As the importance of bacteriophages as novel antimicrobials and potential diagnostics comes increasingly into focus, there is a heightened interest in understanding the mechanisms of how they interact with their bacterial hosts. The first step of a bacteriophage (phage) infection is the recognition of specific moieties on the bacterial cell surface as determined by their phage receptor binding proteins (RBPs). Knowledge of RBPs and how they interact with bacteria has been driven by studies of model phages and of industrially important phages, such as those that impact the dairy industry. Therefore, data from these phage groups constitute the majority of this review. We start with a brief introduction to phages, their life cycles and known receptors. We then review the state-of-the-art knowledge of phage RBPs of Gram-positive bacteria in the context of the better understood Gram-negative bacterial RBPs. In general, more is known about the RBPs of siphoviruses than myoviruses, which is reflected here, but for both virus families, where possible, we show what RBPs are, how they are arranged within phage genomes and what is known about their structures. As RBPs are the key determinant of phage specificity, studying and characterising them is important, for downstream applications such as diagnostic and therapeutic purposes.

Phages are the most abundant biological entity on Earth. There are many variants in phage virion sizes, morphology, and genome sizes. Large virion sized phages, with genome sizes greater than 200 kbp have been identified and termed as Jumbo phages. These phages exhibit certain characteristics that have not been reported in phages with smaller genomes. In this work, a jumbo phage named MIJ3 (vB_PaeM_MIJ3) that infects Pseudomonas aeruginosa PAO1 was isolated from an equine livery yard in Leicestershire, United Kingdom. The genome and biological characteristics of this phage have been investigated. MIJ3 is a Myovirus with multiple long tail fibers. Assessment of the host range of MIJ3 revealed that it has the ability to infect many clinical isolates of P. aeruginosa. Bioinformatics analysis of the phage genome indicated that MIJ3 is closely related to the Pseudomonas phage, PA5oct. MIJ3 possesses several unusual features that are either rarely present in other phages or have not yet been reported. In particular, MIJ3 encodes a FtsH-like protein, and a putative lysidine synthase, TilS. These two proteins have not been reported in phages. MIJ3 also possesses a split DNA polymerase B with a novel intein. Of particular interest, unlike other jumbo phages infecting Pseudomonas spp., MIJ3 lacks the genetic elements required for the formation of the phage nucleus, which was believed to be conserved across jumbo Pseudomonas phages.

Bacteriophage (phage) therapy is a promising alternative to antibiotics for the treatment of bacterial pathogens, including Clostridium difficile. However, as for many species, in C. difficile the physical interactions between phages and bacterial cells have not been studied in detail. The initial interaction, known as phage adsorption, is initiated by the reversible attachment of phage tail fibers to bacterial cell surface receptors followed by an irreversible binding step. Therefore binding can dictate which strains are infected by the phage. In this study, we investigated the adsorption rates and irreversible binding of three C. difficile myoviruses: CDHM1, CDHM3 and CDHM6 to ten strains that represent ten prevalent C. difficile ribotypes, regardless of their ability to infect. CDHM1 and CDHM3 phage particles adsorbed by ~75% to some strains that they infected. The infection dynamics for CDHM6 are less clear and ~30% of the phage particles bound to all strains, irrespective of whether a successful infection was established. The data highlighted adsorption is phage-host specific. However, it was consistently observed that irreversible binding had to be above 80% for successful infection, which was also noted for another two C. difficile myoviruses. Furthermore, to understand if there is a relationship between infection, adsorption and phage tail fibers, the putative tail fiber protein sequences of CDHM1, CDHM3 and CDHM6 were compared. The putative tail fiber protein sequence of CDHM1 shares 45% homology at the amino acid level to CDHM3 and CDHM6, which are identical to each other. However, CDHM3 and CDHM6 display differences in adsorption, which highlights that there is no obvious relationship between putative tail fiber sequence and adsorption. The importance of adsorption and binding to successful infection is often overlooked, and this study provides useful insights into host-pathogen interactions within this phage-pathogen system.

Phage-bacterial recognition is species-specific, determined by interactions between phage receptor-binding proteins (RBPs) and corresponding bacterial receptors. RBPs are diverse, and we present data demonstrating the identification and characterisation of a novel C. difficile phage RBP. Putative RBP was identified for CDHS-1, and overexpressed, purified, and polyclonal antibodies were raised and used in phage neutralization assays. Anti-gp22 neutralised CDHS-1, indicating it is the RBP. Immunogold-labelling and transmission electron microscopy confirmed this, enabling visualization of the protein locations. A detailed structural understanding was obtained from determining the three-dimensional structure of gp22 by X-ray crystallography. gp22 is a new RBP class consisting of an N-terminal L-shaped α-helical superhelix domain and a C-terminal Mg2+-binding domain. The protein is a stable homodimer in solution mediated via reciprocal contacts between an α-helical hairpin located within the superhelix domain and additional asymmetrical contacts between the ends of the short arm of each L-shaped protomer. The dimer resembles U-shape with a crossbar formed from the hairpin of each partner. C. difficile binding is Mg2+-dependent. CDHS-1 could not infect a C. difficile S-layer mutant suggesting the bacterial receptors are within the S-layer. These findings provide novel insights into phage biology and extend our knowledge of RBPs.

Clostridioides difficile causes antibiotic-induced diarrhoea and pseudomembranous colitis in humans and animals. Current conventional treatment relies solely on antibiotics, but C. difficile infection (CDI) cases remain persistently high with concomitant increased recurrence often due to the emergence of antibiotic-resistant strains. Antibiotics used in treatment also induce gut microbial imbalance; therefore, novel therapeutics with improved target specificity are being investigated. Bacteriophages (phages) kill bacteria with precision, hence are alternative therapeutics for the targeted eradication of the pathogen. Here, we review current progress in C. difficile phage research. We discuss tested strategies of isolating C. difficile phages directly, and via enrichment methods from various sample types and through antibiotic induction to mediate prophage release. We also summarise phenotypic phage data that reveal their morphological, genetic diversity, and various ways they impact their host physiology and pathogenicity during infection and lysogeny. Furthermore, we describe the therapeutic development of phages through efficacy testing in different in vitro, ex vivo and in vivo infection models. We also discuss genetic modification of phages to prevent horizontal gene transfer and improve lysis efficacy and formulation to enhance stability and delivery of the phages. The goal of this review is to provide a more in-depth understanding of C. difficile phages and theoretical and practical knowledge on pre-clinical, therapeutic evaluation of the safety and effectiveness of phage therapy for CDI.