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Tape measure (TM) proteins are essential for the formation of long-tailed phages. TM protein assembly into tails requires the action of tail assembly chaperones (TACs). TACs (e.g. gpG and gpT of E. coli phage lambda) are usually produced in a short (TAC-N) and long form (TAC-NC) with the latter comprised of TAC-N with an additional C-terminal domain (TAC-C). TAC-NC is generally synthesized through a ribosomal frameshifting mechanism. TAC encoding genes have never been identified in the intensively studied Escherichia coli phage T4, or any related phages. Here, we have bioinformatically identified putative TAC encoding genes in diverse T4-like phage genomes. The frameshifting mechanism for producing TAC-NC appears to be conserved in several T4-like phage groups. However, the group including phage T4 itself likely employs a different strategy whereby TAC-N and TAC-NC are encoded by separate genes (26 and 51 in phage T4).

At the first step of phage infection, the receptor-binding proteins (RBPs) such as tail fibers are responsible for recognizing specific host surface receptors. The proper folding and assembly of tail fibers usually requires a chaperone encoded by the phage genome. Despite extensive studies on phage structures, the molecular mechanism of phage tail fiber assembly remains largely unknown. Here, using a minimal myocyanophage, termed Pam3, isolated from Lake Chaohu, we demonstrate that the chaperone gp25 forms a stable complex with the tail fiber gp24 at a stoichiometry of 3:3. The 3.1-Å cryo-electron microscopy structure of this complex revealed an elongated structure with the gp25 trimer embracing the distal moieties of gp24 trimer at the center. Each gp24 subunit consists of three domains: the N-terminal α-helical domain required for docking to the baseplate, the tumor necrosis factor (TNF)-like and glycine-rich domains responsible for recognizing the host receptor. Each gp25 subunit consists of two domains: a non-conserved N-terminal β-sandwich domain that binds to the TNF-like and glycine-rich domains of the fiber, and a C-terminal α-helical domain that mediates trimerization/assembly of the fiber. Structural analysis enabled us to propose the assembly mechanism of phage tail fibers, in which the chaperone first protects the intertwined and repetitive distal moiety of each fiber subunit, further ensures the proper folding of these highly plastic structural elements, and eventually enables the formation of the trimeric fiber. These findings provide the structural basis for the design and engineering of phage fibers for biotechnological applications.

The first critical step in a virus's infection cycle is attachment to its host. This interaction is precise enough to ensure the virus will be able to productively infect the cell, but some flexibility can be beneficial to enable coevolution and host range switching or expansion. Bacteriophage Sf6 utilizes a two-step process to recognize and attach to its host Shigella flexneri. Sf6 first recognizes the lipopolysaccharide (LPS) of S. flexneri and then binds outer membrane protein (Omp) A or OmpC. This phage infects serotype Y strains but can also form small, turbid plaques on serotype 2a

Bacterial transduction particles were critical to early advances in molecular biology and are currently experiencing a resurgence in interest within the diagnostic and therapeutic fields. The difficulty of developing a robust and specific transduction reagent capable of delivering a genetic payload to the diversity of strains constituting a given bacterial species or genus is a major impediment to their expanded utility as commercial products. While recent advances in engineering the reactivity of these reagents have made them more attractive for product development, considerable improvements are still needed. Here, we demonstrate a synthetic biology platform derived from bacteriophage P1 as a chassis to target transduction reagents against four clinically prevalent species within the Enterobacterales order. Bacteriophage P1 requires only a single receptor binding protein to enable attachment and injection into a target bacterium. By engineering and screening particles displaying a diverse array of chimeric receptor binding proteins, we generated a potential transduction reagent for a future rapid phenotypic carbapenem-resistant Enterobacterales diagnostic assay.

The genome of the thermophilic bacteriophage GVE2 encodes a putative tailspike protein (GVE2 TSP). Here we report the crystal structure of the truncated GVE2 TSP at 2.0-Å resolution lacking 204 amino acid residues at its N-terminus (ΔnGVE2 TSP), possessing a "vase" outline similar to other TSP's structures. However, ΔnGVE2 TSP displays structural characteristics distinct from other TSPs. Despite lacking 204 amino acid residues, the head domain forms an asymmetric trimer compared to symmetric in other TSPs, suggesting that its long N-terminus may be unique to the long-tailed bacteriophages. Furthermore, the α-helix of the neck is 5-7 amino acids longer than that of other TSPs. The most striking feature is that its binding domain consists of a β-helix with 10 turns, whereas other TSPs have 13 turns, even including the phage Sf6 TSP, which is the closest homologue of GVE2 TSP. The C-terminal structure is also quite different with those of other TSPs. Furthermore, we observed that ΔnGVE2 TSP can slow down growth of its host, demonstrating that this TSP is essential for the phage GVE2 to infect its host. Overall, the structural characteristics suggest that GVE2 TSP may be more primitive than other phage TSPs.

Pseudaminic acid (Pse), a unique carbohydrate in surface-associated glycans of pathogenic bacteria, has pivotal roles in virulence. Owing to its significant antigenicity and absence in mammals, Pse is considered an attractive target for vaccination or antibody-based therapies against bacterial infections. However, a specific and universal probe for Pse, which could also be used in immunotherapy, has not been reported. In a prior study, we used a tail spike protein from a bacteriophage (ΦAB6TSP) that digests Pse-containing exopolysaccharide (EPS) from Acinetobacter baumannii strain 54149 (Ab-54149) to form a glycoconjugate for preparing anti-Ab-54149 EPS serum. We report here that a catalytically inactive ΦAB6TSP (I-ΦAB6TSP) retains binding ability toward Pse. In addition, an I-ΦAB6TSP-DyLight-650 conjugate (Dy-I-ΦAB6TSP) was more sensitive in detecting Ab-54149 than an antibody purified from anti- Ab-54149 EPS serum. Dy-I-ΦAB6TSP also cross-reacted with other pathogenic bacteria containing Pse on their surface polysaccharides (e.g., Helicobacter pylori and Enterobacter cloacae), revealing it to be a promising probe for detecting Pse across bacterial species. We also developed a detection method that employs I-ΦAB6TSP immobilized on microtiter plate. These results suggested that the anti-Ab-54149 EPS serum would exhibit cross-reactivity to Pse on other organisms. When this was tested, this serum facilitated complement-mediated killing of H. pylori and E. cloacae, indicating its potential as a cross-species antibacterial agent. This work opens new avenues for diagnosis and treatment of multidrug resistant (MDR) bacterial infections.

