Development of an enzyme-linked phage receptor-binding protein assay (ELPRA) based on a novel biorecognition molecule- receptor-binding protein Gp130 of Pseudomonas aeruginosa bacteriophage Henu5.

Pseudomonas aeruginosa is a Gram-negative bacterium associated with life-threatening healthcare-associated infections (HAIs), including burn wound infections, pneumonia and sepsis. Moreover, P. aeruginosa has been considered a pathogen of global concern due to its rising antibiotic resistance. Efficient identification of P. aeruginosa would significantly benefit the containment of bacterial infections, prevent pathogen transmission, and provide orientated treatment options. The accuracy and specificity of bacterial detection are primarily dictated by the biorecognition molecules employed. Lytic bacteriophages (or phages) could specifically attach to and lyse host bacterial cells. Phages' host specificity is typically determined by their receptor-binding proteins (RBPs), which recognize and adsorb phages to particular bacterial host receptors. This makes RBPs promising biorecognition molecules in bacterial detection. This study identified a novel RBP (Gp130) from the P. aeruginosa phage Henu5. A modified enzyme-linked phage receptor-binding protein assay (ELPRA) was developed for P. aeruginosa detection employing Gp130 as biorecognition molecules. Optimized conditions provided a calibration curve for P. aeruginosa with a range from 1.0 × 103 to 1.0 × 107 CFU/mL, with a limit of detection as low as 10 CFU/mL in phosphate-buffered saline (PBS). With VITEKⓇ 2 Compact system identification (40 positives and 21 negatives) as the gold standard, the sensitivity of ELPRA was 0.950 (0.818-0.991), and the specificity was 0.905 (0.682-0.983) within a 95 %confidence interval. Moreover, the recovery test in spiked mouse serum showed recovery rates ranging from 82.79 %to 98.17%, demonstrating the prospect of the proposed ELPRA for detecting P. aeruginosa in biological samples.

Tentaclins-A Novel Family of Phage Receptor-Binding Proteins That Can Be Hypermutated by DGR Systems.

Diversity-generating retroelements (DGRs) are prokaryotic systems providing rapid modification and adaptation of target proteins. In phages, the main targets of DGRs are receptor-binding proteins that are usually parts of tail structures and the variability of such host-recognizing structures enables phage adaptation to changes on the bacterial host surface. Sometimes, more than one target gene containing a hypermutated variable repeat (VR) can be found in phage DGRs. The role of mutagenesis of two functionally different genes is unclear. In this study, several phage genomes that contain DGRs with two target genes were found in the gut virome of healthy volunteers. Bioinformatics analysis of these genes indicated that they encode proteins with different topology; however, both proteins contain the C-type lectin (C-lec) domain with a hypermutated beta-hairpin on its surface. One of the target proteins belongs to a new family of proteins with a specific topology: N-terminal C-lec domain followed by one or more immunoglobulin domains. Proteins from the new family were named tentaclins after TENTACLe + proteIN. The genes encoding such proteins were found in the genomes of prophages and phages from the gut metagenomes. We hypothesized that tentaclins are involved in binding either to bacterial receptors or intestinal/immune cells.

Comparative Genomics of a Polyvalent Escherichia-Salmonella Phage fp01 and In Silico Analysis of Its Receptor Binding Protein and Conserved Enterobacteriaceae Phage Receptor.

The polyvalent bacteriophage fp01, isolated from wastewater in Valparaiso, Chile, was described to have lytic activity across bacterial species, including Escherichia coli and Salmonella enterica serovars. Due to its polyvalent nature, the bacteriophage fp01 has potential applications in the biomedical, food and agricultural industries. Also, fundamental aspects of polyvalent bacteriophage biology are unknown. In this study, we sequenced and described the complete genome of the polyvalent phage fp01 (MH745368.2) using long- (MinION, Nanopore) and short-reads (MiSeq, Illumina) sequencing. The bacteriophage fp01 genome has 109,515 bp, double-stranded DNA with an average G+C content of 39%, and 158 coding sequences (CDSs). Phage fp01 has genes with high similarity to Escherichia coli, Salmonella enterica, and Shigella sp. phages. Phylogenetic analyses indicated that the phage fp01 is a new Tequintavirus fp01 specie. Receptor binding protein gp108 was identified as potentially responsible for fp01 polyvalent characteristics, which binds to conserved amino acid regions of the FhuA receptor of Enterobacteriaceae.

