Tail Fiber Research Articles

Effect of a Depolymerase Encoded by Phage168 on a Carbapenem-Resistant Klebsiella pneumoniae and Its Biofilm.

Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are becoming increasingly common within clinical settings, requiring the development of alternative therapies. In this study, we isolated, characterized, and sequenced the genome of a CRKP phage, Phage168. The total genomic DNA of Phage168 was 40,222 bp in length, encoding 49 predicted proteins. Among these proteins, Dep40, the gene product of ORF40, is a putative tail fiber protein that exhibits depolymerase activity based on the result of bioinformatics analyses. In vitro, we confirmed that the molecular weight of the Phage168 depolymerase protein was about 110 kDa, the concentration of the produced phage 168 depolymerase protein was quantified as being 1.2 mg/mL, and the depolymerase activity was still detectable after the dilution of 1.2 µg/mL. This recombinant depolymerase exhibited enzyme activity during the depolymerization of the formed CRKP biofilms. We also found that depolymerase, when combined with polymyxin B, was able to enhance the bactericidal effect of polymyxin B on CRKP strains by disrupting their biofilm. When recombinant depolymerase was used in combination with human serum, it enhanced the sensitivity of the CRKP strain UA168 to human serum, and the synergistic bactericidal effect reached the strongest level when the ratio of depolymerase to human serum was 3:1. Our results indicated that depolymerase encoded by Phage168 may be a promising strategy for combating infections caused by drug-resistant CRKP formed within the biofilm.

Coevolutionary phage training andJoint application delays the emergence ofphage resistance inPseudomonas aeruginosa.

Antibiotic-resistant bacteria are current threats to available antibiotic therapies, and this has renewed interest in the therapeutic use of phage as an alternative. However, development of phage resistance has led to unsuccessful therapeutic outcomes. In the current study, we applied phage training to minimize bacterial phage resistance and to improve treatment outcome by adapting the phage to their target hosts during co-evolution. We isolated and characterized a novel Pseudomonas aeruginosa N4-like lytic phage (PWJ) from wastewater in Yangzhou, China. PWJ is a double-stranded DNA podovirus that can efficiently lyse the model strain ATCC 27,853 and opportunistic pathogen PAO1. Genome sequencing of PWJ revealed features similar to those of the N4-like P. aeruginosa phage YH6. We used PWJ to screen for an evolved trained phage (WJ_Ev14) that restored infectivity to PWJ phage bacterial resisters. BLASTN analysis revealed that WJ_Ev14 is identical to its ancestor PWJ except for the amino acid substitution R1051S in its tail fiber protein. Moreover, phage adsorption tests and transmission electron microscopy of resistant bacteria demonstrated that the R1051S substitution was most likely the reason WJ_Ev14 could re-adsorb and regain infectivity. Furthermore, phage therapy assays in vitro and in a mouse P. aeruginosa lung infection model demonstrated that PWJ treatment resulted in improved clinical results and a reduction in lung bacterial load whereas the joint phage cocktail (PWJ+ WJ_Ev14) was better able to delay the emergence of resister bacteria. The phage cocktail (PWJ +WJ_Ev14) represents a promising candidate for inclusion in phage cocktails developed for clinical applications.

A Novel Subcluster of Closely Related Bacillus Phages with Distinct Tail Fiber/Lysin Gene Combinations.

Bacteriophages (phages) are the most numerous entities on Earth, but we have only scratched the surface of describing phage diversity. We isolated seven Bacillus subtilis phages from desert soil in the southwest United States and then sequenced and characterized their genomes. Comparative analyses revealed high nucleotide and amino acid similarity between these seven phages, which constitute a novel subcluster. Interestingly, the tail fiber and lysin genes of these phages seem to come from different origins and carry out slightly different functions. These genes were likely acquired by this subcluster of phages via horizontal gene transfer. In conjunction with host range assays, our data suggest that these phages are adapting to hosts with different cell walls.

Phage Milagro: a platform for engineering a broad host range virulent phage for Burkholderia.

Burkholderia infections are a significant concern in people with CF and other immunocompromising disorders, and are difficult to treat with conventional antibiotics due to their inherent drug resistance. Bacteriophages, or bacterial viruses, are now seen as a potential alternative therapy for these infections, but most of the naturally occurring phages are temperate and have narrow host ranges, which limit their utility as therapeutics. Here we describe the temperate Burkholderia phage Milagro and our efforts to engineer this phage into a potential therapeutic by expanding the phage host range and selecting for phage mutants that are strictly virulent. This approach may be used to generate new therapeutic agents for treating intractable infections in CF patients.

