Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay

Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage’s ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage’s “adsorptive host range.” The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage’s adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein’s targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage’s adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4’s long tail fiber binding tip (gp37) to evaluate and quantify gp37’s relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay’s ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins.

Manipulating interactions between T4 phage long tail fibers and Escherichia coli receptors

Bacteriophages are the most abundant and diverse biological entities on Earth. Phages exhibit strict host specificity that is largely conferred by adsorption. However, the mechanism underlying this phage–host specificity remains poorly understood. In this study, we examined the interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the long tail fibers of bacteriophage T4. T4 phage uses OmpC of the K12 strain, but not of the O157 strain, for adsorption, even though OmpC from the two E. coli strains share 94% homology. We identified amino acids P177 and F182 in Loop 4 of the K12 OmpC as essential for T4 phage adsorption in the copresence of Loop 1 and Loop 5. Analyses of phage mutants capable of adsorbing to OmpC mutants demonstrated that amino acids at positions 937 and 942 of the gp37 protein, which is present in the digital tip (DT) region of the T4 long tail fibers, play an important role in adsorption. Furthermore, we created a T4 phage mutant library with artificial modifications in the DT region and isolated and characterized multiple phage mutants capable of adsorbing to OmpC of the O157 strain or lipopolysaccharide of the K12 strain. These results shed light on the mechanism underlying the phage–host specificity mediated by gp37 and OmpC and may be useful in the development of phage therapy via artificial modifications of the DT region of T4 phage. IMPORTANCE Understanding the host specificity of phages will lead to the development of phage therapy. The interaction between outer membrane protein C (OmpC), one of the Escherichia coli receptors, and the gp37 protein composing the digital tip (DT) region of the long tail fibers of bacteriophage T4 largely determines its host specificity. Here, we elucidated the amino acid residues important for the interaction between gp37 and OmpC. This result suggests that the shapes of both proteins at the binding interface play important roles in their interactions, which is likely mediated by multiple residues of both binding partners. Additionally, we successfully isolated multiple phage mutants capable of adsorbing to a variety of E. coli receptors using a mutant T4 phage library with artificial modifications in the DT region, providing a foundation for the alteration of the host specificity.

Development of a Bacteriophage Tail Fiber-Based Latex Agglutination Assay for Rapid Clinical Screening of Burkholderia pseudomallei

Melioidosis is a life-threatening disease in humans caused by the Gram- negative bacterium Burkholderia pseudomallei. As severe septicemic melioidosis can lead to death within 24 to 48 hours, a rapid diagnosis of melioidosis is critical for ensuring an optimal antibiotic course is prescribed to patients. Here, we report the development and evaluation of a bacteriophage tail fiber-based latex agglutination assay for rapid detection of B. pseudomallei infection. Burkholderia phage E094 was isolated from rice paddy fields in northeast Thailand, and whole genome sequenced to identify its tail fiber (94TF). The 94TF complex was structurally characterized, which involved identification of a tail assembly protein that forms an essential component of the mature fiber. Recombinant 94TF was conjugated to latex beads and developed into an agglutination-based assay (94TF-LAA). 94TF-LAA was initially tested against a large library of Burkholderia and other bacterial strains before a field evaluation was performed during routine clinical testing. The sensitivity and specificity of the 94TF-LAA were assessed alongside standard biochemical analyses on 300 patient specimens collected from an endemic area of melioidosis over 11 months. The 94TF-LAA took less than 5 minutes to produce positive agglutination, demonstrating 98% (95% CI; 94.2%−99.59%) sensitivity and 83% (95% CI; 75.64%−88.35%) specificity when compared to biochemical-based detection. Overall, we show how a Burkholderia-specific phage tail fiber can be exploited for rapid detection of B. pseudomallei. The 94TF-LAA has the potential for further development as a supplementary diagnostic to assist in clinical identification of this life-threatening pathogen. IMPORTANCE Rapid diagnosis of melioidosis is essential for ensuring optimal antibiotic courses are prescribed to patients, and thus warrants the development of cost-effective and easy-to-use tests for implementation in under-resourced areas such as Northeast Thailand and other tropical regions. Phage tail fibers are an interesting alternative to antibodies for use in various diagnostic assays for different pathogenic bacteria. As exposed appendages of phages, tail fibers are physically robust, easy to manufacture, and critically many tail fibers (such as 94TF investigated here) can target a given bacterial species with remarkable specificity. Here, we demonstrate the effectiveness of a latex agglutination assay using a Burkholderia-specific tail fiber 94TF against biochemical-based detection methods that are the standard diagnostic in many endemic areas of meilodosis.

Complete Genome Sequence of Klebsiella pneumoniae Myophage Muenster

ABSTRACT Klebsiella pneumoniae is associated with antibiotic-resistant nosocomial infections. Here, we present the annotated genome sequence of the Klebsiella jumbo phage Muenster. The Muenster genome sequence (346,937 bp) encodes 6 tRNAs and 561 putative protein-coding genes, including 9 tail fibers, suggesting a genetic mechanism to broaden the host range.