A Novel Bacteriophage with Broad Host Range against Clostridioides difficile Ribotype 078 Supports SlpA as the Likely Phage Receptor.

Bacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ΦCD08011, ΦCD418, ΦCD1801, and ΦCD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ΦCD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ΦCD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI. IMPORTANCE Research into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridioides difficile infection (CDI) is in its infancy, where an optimal "one size fits all" phage cocktail is yet to be derived. The pursuit thus far has aimed to find phages with the broadest possible host range. However, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad host range activity are yet to be discovered. In this study, we isolate four novel myoviruses, including ΦCD1801, which exerts the broadest host range activity toward RT 078 reported in the literature. Through the application of ΦCD1801 to phage-binding assays, we provide data to support the prior notion that SlpA represents the likely phage receptor on the bacterial cell surface. Our finding directs research attention toward the isolation of phages with activity toward strains possessing defined S-layer cassette types.

A combination therapy of Phages and Antibiotics: Two is better than one.

Emergence of antibiotic resistance presents a major setback to global health, and shortage of antibiotic pipelines has created an urgent need for development of alternative therapeutic strategies. Bacteriophage (phage) therapy is considered as a potential approach for treatment of the increasing number of antibiotic-resistant pathogens. Phage-antibiotic synergy (PAS) refers to sublethal concentrations of certain antibiotics that enhance release of progeny phages from bacterial cells. A combination of phages and antibiotics is a promising strategy to reduce the dose of antibiotics and the development of antibiotic resistance during treatment. In this review, we highlight the state-of-the-art advancements of PAS studies, including the analysis of bacterial-killing enhancement, bacterial resistance reduction, and anti-biofilm effect, at both in vitro and in vivo levels. A comprehensive review of the genetic and molecular mechanisms of phage antibiotic synergy is provided, and synthetic biology approaches used to engineer phages, and design novel therapies and diagnostic tools are discussed. In addition, the role of engineered phages in reducing pathogenicity of bacteria is explored.

Enzyme-Linked Phage Receptor Binding Protein Assays (ELPRA) Enable Identification of Bacillus anthracis Colonies.

Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the etiological agent of anthrax, can be difficult because of the bacterium's close relationship with other species of the Bacillus cereussensu lato group. Here, we facilitated the identification of B. anthracis using two implementations of enzyme-linked phage receptor binding protein assays (ELPRA). We developed a single-tube centrifugation assay simplifying the rapid analysis of suspect colonies. A second assay enables identification of suspect colonies from mixed overgrown solid (agar) media derived from the complex matrix soil. Thus, these tests identified vegetative cells of B. anthracis with little processing time and may support or confirm pathogen detection by molecular methods such as polymerase chain reaction.

Characterization and Food Application of the Novel Lytic Phage BECP10: Specifically Recognizes the O-polysaccharide of Escherichia coli O157:H7.

Escherichia coli O157:H7 is a global concern that causes serious diseases, such as hemolytic uremic syndrome and bloody diarrhea. To control E. coli O157:H7 in food, a novel siphophage, BECP10, that targets the O157 serotype was isolated and characterized. Unlike other E. coli phages, BECP10 can only infect E. coli O157 strains, and thus, did not infect other strains. The 48 kbp genome of BECP10 contained 76 open reading frames (ORFs), including 33 putative functional ORFs. The phage did not contain lysogeny-related modules or toxin-associated genes, suggesting that the phage might be strictly lytic. The tail spike protein (TSP) sequence had very low homology with the reported T1-like phages, indicating that TSP might be related to this unique host spectrum. The specific O-antigen residue of E. coli O157:H7 may be a key factor for phage infection by adsorption and receptor identification. The phage exhibited strong antibacterial activity against E. coli O157:H7 over a broad pH range and showed little development of phage-insensitive mutants. The phage sustained viability on the burger patties and reduced E. coli O157:H7 to a non-detectable level without the emergence of resistant cells at low temperatures for five days. Therefore, phage BECP10 might be a good biocontrol agent for E. coli O157:H7-contaminated food matrices.

How does feedback from phage infections influence the evolution of phase variation in Campylobacter?