Summary Available 3D structures of bacteriophage modules combined with predictive bioinformatic algorithms enabled the identification of adhesion modules in 57 siphophages infecting Streptococcus thermophilus (St). We identified several carbohydrate‐binding modules (CBMs) in so‐called evolved distal tail (Dit) and tail‐associated lysozyme (Tal) proteins of St phage baseplates. We examined the open reading frame (ORF) downstream of the Tal‐encoding ORF and uncovered the presence of a putative p2‐like receptor‐binding protein (RBP). A 21 Å resolution electron microscopy structure of the baseplate of cos‐phage STP1 revealed the presence of six elongated electron densities, surrounding the core of the baseplate, that harbour the p2‐like RBPs at their tip. To verify the functionality of these modules, we expressed GFP‐ or mCherry‐coupled Tal and putative RBP CBMs and observed by fluorescence microscopy that both modules bind to their corresponding St host, the putative RBP CBM with higher affinity than the Tal‐associated one. The large number of CBM functional domains in St phages suggests that they play a contributory role in the infection process, a feature that we previously described in lactococcal phages and beyond, possibly representing a universal feature of the siphophage host‐recognition apparatus., We found that for each streptococcal phage several CBMs are present in the virions and are involved in phage adhesion. Moreover, we uncovered an additional ORF that harbours a predicted Receptor Binding Protein (RBP) at its C‐terminus. To confirm these predictions, we determined by electron microscopy the structure of streptococcal phage STP1 baseplate and assessed the binding capability of the CBM and the RBP‐like domain to their specific host using fluorescent binding assays.

The long flexible tail tube of bacteriophage lambda connects its capsid to the tail tip. On infection, a DNA ejection signal is passed from the tip, along the tube to the capsid that triggers passage of the DNA down the tube and into the host bacterium. The tail tube is built from repeating units of the major tail protein, gpV, which has two distinctive domains. Its N-terminal domain has the same fold as proteins that form the rigid inner tubes of contractile tail phages, such as T4, and its C-terminal domain adopt an Ig-like fold of unknown function. We determined structures of the lambda tail tube in free tails and in virions before and after DNA ejection using cryoelectron microscopy. Modeling of the density maps reveals how electrostatic interactions and a mobile loop participate in assembly and also impart flexibility to the tube while maintaining its integrity. We also demonstrate how a common protein fold produces rigid tubes in some phages but flexible tubes in others.

Four tailspike proteins (TSP1-4) of Escherichia coli O157:H7 bacteriophage CBA120 enable infection of multiple hosts. They form a branched complex that attaches to the tail baseplate. Each TSP recognizes a different lipopolysaccharide on the membrane of a different bacterial host. The 335 N-terminal residues of TSP4 promote the assembly of the TSP complex and anchor it to the tail baseplate. The crystal structure of TSP4-N335 reveals a trimeric protein comprising four domains. The baseplate anchor domain (AD) contains an intertwined triple-stranded β-helix. The ensuing XD1, XD2 and XD3 β-sheet containing domains mediate the binding of TSP1-3 to TSP4. Each of the XD domains adopts the same fold as the respective XD domains of bacteriophage T4 gp10 baseplate protein, known to engage in protein–protein interactions via its XD2 and XD3 domains. The structural similarity suggests that XD2 and XD3 of TSP4 also function in protein–protein interactions. Analytical ultracentrifugation analyses of TSP4-N335 and of domain deletion proteins showed how TSP4-N335 promotes the formation of the TSP quaternary complex. TSP1 and TSP2 bind directly to TSP4 whereas TSP3 binding requires a pre-formed TSP4-N335:TSP2 complex. A 3-dimensional model of the bacteriophage CBA120 TSP complex has been developed based on the structural and ultracentrifuge information.

The high specificity of bacteriophages is driven by their receptor-binding proteins (RBPs). Many Klebsiella bacteriophages target the capsular exopolysaccharide as the receptor and encode RBPs with depolymerase activity. The modular structure of these RBPs with an N-terminal structural module to attach the RBP to the phage tail, and a C-terminal specificity module for exopolysaccharide degradation, supports horizontal transfer as a major evolutionary driver for Klebsiella phage RBPs. We mimicked this natural evolutionary process by the construction of modular RBP chimeras, exchanging N-terminal structural modules and C-terminal specificity modules. All chimeras strictly follow the capsular serotype specificity of the C-terminal module. Transplanting chimeras with a K11 N-terminal structural RBP module in a Klebsiella phage K11 scaffold results in a capsular serotype switch and corresponding host range modification of the synthetic phages, demonstrating that horizontal transfer of C-terminal specificity modules offers Klebsiella phages an evolutionary highway for rapid adaptation to new capsular serotypes. IMPORTANCE The antimicrobial resistance crisis has rekindled interest in bacteriophage therapy. Phages have been studied over a century as therapeutics to treat bacterial infections, but one of the biggest challenges for the use of phages in therapeutic interventions remains their high specificity. In particular, many Klebsiella phages have a narrow spectrum constrained by the high diversity of exopolysaccharide capsules that shield access to the cells. In this work, we have elaborated how Klebsiella phages deal with this high diversity by exchanging building blocks of their receptor-binding proteins.

Background The gene transduction efficiency of adenovirus to hematopoietic cells, especially T lymphocytes, is needed to be improved. The purpose of this study is to improve the transduction efficiency of T lymphocytes by using fiber-modified human adenovirus 5 (HAdV-5) vectors. Results Four fiber-modified human adenovirus 5 (HAdV-5) vectors were investigated to transduce hematopoietic cells. F35-EG or F11p-EG were HAdV-35 or HAdV-11p fiber pseudotyped HAdV-5, and HR-EG or CR-EG vectors were generated by incorporating RGD motif to the HI loop or to the C-terminus of F11p-EG fiber. All vectors could transduce more than 90% of K562 or Jurkat cells at an multiplicity of infection (MOI) of 500 viral particle per cell (vp/cell). All vectors except HR-EG could transduce nearly 90% cord blood CD34+ cells or 80% primary human T cells at the MOI of 1000, and F11p-EG showed slight superiority to F35-EG and CR-EG. Adenoviral vectors transduced CD4+ T cells a little more efficiently than they did to CD8+ T cells. These vectors showed no cytotoxicity at an MOI as high as 1000 vp/cell because the infected and uninfected T cells retained the same CD4/CD8 ratio and cell growth rate. Conclusions HAdV-11p fiber pseudotyped HAdV-5 could effectively transduce human T cells when human EF1a promoter was used to control the expression of transgene, suggesting its possible application in T cell immunocellular therapy.