Distantly related Alteromonas bacteriophages share tail fibers exhibiting properties of transient chaperone caps

The host recognition modules encoding the injection machinery and receptor binding proteins (RBPs) of bacteriophages are predisposed to mutation and recombination to maintain infectivity towards co-evolving bacterial hosts. In this study, we reveal how Alteromonas mediterranea schitovirus A5 shares its host recognition module, including tail fiber and cognate chaperone, with phages from distantly related families including Alteromonas myovirus V22. While the V22 chaperone is essential for producing active tail fibers, here we demonstrate production of functional A5 tail fibers regardless of chaperone co-expression. AlphaFold-generated models of tail fiber and chaperone pairs from phages A5, V22, and other Alteromonas phages reveal how amino acid insertions within both A5-like proteins results in a knob domain duplication in the tail fiber and a chaperone β-hairpin “tentacle” extension. These structural modifications are linked to differences in chaperone dependency between the A5 and V22 tail fibers. Structural similarity between the chaperones and intramolecular chaperone domains of other phage RBPs suggests an additional function of these chaperones as transient fiber “caps”. Finally, our identification of homologous host recognition modules from morphologically distinct phages implies that horizontal gene transfer and recombination events between unrelated phages may be a more common process than previously thought among Caudoviricetes phages.

The Isolation and Characterization of Bacteriophages Infecting Avian Pathogenic Escherichia coli O1, O2 and O78 Strains.

Avian pathogenic Escherichia coli (APEC), such as O1, O2 and O78, are important serogroups relating to chicken health, being responsible for colibacillosis. In this study, we isolated and characterized bacteriophages (phages) from hen feces and human sewage in Alberta with the potential for controlling colibacillosis in laying hens. The lytic profile, host range, pH tolerance and morphology of seven APEC-infecting phages (ASO1A, ASO1B, ASO2A, ASO78A, ASO2B, AVIO78A and ASO78B) were assessed using a microplate phage virulence assay and transmission electron microscopy (TEM). The potential safety of phages at the genome level was predicted using AMRFinderPlus and the Virulence Factor Database. Finally, phage genera and genetic relatedness with other known phages from the NCBI GenBank database were inferred using the virus intergenomic distance calculator and single gene-based phylogenetic trees. The seven APEC-infecting phages preferentially lysed APEC strains in this study, with ECL21443 (O2) being the most susceptible to phages (n = 5). ASO78A had the broadest host range, lysing all tested strains (n = 5) except ECL20885 (O1). Phages were viable at a pH of 2.5 or 3.5-9.0 after 4 h of incubation. Based on TEM, phages were classed as myovirus, siphovirus and podovirus. No genes associated with virulence, antimicrobial resistance or lysogeny were detected in phage genomes. Comparative genomic analysis placed six of the seven phages in five genera: Felixounavirus (ASO1A and ASO1B), Phapecoctavirus (ASO2A), Tequatrovirus (ASO78A), Kayfunavirus (ASO2B) and Sashavirus (AVIO78A). Based on the nucleotide intergenomic similarity (<70%), phage ASO78B was not assigned a genus in the siphovirus and could represent a new genus in class Caudoviricetes. The tail fiber protein phylogeny revealed variations within APEC-infecting phages and closely related phages. Diverse APEC-infecting phages harbored in the environment demonstrate the potential to control colibacillosis in poultry.

The Novel Yersinia enterocolitica Telomere Phage vB_YenS_P840 Is Closely Related to PY54, but Reveals Some Striking Differences.