Phage S144, a New Polyvalent Phage Infecting Salmonella spp. and Cronobacter sakazakii

Phages are generally considered species- or even strain-specific, yet polyvalent phages are able to infect bacteria from different genera. Here, we characterize the novel polyvalent phage S144, a member of the Loughboroughvirus genus. By screening 211 Enterobacteriaceae strains, we found that phage S144 forms plaques on specific serovars of Salmonella enterica subsp. enterica and on Cronobacter sakazakii. Analysis of phage resistant mutants suggests that the O-antigen of lipopolysaccharide is the phage receptor in both bacterial genera. The S144 genome consists of 53,628 bp and encodes 80 open reading frames (ORFs), but no tRNA genes. In total, 32 ORFs coding for structural proteins were confirmed by ESI-MS/MS analysis, whereas 45 gene products were functionally annotated within DNA metabolism, packaging, nucleotide biosynthesis and phage morphogenesis. Transmission electron microscopy showed that phage S144 is a myovirus, with a prolate head and short tail fibers. The putative S144 tail fiber structure is, overall, similar to the tail fiber of phage Mu and the C-terminus shows amino acid similarity to tail fibers of otherwise unrelated phages infecting Cronobacter. Since all phages in the Loughboroughvirus genus encode tail fibers similar to S144, we suggest that phages in this genus infect Cronobacter sakazakii and are polyvalent.

Engineering of receptor-binding proteins in bacteriophages and phage tail-like bacteriocins

Bacteriophages and phage tail-like bacteriocins (PTLBs) rely on receptor-binding proteins (RBPs) located in tail fibers or spikes for an initial and specific interaction with susceptible bacteria. Bacteriophages kill bacteria through a lytic, replicative cycle, whereas PTLBs kill the target through membrane depolarization in a single hit mechanism. Extensive efforts in the engineering of RBPs of both phages and PTLBs have been undertaken to obtain a greater understanding of the structural organization of RBPs. In addition, a major goal of engineering RBPs of phages and PTLBs is the production of antibacterials with a customized spectrum. Swapping of the RBP of phages and PTLBs results in a shift in activity spectrum in accordance with the spectrum of the new RBP. The engineering of strictly virulent phages with new RBPs required significant technical advances in the past decades, whereas the engineering of RBPs of PTLBs relied on the traditional molecular techniques used for the manipulation of bacteria and was thus relatively straightforward. While phages and PTLBs share their potential for specificity tuning, specific features of phages such as their lytic killing mechanism, their self-replicative nature and thus different pharmacokinetics and their potential to co-evolve are clear differentiators compared with PTLBs in terms of their antibacterial use.

Unravelling the Links between Phage Adsorption and Successful Infection in Clostridium difficile

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.

Pseudomonas chlororaphisProduces Multiple R-Tailocin Particles That Broaden the Killing Spectrum and Contribute to Persistence in Rhizosphere Communities

ABSTRACTR-tailocins are high-molecular-weight bacteriocins resembling bacteriophage tails.Pseudomonas chlororaphis30-84 is a plant growth-promoting rhizobacterial (PGPR) strain that produces two distinct R-tailocin particles with different killing spectra. The two R-tailocins have different evolutionary histories but are released by the same lysis cassette. A previous study showed that both tailocins are important for pairwise competition with susceptible rhizosphere-colonizing strains; however, the broader role of tailocins in competition with the native rhizosphere microbiome was not tested. Genomic analysis of theP. chlororaphis30-84 R-tailocin gene cluster uncovered the presence of three tail fiber genes in the tailocin 2 genetic module that could potentially result in tailocin 2 particles having different tail fibers and thus a wider killing spectrum. In this study, the tail fibers were found to incorporate onto different tailocin 2 particles, each with a distinct killing spectrum. A loss of production of one or both tailocins resulted in decreasedP. chlororaphis30-84 persistence within the wheat rhizosphere when in competition with the native microflora but not bulk soil. The capacity to produce three different versions of a single tailocin, each having one of three different types of tail fibers, is a previously unreported mechanism that leads to a broader R-tailocin killing spectrum. This study also provides evidence for the function of R-tailocins in competition with rhizosphere microbiome communities but not in bulk soil.IMPORTANCEAlthough R-tailocin gene clusters typically encode one tail fiber protein, three tail fiber-resembling genes were identified in association with one of the two sets of R-tailocin genes within the tailocin cluster ofP. chlororaphis30-84 and other sequencedP. chlororaphisstrain genomes. This study confirmed thatP. chlororaphis30-84 not only produces two distinct tailocins, but that one of them is produced with three different types of tail fibers. This is a previously unreported strategy to increase the breadth of strains targeted by an R-tailocin. Our finding that R-tailocins produced by a PGPRPseudomonasstrain enhanced its persistence within the wheat rhizosphere microbiome confirms that R-tailocin production contributes to the population dynamics of rhizobacterial communities.