For successful infection, bacteriophages must overcome multiple barriers to transport their genome and proteins across the bacterial cell envelope. We use cryo-electron tomography to study the infection initiation of phage P22 in Salmonella enterica serovar Typhimurium, revealing how a channel forms to allow genome translocation into the cytoplasm. Our results show free phages that initially attach obliquely to the cell through interactions between the O antigen and two of the six tailspikes; the tail needle also abuts the cell surface. The virion then orients perpendicularly and the needle penetrates the outer membrane. The needle is released and the internal head protein gp7* is ejected and assembles into an extracellular channel that extends from the gp10 baseplate to the cell surface. A second protein, gp20, is ejected and assembles into a structure that extends the extracellular channel across the outer membrane into the periplasm. Insertion of the third ejected protein, gp16, into the cytoplasmic membrane probably completes the overall trans-envelope channel into the cytoplasm. Construction of a trans-envelope channel is an essential step during infection of Gram-negative bacteria by all short-tailed phages, because such virions cannot directly deliver their genome into the cell cytoplasm. Cryo-electron tomography was used to study the initial steps of infection of Salmonella enterica serovar Salmonella Typhimurium with phage P22 and reveals how the phage forms a channel through the host outer and inner membranes to translocate its genome into the bacterial cytoplasm.

ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host range mutants within infant rabbits infected with a mixture of wild-type and OmpU mutant strains. ICP2 host range mutants that can now infect OmpU mutant strains have missense mutations in the putative tail fiber gene gp25 and the putative adhesin gene gp23. Using site-specific mutagenesis, we show that single or double mutations in gp25 are sufficient to generate the host range mutant phenotype. However, at least one additional mutation in gp23 is required for robust plaque formation on specific OmpU mutants. Mutations in gp23 alone were insufficient to produce a host range mutant phenotype. All ICP2 host range mutants retained the ability to form plaques on wild-type V. cholerae cells. The strength of binding of host range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in gp25 and gp23 restore molecular interactions with the receptor. We propose that ICP2 host range mutants evolve by a two-step process. First, gp25 mutations are selected for their broad host range, albeit accompanied by low-level phage adsorption. Subsequent selection occurs for gp23 mutations that further increase productive binding to specific OmpU alleles, allowing for near-wild-type efficiencies of adsorption and subsequent phage multiplication. IMPORTANCE Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to renewed interest in phage biology and the potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail that have been shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular coevolutionary arms race presents fitness costs to both ICP2 and V. cholerae.

The overuse of antibiotics has led to emergence of antimicrobial resistance, and as a result, antibacterial peptides (ABPs) are receiving significant attention as an alternative. Identification of effective ABPs in lab from natural sources is a cost-intensive and time-consuming process. Therefore, there is a need for the development of in silico models, which can identify novel ABPs in protein sequences for chemical synthesis and testing. In this study, we propose a deep learning classifier named Deep-ABPpred that can identify ABPs in protein sequences. We developed Deep-ABPpred using bidirectional long short-term memory algorithm with amino acid level features from word2vec. The results show that Deep-ABPpred outperforms other state-of-the-art ABP classifiers on both test and independent datasets. Our proposed model achieved the precision of approximately 97 and 94% on test dataset and independent dataset, respectively. The high precision suggests applicability of Deep-ABPpred in proposing novel ABPs for synthesis and experimentation. By utilizing Deep-ABPpred, we identified ABPs in the tail protein sequences of Streptococcus bacteriophages, chemically synthesized identified peptides in lab and tested their activity in vitro. These ABPs showed potent antibacterial activity against selected Gram-positive and Gram-negative bacteria, which confirms the capability of Deep-ABPpred in identifying novel ABPs in protein sequences. Based on the proposed approach, an online prediction server is also developed, which is freely accessible at https://abppred.anvil.app/. This web server takes the protein sequence as input and provides ABPs with high probability (>0.95) as output.

Invasive pathobionts or microbes capable of causing disease can reside deep within the mucosal epithelium of our gastrointestinal tract. Targeted effective antibacterial therapies are needed to combat these disease-causing organisms, many of which may be multidrug resistant., The human gastrointestinal mucosal surface consists of a eukaryotic epithelium, a prokaryotic microbiota, and a carbohydrate-rich interface that separates them. In the gastrointestinal tract, the interaction of bacteriophages (phages) and their prokaryotic hosts influences the health of the mammalian host, especially colonization with invasive pathobionts. Antibiotics may be used, but they also kill protective commensals. Here, we report a novel phage whose lytic cycle is enhanced in intestinal environments. The tail fiber gene, whose protein product binds human heparan sulfated proteoglycans and localizes the phage to the epithelial cell surface, positions it near its bacterial host, a type of locational targeting mechanism. This finding offers the prospect of developing mucosal targeting phage to selectively remove invasive pathobiont species from mucosal surfaces.

Nowadays, bacteriophages are increasingly considered as an alternative treatment for a variety of bacterial infections in cases where classical antibiotics have become ineffective. However, characterizing the host specificity of phages remains a labor- and time-intensive process. In order to alleviate this burden, we have developed a new machine-learning-based pipeline to predict bacteriophage hosts based on annotated receptor-binding protein (RBP) sequence data. We focus on predicting bacterial hosts from the ESKAPE group, Escherichia coli, Salmonella enterica and Clostridium difficile. We compare the performance of our predictive model with that of the widely used Basic Local Alignment Search Tool (BLAST). Our best-performing predictive model reaches Precision-Recall Area Under the Curve (PR-AUC) scores between 73.6 and 93.8% for different levels of sequence similarity in the collected data. Our model reaches a performance comparable to that of BLASTp when sequence similarity in the data is high and starts outperforming BLASTp when sequence similarity drops below 75%. Therefore, our machine learning methods can be especially useful in settings in which sequence similarity to other known sequences is low. Predicting the hosts of novel metagenomic RBP sequences could extend our toolbox to tune the host spectrum of phages or phage tail-like bacteriocins by swapping RBPs.