Telomere phages are a small group of temperate phages, whose prophages replicate as a linear plasmid with covalently closed ends. They have been isolated from some Enterobacteriaceae and from bacterial species living in aquatic environments. Phage PY54 was the first Yersinia (Y.) enterocolitica telomere phage isolated from a nonpathogenic O:5 strain, but recently a second telomeric Yersinia phage (vB_YenS_P840) was isolated from a tonsil of a wild boar in Germany. Both PY54 and vB_YenS_P840 (P840) have a siphoviridal morphology and a similar genome organization including the primary immunity region immB and telomere resolution site telRL. However, whereas PY54 only possesses one prophage repressor for the lysogenic cycle, vB_YenS_P840 encodes two. The telRL region of this phage was shown to be processed by the PY54 protelomerase under in vivo conditions, but unlike with PY54, a flanking inverted repeat was not required for processing. A further substantial difference between the phages is their host specificity. While PY54 infects Y. enterocolitica strains belonging to the serotypes O:5 and O:5,27, vB_YenS_P840 exclusively lyses O:3 strains. As the tail fiber and tail fiber assembly proteins of the phages differ significantly, we introduced the corresponding genes of vB_YenS_P840 by transposon mutagenesis into the PY54 genome and isolated several mutants that were able to infect both serotypes, O:5,27 and O:3.

Efficient screening of adsorbed receptors for Salmonella phage LP31 and identification of receptor-binding protein.

The adsorption process is the first step in the lifecycle of phages and plays a decisive role in the entire infection process. Identifying the adsorption mechanism of phages not only makes phage therapy more precise and efficient but also enables the exploration of other potential applications and modifications of phages. Phage LP31 can lyse multiple Salmonella serotypes, efficiently clearing biofilms formed by Salmonella enterica serovar Enteritidis (S. Enteritidis) and significantly reducing the concentration of S. Enteritidis in chicken feces. Therefore, LP31 has great potential for many practical applications. In this study, we established an efficient screening method for phage infection-related genes and identified a total of 10 genes related to the adsorption process of phage LP31. After the construction of strain C50041ΔrfaL 58-358, it was found that the knockout strain had a rough phenotype as an O-antigen-deficient strain. Adsorption rate and transmission electron microscopy experiments showed that the receptor for phage LP31 was the O9 antigen of S. Enteritidis. Homology comparison and adsorption experiments confirmed that the tail fiber protein Lp35 of phage LP31 participated in the adsorption process as a receptor-binding protein. IMPORTANCE A full understanding of the interaction between phages and their receptors can help with the development of phage-related products. Phages like LP31 with the tail fiber protein Lp35, or a closely related protein, have been reported to effectively recognize and infect multiple Salmonella serotypes. However, the role of these proteins in phage infection has not been previously described. In this study, we established an efficient screening method to detect phage adsorption to host receptors. We found that phage LP31 can utilize its tail fiber protein Lp35 to adsorb to the O9 antigen of S. Enteritidis, initiating the infection process. This study provides a great model system for further studies of how a phage-encoded receptor-binding protein (RBP) interacts with its host's RBP binding target, and this new model offers opportunities for further theoretical and experimental studies to understand the infection mechanism of phages.

Asymmetric Structure of Podophage GP4 Reveals a Novel Architecture of Three Types of Tail Fibers

Bacteriophage tail fibers (or called tail spikes) play a critical role in the early stage of infection by binding to the bacterial surface. Podophages with known structures usually possess one or two types of fibers. Here, we resolved an asymmetric structure of the podophage GP4 to near-atomic resolution by cryo-EM. Our structure revealed a symmetry-mismatch relationship between the components of the GP4 tail with previously unseen topologies. In detail, two dodecameric adaptors (adaptors I and II), a hexameric nozzle, and a tail needle form a conserved tail body connected to a dodecameric portal occupying a unique vertex of the icosahedral head. However, five chain-like extended fibers (fiber I) and five tulip-like short fibers (fiber II) are anchored to a 15-fold symmetric fiber-tail adaptor, encircling the adaptor I, and six bamboo-like trimeric fibers (fiber III) are connected to the nozzle. Five fibers I, each composed of five dimers of the protein gp80 linked by an elongated rope protein, are attached to the five edges of the tail vertex of the icosahedral head. In this study, we identified a new structure of the podophage with three types of tail fibers, and such phages with different types of fibers may have a broad host range and/or infect host cells with considerably high efficiency, providing evolutionary advantages in harsh environments.

Isolation, characterization and application of a lytic phage vB_VspM_VS1 against Vibrio splendidus biofilm.

