillus subtilis phage GA-1 neck appendage protein CIMCD. Furthermore...

SummaryBacterial capsules are not only important virulence factors, but also provide attachment sites for bacteriophages that possess capsule degrading enzymes as tailspike proteins. To gain insight into the evolution of these specialized viruses, we studied a panel of tailed phages specific for Escherichia coli K1, a neuroinvasive pathogen with a polysialic acid capsule. Genome sequencing of two lytic K1-phages and comparative analyses including a K1-prophage revealed that K1-phages did not evolve from a common ancestor. By contrast, each phage is related to a different progenitor type, namely T7-, SP6-, and P22-like phages, and gained new host specificity by horizontal uptake of an endosialidase gene. The new tailspikes emerged by combining endosialidase domains with the capsid binding module of the respective ancestor. For SP6-like phages, we identified a degenerated tailspike protein which now acts as versatile adaptor protein interconnecting tail and newly acquired tailspikes and demonstrate that this adapter utilizes an N-terminal undecapeptide interface to bind otherwise unrelated tailspikes. Combining biochemical and sequence analyses with available structural data, we provide new molecular insight into basic mechanisms that allow changes in host specificity while a conserved head and tail architecture is maintained. Thereby, the present study contributes not only to an improved understanding of phage evolution and hostrange extension but may also facilitate the on purpose design of therapeutic phages based on wellcharacterized template phages.

Folding and assembly of endosialidases, the trimeric tail spike proteins of Escherichia coli K1-specific bacteriophages, crucially depend on their C-terminal domain (CTD). Homologous CTDs were identified in phage proteins belonging to three different protein families: neck appendage proteins of several Bacillus phages, L-shaped tail fibers of coliphage T5, and K5 lyases, the tail spike proteins of phages infecting E. coli K5. By analyzing a representative of each family, we show that in all cases, the CTD is cleaved off after a strictly conserved serine residue and alanine substitution prevented cleavage. Further structural and functional analyses revealed that (i) CTDs are autonomous domains with a high ␣-helical content; (ii) proteolytically released CTDs assemble into hexamers, which are most likely dimers of trimers; (iii) highly conserved amino acids within the CTD are indispensable for CTD-mediated folding and complex formation; (iv) CTDs can be exchanged between proteins of different families; and (v) proteolytic cleavage is essential to stabilize the native protein complex. Data obtained for full-length and proteolytically processed endosialidase variants suggest that release of the CTD increases the unfolding barrier, trapping the mature trimer in a kinetically stable conformation. In summary, we characterize the CTD as a novel C-terminal chaperone domain, which assists folding and assembly of unrelated phage proteins.

Bacteriophages with contractile tails and the bacterial type VI secretion system have been proposed to use a special protein to create an opening in the host cell membrane during infection. These proteins have a modular architecture but invariably contain an oligonucleotide/oligosaccharide-binding (OB-fold) domain and a long β-helical C-terminal domain, which initiates the contact with the host cell membrane. Using X-ray crystallography and electron microscopy, we report the atomic structure of the membrane-piercing proteins from bacteriophages P2 and ϕ92 and identify the residues that constitute the membrane-attacking apex. Both proteins form compact spikes with a ∼10Å diameter tip that is stabilized by a centrally positioned iron ion bound by six histidine residues. The accumulated data strongly suggest that, in the process of membrane penetration, the spikes are translocated through the lipid bilayer without undergoing major unfolding.

Endosialidases (endoNs), as identified so far, are tailspike proteins of bacteriophages that specifically bind and degrade the ␣2,8-linked polysialic acid (polySia) capsules of their hosts. The crystal structure solved for the catalytic domain of endoN from coliphage K1F (endoNF) revealed a functional trimer. Folding of the catalytic trimer is mediated by an intramolecular C-terminal chaperone domain. Release of the chaperone from the folded protein confers kinetic stability to endoNF. In mutant c(S), the replacement of serine 911 by alanine prevents proteolysis and generates an enzyme that varies in activity from wild type. Using soluble polySia as substrate a 3-times higher activity was detected while evaluation with immobilized polySia revealed a 190-fold reduced activity. Importantly, activity of c(S) did not differ from wild type with tetrameric sialic acid, the minimal endoNF substrate. Furthermore, we show that the presence of the chaperone domain in c(S) destabilizes binding to polySia in a similar way as did selective disruption of a polySia binding site in the stalk domain. The improved catalytic efficiency toward soluble polySia observed in these mutants can be explained by higher dissociation and association probabilities, whereas inversely, an impaired processivity was found. The fact that endoNF is a processive enzyme introduces a new molecular basis to explain capsule degradation by bacteriophages, which until now has been regarded as a result of cooperative interaction of tailspike proteins. Moreover, knowing that release of the chaperone domain confers kinetic stability and processivity, conservation of the proteolytic process can be explained by its importance in phage evolution.

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Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is well studied in the nervous system and described as a dynamic modulator of plastic processes like precursor cell migration, axon fasciculation, and synaptic plasticity. Here, we describe a novel function of polysialylated NCAM (polySia-NCAM) in innate immunity of the lung. In mature lung tissue of healthy donors, polySia was exclusively attached to the transmembrane isoform NCAM-140 and located to intracellular compartments of epithelial cells. In patients with chronic obstructive pulmonary disease, however, increased polySia levels and processing of the NCAM carrier were observed. Processing of polysialylated NCAM was reproduced in a mouse model by bleomycin administration leading to an activation of the inflammasome and secretion of interleukin (IL)-1β. As shown in a cell culture model, polySia-NCAM-140 was kept in the late trans-Golgi apparatus of lung epithelial cells and stimulation by IL-1β or lipopolysaccharide induced metalloprotease-mediated ectodomain shedding, resulting in the secretion of soluble polySia-NCAM. Interestingly, polySia chains of secreted NCAM neutralized the cytotoxic activity of extracellular histones as well as DNA/histone-network-containing "neutrophil extracellular traps", which are formed during invasion of microorganisms. Thus, shedding of polySia-NCAM by lung epithelial cells may provide a host-protective mechanism to reduce tissue damage during inflammatory processes.