Pathogenic microorganisms often reside in glycan-based biofilms. Concentration and chain length distribution of these mostly anionic exopolysaccharides (EPS) determine the overall biophysical properties of a biofilm and result in a highly viscous environment. Bacterial communities regulate this biofilm state via intracellular small-molecule signaling to initiate EPS synthesis. Reorganization or degradation of this glycan matrix, however, requires the action of extracellular glycosidases. So far, these were mainly described for bacteriophages that must degrade biofilms for gaining access to host bacteria. The plant pathogen Pantoea stewartii (P. stewartii) encodes the protein WceF within its EPS synthesis cluster. WceF has homologs in various biofilm forming plant pathogens of the Erwinia family. In this work, we show that WceF is a glycosidase active on stewartan, the main P. stewartii EPS biofilm component. WceF has remarkable structural similarity with bacteriophage tailspike proteins (TSPs). Crystal structure analysis showed a native trimer of right-handed parallel ��-helices. Despite its similar fold, WceF lacks the high stability found in bacteriophage TSPs. WceF is a stewartan hydrolase and produces oligosaccharides, corresponding to single stewartan repeat units. However, compared with a stewartan-specific glycan hydrolase of bacteriophage origin, WceF showed lectin-like autoagglutination with stewartan, resulting in notably slower EPS cleavage velocities. This emphasizes that the bacterial enzyme WceF has a role in P. stewartii biofilm glycan matrix reorganization clearly different from that of a bacteriophage exopolysaccharide depolymerase.

The P22 tailspike endorhamnosidase confers the high specificity of bacteriophage P22 for some serogroups of Salmonella differing only slightly in their O-antigen polysaccharide. We used several biophysical methods to study the binding and hydrolysis of O-antigen fragments of different lengths by P22 tailspike protein. O-Antigen saccharides of defined length labeled with fluorophors could be purified with higher resolution than previously possible. Small amounts of naturally occurring variations of 0antigen fragments missing the nonreducing terminal galactose could be used to determine the contribution of this part to the free energy of binding to be similar to 7 kJ/mol. We were able to show via several independent lines of evidence that an unproductive binding mode is highly favored in binding over all other possible binding modes leading to hydrolysis. This is true even under circumstances under which the O-antigen fragment is long enough to be cleaved efficiently by the enzyme. The high-affinity unproductive binding mode results in a strong self-competitive inhibition in addition to product inhibition observed for this system. Self-competitive inhibition is observed for all substrates that have a free reducing end rhamnose. Naturally occurring O-antigen, while still attached to the bacterial outer membrane, does not have a free reducing end and therefore does not perform self-competitive inhibition.

International audience; Bacteriophages, viruses that infect bacteria, are the most abundant biological entities on Earth. Siphophages, accounting for ∼60% of known phages, bear a long, flexible tail that allows host recognition and safe delivery of the DNA from the capsid to the cytoplasm of the infected cell. Independently from their host (Gram positive or Gram negative) and the nature of their receptor at its surface (polysaccharide or protein), the core tail architecture of all caudophages and of bacterial phage-derived contractile injection systems share the same structural organisation and are thought to be homologous. Here, we review the recent advances in the structure, function and assembly of the core tail architecture of siphophages.

Infecting bacteriophage T4 uses a contractile tail structure to breach the envelope of the Escherichia coli host cell. During contraction, the tail tube headed with the &ldquo, central spike complex&rdquo, is thought to mechanically puncture the outer membrane. We show here that a purified tip fragment of the central spike complex interacts with periplasmic chaperone PpiD, which is anchored to the cytoplasmic membrane. PpiD may be involved in the penetration of the inner membrane by the T4 injection machinery, resulting in a DNA-conducting channel to translocate the phage DNA into the interior of the cell. Host cells with the ppiD gene deleted showed partial reduction in the plating efficiency of T4, suggesting a supporting role of PpiD to improve the efficiency of the infection process.

The genome of Escherichia coli O157:H7 bacteriophage vB_EcoM_CBA120 encodes four distinct tailspike proteins (TSPs). The four TSPs, TSP1-4, attach to the phage baseplate forming a branched structure. We report the 1.9 Å resolution crystal structure of TSP2 (ORF211), the TSP that confers phage specificity towards E. coli O157:H7. The structure shows that the N-terminal 168 residues involved in TSPs complex assembly are disordered in the absence of partner proteins. The ensuing head domain contains only the first of two fold modules seen in other phage vB_EcoM_CBA120 TSPs. The catalytic site resides in a cleft at the interface between adjacent trimer subunits, where Asp506, Glu568, and Asp571 are located in close proximity. Replacement of Asp506 and Asp571 for alanine residues abolishes enzyme activity, thus identifying the acid/base catalytic machinery. However, activity remains intact when Asp506 and Asp571 are mutated into asparagine residues. Analysis of additional site-directed mutants in the background of the D506N:D571N mutant suggests engagement of an alternative catalytic apparatus comprising Glu568 and Tyr623. Finally, we demonstrate the catalytic role of two interacting glutamate residues of TSP1, located in a cleft between two trimer subunits, Glu456 and Glu483, underscoring the diversity of the catalytic apparatus employed by phage vB_EcoM_CBA120 TSPs.

International audience; Intestinal microbiota have been proposed to induce commensal-specific memory T cells that cross-react with tumor-associated antigens. We identified major histocompatibility complex (MHC) class I-binding epitopes in the tail length tape measure protein (TMP) of a prophage found in the genome of the bacteriophage Enterococcus hirae Mice bearing E. hirae harboring this prophage mounted a TMP-specific H-2Kb-restricted CD8+ T lymphocyte response upon immunotherapy with cyclophosphamide or anti-PD-1 antibodies. Administration of bacterial strains engineered to express the TMP epitope improved immunotherapy in mice. In renal and lung cancer patients, the presence of the enterococcal prophage in stools and expression of a TMP-cross-reactive antigen by tumors correlated with long-term benefit of PD-1 blockade therapy. In melanoma patients, T cell clones recognizing naturally processed cancer antigens that are cross-reactive with microbial peptides were detected.

International audience; Virulent phages infecting L. lactis, an industry-relevant bacterium, pose a significant risk to the quality of the fermented milk products. Phages of the Skunavirus genus are by far the most isolated lactococcal phages in the cheese environments and phage p2 is the model siphophage for this viral genus. The baseplate of phage p2, which is used to recognize its host, was previously shown to display two conformations by X-ray crystallography, a rested state and an activated state ready to bind to the host. The baseplate became only activated and opened in the presence of Ca 2+. However, such an activated state was not previously observed in the virion. Here, using nanobodies binding to the baseplate, we report on the negative staining electron microscopy structure of the activated form of the baseplate directly observed in the p2 virion, that is compatible with the activated baseplate crystal structure. Analyses of this new structure also established the presence of a second distal tail (Dit) hexamer as a component of the baseplate, the topology of which differs largely from the first one. We also observed an uncoupling between the baseplate activation and the tail tip protein (Tal) opening, suggesting an infection mechanism more complex than previously expected.