Vibrio splendidus is a common pathogen in the ocean that infects Apostichopus japonicus, Atlantic salmon and Crassostrea gigas, leading to a variety of diseases. In this study, a virulent phage vB_VspM_VS1, which infects V. splendidus, was isolated from aquaculture ponds in Dalian, China, and it belongs to the family Straboviridae in the order Caudoviricetes. vB_VspM_VS1 had an adsorption rate of 96% in 15 min, a latent period of 65 min, and a burst size of 140 ± 6 PFU/cell. The complete genome of phage vB_VspM_VS1 consists of a linear double-stranded DNA that is 248,270 bp in length with an average G + C content of 42.5% and 389 putative protein-coding genes; 116 genes have known functions. There are 4 tail fiber genes in the positive and negative strands of the phage vB_VspM_VS1 genome. The protein domain of the phage vB_VspM_VS1 tail fibers was obtained from the Protein Data Bank and the SMART (http://smart.embl.de) database. Bacterial challenge tests revealed that the growth of V. splendidus HS0 was apparently inhibited (OD600 < 0.01) in 12 h at an MOI of 10. In against biofilms, we also showed that the OD570 value of the vB_VspM_VS1-treated group (MOI = 1) decreased significantly to 0.04 ± 0.01 compared with that of the control group (0.48 ± 0.08) at 24 h. This study characterizes the genome of the phage vB_VspM_VS1 that infects the pathogenic bacterium V. splendidus of A. japonicus.

Isolation and Characterization of the Acadevirus Members BigMira and MidiMira Infecting a Highly Pathogenic Proteus mirabilis Strain.

Proteus mirabilis is an opportunistic pathogen and is responsible for more than 40% of all cases of catheter-associated urinary tract infections (CAUTIs). Healthcare-associated infections have been aggravated by the constant emergence of antibiotic-resistant bacterial strains. Because of this, the use of phages to combat bacterial infections gained renewed interest. In this study, we describe the biological and genomic features of two P. mirabilis phages, named BigMira and MidiMira. These phages belong to the Acadevirus genus (family Autographiviridae). BigMira and MidiMira are highly similar, differing only in four missense mutations in their phage tail fiber. These mutations are sufficient to impact the phages' depolymerase activity. Subsequently, the comparative genomic analysis of ten clinical P. mirabilis strains revealed differences in their antibiotic resistance profiles and lipopolysaccharide locus, with the latter potentially explaining the host range data of the phages. The massive presence of antimicrobial resistance genes, especially in the phages' isolation strain P. mirabilis MCS, highlights the challenges in treating infections caused by multidrug-resistant bacteria. The findings reinforce BigMira and MidiMira phages as candidates for phage therapy purposes.

Paper-based analytical device integrated with bacteriophage tail fiber protein for bacteria detection and antimicrobial susceptibility test

Severe antimicrobial resistance calls for developing rapid, sensitive and affordable methodological platform for clinical diagnosis of bacterial infection. Herein, a paper-based analytical device (PAD) for fluorescent (FL) detection and antimicrobial susceptibility test (AST) of Pseudomonas aeruginosa (P. aeruginosa) was fabricated by defining 96 hydrophilic microzones on a filter paper using a handheld stamp dipped with liquid wax. The size of microzones was designed to be consistent with traditional 96-well microplate, thus the FL signals can be collected by a commercialized microplate reader. Streptavidin was immobilized into the microzones by chitosan-glutaraldehyde crosslinking reaction, and then biotinylated bacteriophage tail fiber protein (TFP) was conjugated through biotin-streptavidin affinity system. TFP and fluorescein isothiocyanate (FITC) labeled antimicrobial peptide were used as capture agent and signal probe, respectively, for FL detection of P. aeruginosa on the PAD. The linear range for quantifying P. aeruginosa is 1.0 × 103 CFU/mL to 1.0 × 107 CFU/mL, with a detection limit of 137 CFU/mL. The PAD was also applied to conduct AST of P. aeruginosa for imipenem, meropenem, cefepime, amikacin, and gentamicin, and the results are consistent with the traditional broth dilution method. The PAD provides an affordable diagnosis platform for bacterial infection, especially in resource-limited institutes and countries.