Campylobacter jejuni (C. jejuni) causes gastroenteritis following the consumption of contaminated poultry meat, resulting in a large health and economic burden worldwide. Phage therapy is a promising technique for eradicating C. jejuni from poultry flocks and chicken carcasses. However, C. jejuni can resist infections by some phages through stochastic, phase-variable ON/OFF switching of the phage receptors mediated by simple sequence repeats (SSR). While selection strength and exposure time influence the evolution of SSR-mediated phase variation (PV), phages offer a more complex evolutionary environment as phage replication depends on having a permissive host organism. Here, we build and explore several continuous culture bacteria-phage computational models, each analysing different phase-variable scenarios calibrated to the experimental SSR rates of C. jejuni loci and replication parameters for the F336 phage. We simulate the evolution of PV rates via the adaptive dynamics framework for varying levels of selective pressures that act on the phage-resistant state. Our results indicate that growth reducing counter-selection on a single PV locus results in the stable maintenance of the phage, while compensatory selection between bacterial states affects the evolutionary stable mutation rates (i.e. very high and very low mutation rates are evolutionarily disadvantageous), whereas, in the absence of either selective pressure the evolution of PV rates results in mutation rates below the basal values. Contrastingly, a biologically-relevant model with two phase-variable loci resulted in phage extinction and locking of the bacteria into a phage-resistant state suggesting that another counter-selective pressure is required, instance, the use of a distinct phage whose receptor is an F336-phage-resistant state. We conclude that a delicate balance between counter-selection and phage-attack can result in both the evolution of phase-variable phage receptors and persistence of PV-receptor-specific phage.

A PolyQ Membrane Protein of Vibrio cholerae Acts as the Receptor for Phage Infection.

Bacteriophage VP1 is a typing phage used for the phage subtyping of Vibrio cholerae O1 biotype El Tor, but the molecular mechanisms of its receptor recognition and the resistance of its host to infection are mostly unknown. In this study, we aimed to identify the host receptor and its role in resistance in natural VP1-resistant strains. Generating spontaneous resistance mutations and genome sequencing mutant strains found the polyQ protein VcpQ, which carries 46 glutamine residues in its Q-rich region, to be responsible for infection by VP1. VcpQ is a membrane protein and possibly forms homotrimers. VP1 adsorbed to V. cholerae through VcpQ. Sequence comparisons showed that 72% of natural VP1-resistant strains have fewer glutamines in the VcpQ Q-rich stretch than VP1-sensitive strains. This difference did not affect the membrane location and oligomer of VcpQ but abrogated VP1 adsorption. These mutant VcpQs did not recover VP1 infection sensitivity in a V. cholerae strain with vcpQ deleted. Our study revealed that the polyQ protein VcpQ is responsible for the binding of VP1 during its infection of V. cholerae and that glutamine residue reduction in VcpQ affects VP1 adsorption to likely be the main cause of VP1 resistance in natural resistant strains. The physiological functions of this polyQ protein in bacteria need further clarification; however, mutations in the polyQ stretch may endow V. cholerae with phage resistance and enhance survival against VP1 or related phages.IMPORTANCE Receptor recognition and binding by bacteriophage are the first step for its infection of bacterial cells. In this study, we found the Vibrio cholerae subtyping phage VP1 uses a polyQ protein named VcpQ (V. cholerae polyQ protein) as the receptor for VP1 infection. Our study reveals the receptor's recognition of phage VP1 during its adsorption and the VP1 resistance mechanism of the wild resistant V. cholerae strains bearing the mutagenesis in the receptor VcpQ. These mutations may confer the survival advantage on these resistant strains in the environment containing VP1 or its similar phages.

Compilation of Escherichia coli K-12 outer membrane phage receptors - their function and some historical remarks.

Many Escherichia coli phages have been sequenced, but in most cases their sequences alone do not suffice to predict their host specificity. Analysis of phage resistant E. coli K-12 mutants have uncovered a certain set of outer membrane proteins and polysaccharides as receptors. In this review, a compilation of E. coli K12 phage receptors is provided and their functional characterization, often driven by studies on phage resistant mutants, is discussed in the historical context. While great progress has been made in this field thus far, several proteins in the outer membrane still await characterization as phage receptors.