International audience; Bacteriophages can play beneficial roles in phage therapy and destruction of food pathogens. Conversely, they play negative roles as they infect bacteria involved in fermentation, resulting in serious industrial losses. Siphoviridae phages possess a long non-contractile tail and use a mechanism of infection whose first step is host recognition and binding. They have evolved adhesion devices at their tails' distal end, tuned to recognize specific proteinaceous or saccharidic receptors on the host's surface that span a large spectrum of shapes. In this review, we aimed to identify common patterns beyond this apparent diversity. To this end, we analyzed siphophage tail tips or baseplates, evaluating their known structures, where available, and uncovering patterns with bioinformatics tools when they were not. It was thereby identified that a triad formed by three proteins in complex, i.e., the tape measure protein (TMP), the distal tail protein (Dit), and the tail-associated lysozyme (Tal), is conserved in all phages. This common scaffold may harbor various functional extensions internally while it also serves as a platform for plug-in ancillary or receptor-binding proteins (RBPs). Finally, a group of siphophage baseplates involved in saccharidic receptor recognition exhibits an activation mechanism reminiscent of that observed in Myoviridae.

Productive virus infection depends upon delivery of viral genomic material into the host cell cytoplasm. The tails of bacteriophages recognize host cells and mediate host cell wall and membrane penetration. Recent cryo-electron microscopy studies have revealed near atomic-resolutions structures of the entire or almost entire bacteriophage particles of model systems including phi29, P22, P68, and T4. These structures allow comparisons between not only different states of the same phage but also between distantly related phages. In this review, we summarize the findings from recent structural studies of the bacteriophages that target Gram-positive bacteria, for a better understanding of the interactions between host cells and bacteriophages.

Summary Capsule polysaccharide is a major virulence factor of Klebsiella pneumoniae, a nosocomial pathogen associated with a wide range of infections. It protects bacteria from harsh environmental conditions, immune system response, and phage infection. To access cell wall-located receptors, some phages possess tailspike depolymerases that degrade the capsular polysaccharide. Here, we present the crystal structure of a tailspike against Klebsiella, KP32gp38, whose primary sequence shares no similarity to other proteins of known structure. In the trimeric structure of KP32gp38, each chain contains a flexible N-terminal domain, a right-handed parallel β helix domain and two β sandwiches with carbohydrate binding features. The crystal structure and activity assays allowed us to locate the catalytic site. Also, our data provide experimental evidence of a branching architecture of depolymerases in KP32 Klebsiella viruses, as KP32gp38 displays nanomolar affinity to another depolymerase from the same phage, KP32gp37. Results provide a structural framework for enzyme engineering to produce serotype-broad-active enzyme complexes against K. pneumoniae.

Bacteriophages (phages), viruses that infect bacteria, are considered to be highly host-specific. To add to the knowledge about the evolution and development of bacteriophage speciation toward its host, we conducted a 21-day experiment with the broad host-range bacteriophage Aquamicrobium phage P14. We incubated the phage, which was previously isolated and enriched with the Alphaproteobacteria Aquamicrobium H14, with the Betaproteobacteria Alcaligenaceae H5. During the experiment, we observed an increase in the phage&rsquo, s predation efficacy towards Alcaligenaceae H5. Furthermore, genome analysis and the comparison of the bacteriophage&rsquo, s whole genome indicated that rather than being scattered evenly along the genome, mutations occur in specific regions. In total, 67% of the mutations with a frequency higher than 30% were located in genes that encode tail proteins, which are essential for host recognition and attachment. As control, we incubated the phage with the Alphaproteobacteria Aquamicrobium H8. In both experiments, most of the mutations appeared in the gene encoding the tail fiber protein. However, mutations in the gene encoding the tail tubular protein B were only observed when the phage was incubated with Alcaligenaceae H5. This highlights the phage&rsquo, s tail as a key player in its adaptation to different hosts. We conclude that mutations in the phage&rsquo, s genome were mainly located in tail-related regions. Further investigation is needed to fully characterize the adaptation mechanisms of the Aquamicrobium phage P14.

The adsorption of phages to hosts is the first step of phage infection. Studies have shown that tailed phages use tail fibres or spikes to recognize bacterial receptors and mediate adsorption. However, whether other phage tail components can also recognize host receptors is unknown. To identify potential receptors, we screened a transposon mutagenesis library of the marine pathogen Vibrio parahaemolyticus and discovered that a vp0980 mutant (vp0980 encodes a predicted transmembrane protein) could not be lysed by phage OWB. Complementation of this mutant with wild-type vp0980 in trans restored phage-mediated lysis. Phage adsorption and confocal microscopy assays demonstrated that phage OWB had dramatically reduced adsorption to the vp0980 mutant compared to that to the wild type. Pulldown assays showed that phage tail tubular proteins A and B (TTPA and TTPB) interact with Vp0980, suggesting that Vp0980 is a TTPA and TTPB receptor. Vp0980 lacking the outer membrane region (aa 114–127) could not bind to TTPA and TTPB, resulting in reduced phage adsorption. These results strongly indicated that TTPA and TTPB binding with their receptor Vp0980 mediates phage adsorption and subsequent bacterial lysis. To the best of our knowledge, this study is the first report of a bacterial receptor for phage tail tubular proteins.

Rapid and accurate bacterial detection is crucial to an early diagnosis for treating various infectious diseases. A recombinant tail fiber protein (P069) of the Pseudomonas aeruginosa ( P. aeruginosa) phage was expressed in Escherichia coli. After renaturation at a low temperature, the inclusion body of P069 was successfully transformed to an aqueous soluble protein that retained the capacity for recognizing P. aeruginosa. The recombinant P069 did not show lytic activity to P. aeruginosa, which facilitated the capture and manipulation of bacterial whole cells with a high flexibility for downstream identification and detection. Bioluminescent and fluorescent methods using this biorecognition element allowed P. aeruginosa detection with the detection limits of 6.7 × 102 CFU mL-1 and 1.7 × 102 CFU mL-1, respectively. Moreover, the specificity investigations showed that P069 was a species-specific protein. Therefore, it avoided the potential false negative results originating from the excessive high specificity of phage toward a given strain. It has been successfully applied to detect P. aeruginosa in spiked samples with acceptable recovery values ranging from 88% to 98%. The above results demonstrate that P069 is an ideal biorecognition element for the detection of P. aeruginosa in complicated sample matrixes.