Phage tailspike modularity and horizontal gene transfer reveals specificity towards E. coli O-antigen serogroups

BackgroundThe interaction between bacteriophages and their hosts is intricate and highly specific. Receptor-binding proteins (RBPs) of phages such as tail fibers and tailspikes initiate the infection process. These RBPs bind to diverse outer membrane structures, including the O-antigen, which is a serogroup-specific sugar-based component of the outer lipopolysaccharide layer of Gram-negative bacteria. Among the most virulent Escherichia coli strains is the Shiga toxin-producing E. coli (STEC) pathotype dominated by a subset of O-antigen serogroups.MethodsExtensive phylogenetic and structural analyses were used to identify and validate specificity correlations between phage RBP subtypes and STEC O-antigen serogroups, relying on the principle of horizontal gene transfer as main driver for RBP evolution.ResultsWe identified O-antigen specific RBP subtypes for seven out of nine most prevalent STEC serogroups (O26, O45, O103, O104, O111, O145 and O157) and seven additional E. coli serogroups (O2, O8, O16, O18, 4s/O22, O77 and O78). Eight phage genera (Gamaleya-, Justusliebig-, Kaguna-, Kayfuna-, Kutter-, Lederberg-, Nouzilly- and Uetakeviruses) emerged for their high proportion of serogroup-specific RBPs. Additionally, we reveal sequence motifs in the RBP region, potentially serving as recombination hotspots between lytic phages.ConclusionThe results contribute to a better understanding of mosaicism of phage RBPs, but also demonstrate a method to identify and validate new RBP subtypes for current and future emerging serogroups.

Phages on filaments: A genetic screen elucidates the complex interactions between Salmonella enterica flagellin and bacteriophage Chi.

The bacterial flagellum is a rotary motor organelle and important virulence factor that propels motile pathogenic bacteria, such as Salmonella enterica, through their surroundings. Bacteriophages, or phages, are viruses that solely infect bacteria. As such, phages have myriad applications in the healthcare field, including phage therapy against antibiotic-resistant bacterial pathogens. Bacteriophage χ (Chi) is a flagellum-dependent (flagellotropic) bacteriophage, which begins its infection cycle by attaching its long tail fiber to the S. enterica flagellar filament as its primary receptor. The interactions between phage and flagellum are poorly understood, as are the reasons that χ only kills certain Salmonella serotypes while others entirely evade phage infection. In this study, we used molecular cloning, targeted mutagenesis, heterologous flagellin expression, and phage-host interaction assays to determine which domains within the flagellar filament protein flagellin mediate this complex interaction. We identified the antigenic N- and C-terminal D2 domains as essential for phage χ binding, with the hypervariable central D3 domain playing a less crucial role. Here, we report that the primary structure of the Salmonella flagellin D2 domains is the major determinant of χ adhesion. The phage susceptibility of a strain is directly tied to these domains. We additionally uncovered important information about flagellar function. The central and most variable domain, D3, is not required for motility in S. Typhimurium 14028s, as it can be deleted or its sequence composition can be significantly altered with minimal impacts on motility. Further knowledge about the complex interactions between flagellotropic phage χ and its primary bacterial receptor may allow genetic engineering of its host range for use as targeted antimicrobial therapy against motile pathogens of the χ-host genera Salmonella, Escherichia, or Serratia.

High-resolution cryo-EM structure of the Pseudomonas bacteriophage E217

E217 is a Pseudomonas phage used in an experimental cocktail to eradicate cystic fibrosis-associated Pseudomonas aeruginosa. Here, we describe the structure of the whole E217 virion before and after DNA ejection at 3.1 Å and 4.5 Å resolution, respectively, determined using cryogenic electron microscopy (cryo-EM). We identify and build de novo structures for 19 unique E217 gene products, resolve the tail genome-ejection machine in both extended and contracted states, and decipher the complete architecture of the baseplate formed by 66 polypeptide chains. We also determine that E217 recognizes the host O-antigen as a receptor, and we resolve the N-terminal portion of the O-antigen-binding tail fiber. We propose that E217 design principles presented in this paper are conserved across PB1-like Myoviridae phages of the Pbunavirus genus that encode a ~1.4 MDa baseplate, dramatically smaller than the coliphage T4.

The saclayvirus Aci01-1 very long and complex fiber and its receptor at the Acinetobacter baumannii surface.

The Acinetobacter baumannii bacteriophage Aci01-1, which belongs to the genus Saclayvirus of the order Caudoviricetes, has an icosahedral head and a contractile rigid tail. We report that Aci01-1 has, attached to the tail conical tip, a remarkable 146-nm-long flexible fiber with seven beads and a terminal knot. Its putative gene coding for a 241.36-kDa tail fiber protein is homologous to genes in Aci01-1-related and unrelated phages. Analysis of its 3D structure using AlphaFold provides a structural model for the fiber observed by electron microscopy. We also identified a putative receptor of the phage on the bacterial capsule that is hypothesized to interact with the Aci01-1 long fiber.