The prevalence of multidrug-resistant Salmonella is ever increasing and calls for alternatives to antibiotics. The use of phages has been anticipated to reduce the multidrug-resistant human pathogens in food environment. Salmonella phage vB_SalM_PM10 (PM10) was isolated from sewage-polluted river in India. It shows an icosahedral head (94 ± 4 nm) along with a long contractile tail (106 ± 7 × 18 ± 2 nm), a morphotype of family Ackermannviridae. Additionally, the phage displayed the features resembling to existing Cba120viruses. Phage PM10 could infect S. enterica serovars Typhimurium, Typhi, and Enteritidis. The genome sequencing analysis of phage PM10 revealed circular 158.08 kb double-stranded DNA, with the GC content of 44.6%. Two hundred and nine ORFs, 171 putative promoters, 122 rho-independent terminators, and 5 transfer RNA encoding genes were found in the genome. The genome-wide comparisons and phylogenetic analyses showed that phage PM10 is closely related to Salmonella phage PhiSH19. Comparison of the tail-spike protein sequences encoded in PM10 and PhiSH19 genome showed the variation, which might have facilitated PM10's simultaneous infectivity to aforementioned S. enterica serovars. This is a varied host range than that of PhiSH19 or any other Cba120viruses.

A. baumannii is an opportunistic pathogen and a major cause of various community-acquired infections. Strains of this species can be resistant to multiple antimicrobial agents, leaving limited therapeutic options, also lacking in methods for accurate and prompt diagnosis. In this context, AbTJ, a novel phage that infects A. baumannii MDR-TJ, was isolated and characterized, together with its two tail fiber proteins. Morphological analysis revealed that it belongs to Podoviridae family. Its host range, growth characteristics, stability under various conditions, and genomic sequence, were systematically investigated. Bioinformatic analysis showed that AbTJ consists of a circular, double-stranded 42670-bp DNA molecule which contains 62 putative open reading frames (ORFs). Genome comparison revealed that the phage AbTJ is related to the Acinetobacter phage Ab105-1phi (No. KT588074). Tail fiber protein (TFPs) gp52 and gp53 were then identified and confirmed as species-specific proteins. By using a combination of bioluminescent methods and magnetic beads, these TFPs exhibit excellent specificity to detect A. baumannii. The findings of this study can be used to help control opportunistic infections and to provide pathogen-binding modules for further construction of engineered bacteria of diagnosis and treatment.

Broad and unspecific use of antibiotics accelerates spread of resistances. Sensitive and robust pathogen detection is thus important for a more targeted application. Bacteriophages contain a large repertoire of pathogen‐binding proteins. These tailspike proteins (TSP) often bind surface glycans and represent a promising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizes the O‐polysaccharide of dysentery‐causing Shigella flexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnose backbone conformations were obtained from 2D 1H,1H‐trNOESY NMR utilizing methine–methine and methine–methyl correlations. They agreed well with conformations obtained from molecular dynamics (MD), validating the method for further predictions. In a set of mutants, MD predicted ligand flexibilities that were in good correlation with binding strength as confirmed on immobilized S. flexneri O‐polysaccharide (PS) with surface plasmon resonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP‐based pathogen sensor design., The way to go: Antibiotic resistance is increasing worldwide. By understanding and fine‐tuning protein–carbohydrate interaction, improved bacterial sensors for faster diagnostics can be developed for example based on bacteriophage tailspike proteins. We used molecular dynamics simulations, NMR spectroscopy and surface plasmon resonance experiments for the design of affinity improved Shigella flexneri O‐antigen binding tailspike proteins.

In efforts to speed up the assessment of microorganisms, researchers have sought to use bacteriophages as a biosensing tool, due to their host-specificity, wide abundance, and safety. However, the lytic cycle of the phage has limited its efficacy as a biosensor. Here, we cloned a fragment of tail protein J from phage lambda and characterized its binding with the host, E. coli K-12, and other microorganism. The N-terminus of J was fused with a His-tag (6HN-J), overexpressed, purified, and characterized using anti-His monoclonal antibodies. The purified protein demonstrated a size of ∼38 kDa upon SDS-PAGE and bound with the anti-His monoclonal antibodies. ELISA, dot blot, and TEM data revealed that it specifically bound to E. coli K-12, but not to Pseudomonas aeruginosa. The observed protein binding occurred over a concentration range of 0.01-5 μg/ml and was found to inhibit the in vivo adsorption of phage to host cells. This specific binding was exploited by surface plasmon resonance (SPR) to generate a novel 6HN-J-functionalized SPR biosensor. This biosensor showed rapid label-free detection of E. coli K-12 in the range of 2 × 10

Nongenetic variation drives viral evolution Bacteriophage λ is a virus that infects bacteria by exploiting various membrane proteins in a well-characterized manner. Petrie et al. show how the evolution of variable folding conformations of isogenic proteins, which do not differ in their genetic sequences, contributed to λ's ability to exploit an additional host receptor for infection. Because the protein can take on two shapes, this genotype can have two phenotypes. Natural selection may thus be able to act on this nongenetic heterogeneity to connect phenotypic heterogeneity, evolvability, and protein stability. Science , this issue p. 1542

P22 bacteriophage has been studied extensively and has served as a model for many important processes such as in vivo protein folding, protein aggregation and protein-protein interactions. The trimeric tailspike protein (TSP) serves as the receptor-binding protein for the P22 bacteriophage to the bacterial host. The homotrimeric P22 tail consists of three chains of 666aa in which the first 108aa form a trimeric dome-like structure which is called the N-terminal domain (NTD) and is responsible for attachment of the tailspike protein to the rest of the phage particle structure in the phage assembly pathway. Knowledge of this interaction requires information on what amino acids are interacting in the interface and how the NTD structure is maintained. The first 23aa form the "stem peptide" which originates at the dome top and terminates at the dome bottom. It contains a hydrophobic valine patch (V8-V9-V10) located within the dome structure. It is hypothesized that the interaction between the hydrophobic valine patch located on stem peptide and the adjacent polypeptide is critical for the interchain interaction which should be important for the stability of the P22 TSP NTD itself. To test this hypothesis, each amino acid in the valine residues is substituted by an acid, a basic, and a hydrophobic amino acid. The results of such substitutions are presented as well as associated studies. The data strongly suggest that the valine patch is of critical importance in the hydrophobic interaction between stem peptide valine patch and an adjacent chain.