Characterization of the Attachment of Three New Coliphages onto the Ferrichrome Transporter FhuA.

Receptor-binding proteins (RBPs) allow phages to dock onto their host and initiate infection through the recognition of proteinaceous or saccharidic receptors located on the cell surface. FhuA is the ferrichrome hydroxamate transporter in Escherichia coli and serves as a receptor for the well-characterized phages T1, T5, and phi80. To further characterize how other FhuA-dependent phages attach to FhuA, we isolated and published the genomes of three new FhuA-dependent coliphages: JLBYU37, JLBYU41, and JLBYU60. We identified the egions of FhuA involved in phage attachment by testing the effect of mutant fhuA alleles containing single-loop deletions of extracellular loops (L3, L4, L5, L8, L10, and L11) on phage infectivity. Deletion of loop 8 resulted in complete resistance to SO1-like phages JLBYU37 and JLBYU60 and the previously isolated vB_EcoD_Teewinot phage, but no single-loop deletions significantly altered the infection of T1-like JLBYU41. Additionally, lipopolysaccharide (LPS) truncation coupled with the L5 mutant significantly impaired the infectivity of JLBYU37 and JLBYU60. Moreover, significant reductions in the infectivity of JLBYU41 were observed upon LPS truncation in the L8 mutant strain. Analysis of the evolutionary relationships among FhuA-dependent phage RBPs highlights the conservation of L8 dependence in JLBYU37, JLBYU60, Teewinot, T5, and phi80, but also showcases how positive selective pressure and/or homologous recombination also selected for L4 dependence in T1 and even the lack of complete loop dependence in JLBYU41. IMPORTANCE Phage attachment is the first step of phage infection and plays a role in governing host specificity. Characterizing the interactions taking place between phage tail fibers and bacterial receptors that better equip bacteria to survive within the human body may provide insights to aid the development of phage therapeutics.

Nature's Needles: Adapting Bacterial “Syringes” into Programmable Protein Delivery Devices

Nature's Needles: Adapting Bacterial “Syringes” into Programmable Protein Delivery Devices

F-Type Pyocins Are Diverse Noncontractile Phage Tail-Like Weapons for Killing Pseudomonas aeruginosa.

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or noncontractile phage tails known as R- and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3' end of the F-type pyocin cluster are divergent in sequence and likely determine bactericidal specificity. We use sequence similarities among these proteins to define eleven distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly reassorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins may have emerged earlier. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as antibacterial therapeutics. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes antibiotic-resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great need for the development of alternative therapeutics. In this study, we investigate one such potential therapeutic: F-type pyocins, which are bacteriocins naturally produced by P. aeruginosa that resemble noncontractile phage tails. We show that they are potent killers of P. aeruginosa and identify their probable bactericidal specificity determinants, which opens up the possibility of engineering them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well-characterized phage tails will greatly facilitate their development into effective antibacterials.

Insights into the Alcyoneusvirus Adsorption Complex.

The structures of the Caudovirales phage tails are key factors in determining the host specificity of these viruses. However, because of the enormous structural diversity, the molecular anatomy of the host recognition apparatus has been elucidated in only a number of phages. Klebsiella viruses vB_KleM_RaK2 (RaK2) and phiK64-1, which form a new genus Alcyoneusvirus according to the ICTV, have perhaps one of the most structurally sophisticated adsorption complexes of all tailed viruses described to date. Here, to gain insight into the early steps of the alcyoneusvirus infection process, the adsorption apparatus of bacteriophage RaK2 is studied in silico and in vitro. We experimentally demonstrate that ten proteins, gp098 and gp526-gp534, previously designated as putative structural/tail fiber proteins (TFPs), are present in the adsorption complex of RaK2. We show that two of these proteins, gp098 and gp531, are essential for attaching to Klebsiella pneumoniae KV-3 cells: gp531 is an active depolymerase that recognizes and degrades the capsule of this particular host, while gp098 is a secondary receptor-binding protein that requires the coordinated action of gp531. Finally, we demonstrate that RaK2 long tail fibers consist of nine TFPs, seven of which are depolymerases, and propose a model for their assembly.