The sophisticated tail structures of DNA bacteriophages play essential roles in life cycles. Podoviruses P22 and Sf6 have short tails consisting of multiple proteins, among which is a tail adaptor protein that connects the portal protein to the other tail proteins. Assembly of the tail has been shown to occur in a sequential manner to ensure proper molecular interactions, but the underlying mechanism remains to be understood. Here, we report the high-resolution structure of the tail adaptor protein gp7 from phage Sf6. The structure exhibits distinct distribution of opposite charges on two sides of the molecule. A gp7 dodecameric ring model shows an entirely negatively charged surface, suggesting that the assembly of the dodecamer occurs through head-to-tail interactions of the bipolar monomers. The N-terminal helix-loop structure undergoes rearrangement compared with that of the P22 homolog complexed with the portal, which is achieved by repositioning of two consecutive repeats of a conserved octad sequence motif. We propose that the conformation of the N-terminal helix-loop observed in the Sf6-gp7 and P22 portal:gp4 complex represents the pre- and postassembly state, respectively. Such motif repositioning may serve as a conformational switch that creates the docking site for the tail nozzle only after the assembly of adaptor protein to the portal. In addition, the C-terminal portion of gp7 shows conformational flexibility, indicating an induced fit on binding to the portal. These results provide insight into the mechanistic role of the adaptor protein in mediating the sequential assembly of the phage tail.

In this paper, the enzymatic activity, substrate specificity and antibiofilm feature of bacteriophage dual-function tail proteins are presented. So far, tail tubular proteins A–TTPAgp31 and TTPAgp44-have been considered as structural proteins of Klebsiella pneumoniae bacteriophages KP32 and KP34, respectively. Our results show that TTPAgp31 is able to hydrolyze maltose as well as Red-starch. The activity of 1 µM of the protein was calculated as 47.6 milli-Units/assay relating to the α-amylase activity. It degrades capsular polysaccharides (cPS), slime polysaccharides (sPS) and lipopolysaccharide (LPS) of K. pneumoniae PCM 2713 and shows antibiofilm reactivity towards S. aureus PCM 519 and E. faecalis PCM 2673. TTPAgp44 hydrolyses trehalose and cPS of E. faecium PCM 1859. TTPAgp44′s activity was also observed in the antibiofilm test against P. aeruginosa PCM 2710 and B. subtilis PCM 2021. TTPAgp31 has been identified as α-1,4-glucosidase whereas, TTPAgp44 exhibits trehalase-like activity. Both proteins contain aspartate and glutamate residues in the β-stranded region which are essential for catalytic activity of glycoside hydrolases. The significant novelty of our results is that for the first time the bacteriophage tubular proteins are described as the unique enzymes displaying no similarity to any known phage hydrolases. They can be used as antibacterial agents directed against bacterial strains producing exopolysaccharides and forming a biofilm.

The vast majority of phages, bacterial viruses, possess a tail ensuring host recognition, cell wall perforation and safe viral DNA transfer from the capsid to the host cytoplasm. Long flexible tails are formed from the tail tube protein (TTP) polymerised as hexameric rings around and stacked along the tape measure protein (TMP). Here, we report the crystal structure of T5 TTP pb6 at 2.2 Å resolution. Pb6 is unusual in forming a trimeric ring, although structure analysis reveals homology with all classical TTPs and related tube proteins of bacterial puncturing devices (type VI secretion system and R-pyocin). Structures of T5 tail tubes before and after interaction with the host receptor were determined by cryo-electron microscopy at 6 Å resolution. Comparison of these two structures reveals that host-binding information is not propagated to the capsid through conformational changes in the tail tube, suggesting a role of the TMP in this information transduction process., Host cell recognition is mediated by the phage tail tip proteins, which then triggers viral genome delivery via the phage tail. Here, the authors combine crystallography and cryoEM to structurally characterise the bacteriophage T5 tail tube structure before and after interaction with its host receptor.

Phage G is recognized as having a remarkably large genome and capsid size among isolated, propagated phages. Negative stain electron microscopy of the host–phage G interaction reveals tail sheaths that are contracted towards the distal tip and decoupled from the head–neck region. This is different from the typical myophage tail contraction, where the sheath contracts upward, while being linked to the head–neck region. Our cryo-EM structures of the non-contracted and contracted tail sheath show that: (1) The protein fold of the sheath protein is very similar to its counterpart in smaller, contractile phages such as T4 and phi812, (2) Phage G’s sheath structure in the non-contracted and contracted states are similar to phage T4’s sheath structure. Similarity to other myophages is confirmed by a comparison-based study of the tail sheath’s helical symmetry, the sheath protein’s evolutionary timetree, and the organization of genes involved in tail morphogenesis . Atypical phase G tail contraction could be due to a missing anchor point at the upper end of the tail sheath that allows the decoupling of the sheath from the head­–neck region. Explaining the atypical tail contraction requires further investigation of the phage G sheath anchor points.

Due to the emergence of multidrug-resistant bacteria, bacteriophages have become a viable alternative in controlling bacterial growth or biofilm formation. Biofilm is formed by extracellular polymeric substances (EPS) and is one of the factors responsible for increasing bacterial resistance . Bacteriophages have been studied as a bacterial control agent by use of phage enzymes or due to their bactericidal activities . A specific phage against Serratia marcescens was isolated in this work and was evaluated its biological and genomic aspects. The object of this study was UFV01, a bacteriophage belonging to the Podoviridae family, genus Teseptimavirus (group of lytic viruses), specific to the species S. marcescens , which may be related to several amino acid substitutions in the virus tail fibers. Despite this high specificity, the phage reduced the biofilm formation of several Escherichia coli strains without infecting them. UFV01 presents a relationship with phages of the genus Teseptimavirus, although it does not infect any of the E. coli strains evaluated, as these others do. All the characteristics make the phage an interesting alternative in biofilm control in hospital environments since small breaks in the biofilm matrix can lead to a complete collapse.

Bacteriophage SP6 exhibits dual-host adsorption specificity. The SP6 tailspikes are recognized as important in host range determination but the mechanisms underlying dual host specificity are unknown. Cryo-electron tomography and sub-tomogram classification were used to analyze the SP6 virion with a particular focus on the interaction of tailspikes with host membranes. The SP6 tail is surrounded by six V-shaped structures that interconnect in forming a hand-over-hand hexameric garland. Each V-shaped structure consists of two trimeric tailspike proteins: gp46 and gp47, connected through the adaptor protein gp37. SP6 infection of Salmonella enterica serovars Typhimurium and Newport results in distinguishable changes in tailspike orientation, providing the first direct demonstration how tailspikes can confer dual host adsorption specificity. SP6 also infects S. Typhimurium strains lacking O antigen; in these infections tailspikes have no apparent specific role and the phage tail must therefore interact with a distinct host receptor to allow infection.

SP6 is a salmonella phage closely related to coliphage K1-5. K1-5 is notable in that it encodes two polysaccharide-degrading tailspike proteins, an endosialidase that allows it to infect E. coli K1, and a lyase that enables it to infect K5 strains. SP6 is similar to K1-5 except that it encodes a P22-like endorhamnosidase tailspike, gp46, allowing it to infect group B Salmonella. We show here that SP6 can also infect Salmonella serogroups C2 and C3 and that a mutation in a putative second tailspike, gp47, eliminates this specificity. Gene 47 was fused to the coding region of the N-terminal portion of the Pseudomonas aeruginosa R2 pyocin tail fiber and expressed in trans such that the fusion protein becomes incorporated into pyocin particles. These pyocins, termed AvR2-SP47, killed serogroups C2 and C3 Salmonella. We conclude that SP6 encodes two tail proteins providing it a broad host range among Salmonella enterica.

Bacteriophages recognize and bind to their hosts with the help of receptor-binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C - tailspikes gp63.1 and gp66 - are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and function of gp63.1 and show that unlike any previously described RPB, gp63.1 deacetylates surface polysaccharides of E. coli 4s leaving the backbone of the polysaccharide intact. We demonstrate that gp63.1 and gp66 form a stable complex, in which the N-terminal part of gp66 serves as an attachment site for gp63.1 and anchors the gp63.1-gp66 complex to the G7C tail. The esterase domain of gp63.1 as well as domains mediating the gp63.1-gp66 interaction is widespread among all three families of tailed bacteriophages.

Highlights • A virulent G2b PEDV strain KNU-141112 was subjected to Vero cell culture passage. • Deletions (DEL) in S and ORF3 of KNU-141112 at high passage levels were identified. • Cell-adapted KNU-141112 DEL strains were highly attenuated and immunogenic in pigs. • Large deletions in ORF3 appear to be associated with PEDV attenuation in pigs. • High-cell-passage KNU-141112 DEL strains can be live, attenuated vaccine candidates., Porcine epidemic diarrhea virus (PEDV) has emerged or re-emerged worldwide, posing a significant financial threat to major pig-producing countries. In the present study, a virulent Korean pandemic PEDV strain, KNU-141112, was serially propagated in Vero cells for up to 100 passages. Through cell culture adaptation, we obtained four distinct deletion (DEL) mutants by plaque purification followed by nucleotide sequencing of the spike (S)/ORF3 gene-coding region, which were designated KNU-141112-S DEL2, −S DEL5, −S DEL2/ORF3, and −S DEL5/ORF3. Further whole genome sequencing identified 12 or 14 amino acid changes in the cell-adapted DEL strains. Animal inoculation studies revealed that the virulence of both S DEL2/ORF3 and S DEL5/ORF3 viruses with a large 46-nt deletion in the intergenic portion of S and ORF3 was remarkably diminished, indicating viral attenuation in the natural host. Furthermore, these cell-adapted strains elicited potent neutralizing antibody responses in immunized pigs. Taken together, our data indicate that the cell-attenuated S DEL2/ORF3 and S DEL5/ORF3 strains are promising candidates for the development of a safe and effective live PEDV vaccine.

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural &ldquo, bricks&rdquo, to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has spread globally and emerged as an urgent public health threat. Bacteriophages are considered an effective weapon against multidrug-resistant pathogens. In this study, we report a novel lytic phage, kpssk3, which is able to lyse CRKP and degrade exopolysaccharide (EPS). The morphological characteristics of kpssk3 observed by transmission electron microscopy, including a polyhedral head and a short tail, indicate that it belongs to the family Podoviridae. A one-step growth curve revealed that kpssk3 has a latent period of 10 min and a burst size of 200 plaque-forming units (pfu) per cell. kpssk3 was able to lyse 25 out of 27 (92.59%) clinically isolated CRKP strains, and it also exhibited high stability to changes in temperature and pH. kpssk3 has a linear dsDNA genome of 40,539 bp with 52.80% G+C content and 42 putative open reading frames (ORFs). No antibiotic resistance genes, virulence factors, or integrases were identified in the genome. Based on bioinformatic analysis, the tail fiber protein of phage kpssk3 was speculated to possess depolymerase activity towards EPS. By comparative genomics and phylogenetic analysis, it was determined that kpssk3 is a new T7-like virus and belongs to the subfamily Autographivirinae. The characterization and genomic analysis of kpssk3 will promote our understanding of phage biology and diversity and provide a potential strategy for controlling CRKP infection.

Bacteriophage tailspike proteins mediate virion absorption through reversible primary receptor binding, followed by lipopolysaccharide or exopolysaccharide degradation. The Escherichia coli O157:H7 bacteriophage CBA120 genome encodes four distinct tailspike proteins, annotated as ORFs 210 through 213. Previously, we reported the crystal structure of ORF210 (TSP1). Here we describe the crystal structure of ORF212 (TSP3) determined at 1.85 Å resolution. As observed with other tailspike proteins, TSP3 assembles into a trimer. Each subunit of TSP3 has an N-terminal head domain that is structurally similar to that of TSP1, consistent with their high amino acid sequence identity. In contrast, despite sharing a β-helix fold, the overall structure of the C-terminal catalytic domain of TSP3 is quite different when compared to TSP1. The TSP3 structure suggests that the glycosidase active site resides in a cleft at the interface between two adjacent subunits where three acidic residues, Glu362 and Asp383 on one subunit, and Asp426 on a second subunit, are located in close proximity. Comparing the glycosidase activity of wild-type TSP3 to various point mutants revealed that catalysis requires the carboxyl groups of Glu362 and Asp426, and not of Asp383, confirming the enzyme employs two carboxyl groups to degrade lippopolysaccharide using an acid/base mechanism.