Enhanced targeting: Production of filamentous bacteriophage monoclonal antibodies using the major coat protein pVIII as anchor.

Filamentous bacteriophage display technology has been employed in antibody discovery, drug screening, and protein-protein interaction study across various fields, including food safety, agricultural pollution, and environmental monitoring. Antifilamentous bacteriophage antibodies for identifying filamentous bacteriophage are playing a pivotal role in this technology. However, the existing antifilamentous bacteriophage antibodies lack sensitivity and specificity, and the antibodies preparation methods are cumbersome and hyposensitive. The major coat protein pVIII of filamentous bacteriophage has an advantage in quantification, which is benefit for detecting signal amplification but its full potential remains underutilized. In this study, the partial polypeptide CT21 of the major coat protein pVIII of filamentous bacteriophage was intercepted as the targeted immunogen or coating antigen to prepare antifilamentous bacteriophage antibodies. Six filamentous bacteriophage-specific monoclonal antibodies (mAbs) M5G8, M9A2, P6B5, P6D2, P8E4, and P10D4 were obtained. The limit of detections of the prepared six mAbs for detecting filamentous bacteriophage was 1.0 × 107  pfu mL-1 . These mAbs stayed stable under different pH, temperature, and exhibited high specificity in real application. This study not only provides a new idea for simplifying the preparation of antifilamentous bacteriophage antibodies which could apply in filamentous bacteriophage display, but it also presents a novel strategy for preparing antibodies against protein-specific epitopes with high sensitivity.

A New Inovirus from the Human Blood Encodes Proteins with Nuclear Subcellular Localization.

Viruses infecting bacteria (bacteriophages) represent the most abundant viral particles in the human body. They participate in the control of the human-associated bacterial communities and play an important role in the dissemination of virulence genes. Here, we present the identification of a new filamentous single-stranded DNA phage of the family Inoviridae, named Ralstonia Inoviridae Phage 1 (RIP1), in the human blood. Metagenomics and PCR analyses detected the RIP1 genome in blood serum, in the absence of concomitant bacterial infection or contamination, suggesting inovirus persistence in the human blood. Finally, we have experimentally demonstrated that the RIP1-encoded rolling circle replication initiation protein and serine integrase have functional nuclear localization signals and upon expression in eukaryotic cells both proteins were translocated into the nucleus. This observation adds to the growing body of data suggesting that phages could have an overlooked impact on the evolution of eukaryotic cells.

Visualization of Engineered M13 Phages Bound to Bacterial Targets by Transmission Electron Microscopy.

The filamentous phage M13 is one of the most well-studied and characterized phages, particularly since it was introduced as a scaffold for phage display, a technique to express and evolve fusion proteins on the M13 phage's coat to study protein or peptide binding interactions. Since phages can be engineered or evolved to specifically bind to a variety of targets, engineered M13 phages have been explored for applications such as drug delivery, biosensing, and cancer therapy, among others. Specifically, with the rising challenge of antimicrobial resistance among bacteria, chimeric M13 phages have been explored both as detection and therapeutic agents due to the flexibility in tuning target specificity. Transmission electron microscopy (TEM) is a powerful tool enabling researchers to directly visualize and characterize binding of phages to bacterial surfaces. However, the filamentous phage structure poses a challenge for this technique, as the phages have similar morphology to bacterial structures such as pili. In order to differentiate between bacterial structures and the filamentous phages, here we describe a protocol to prepare TEM samples of engineered M13 phages bound to bacterial cells, in which the phage virions have been specifically labeled by decoration of the major capsid proteins with gold nanoparticles. This protocol enables clear visualization and unambiguous identification of attached filamentous phages within the context of bacterial cells expressing numerous pili.

Preparation of Bioconjugates of Chimeric M13 Phage and Gold Nanorods.

Phage-nanomaterial conjugates are functional bio-nanofibers with various applications. While phage display can select for phages with desired genetically encoded functions and properties, nanomaterials can endow the phages with additional features at nanoscale dimensions. Therefore, combining phages with nanotechnology can construct bioconjugates with unique characteristics. One strategy for filamentous phages is to adsorb nanoparticles onto the side wall, composed of pVIII subunits, through electrostatic interactions. However, a noncovalent approach may cause offloading if the environment changes, potentially causing side effects especially for in vivo applications. Therefore, building stable phage-bioconjugates is an important need. We previously reported the construction of chimeric M13 phage conjugated with gold nanorods, named "phanorods," without weakening the binding affinity to the bacterial host cells. Herein, we give a detailed protocol for preparing the chimeric M13 phage and covalently conjugating gold nanorods to the phage.

Advanced detection of cervical cancer biomarkers using engineered filamentous phage nanofibers.

Cervical cancer is a major global health concern, characterized by its high incidence and mortality rates. The detection of tumor markers is crucial for managing cancer, making treatment decisions, and monitoring disease progression. Vascular endothelial growth factor (VEGF) and programmed death-ligand 1 (PDL-1) are key targets in cervical cancer therapy and valuable biomarkers in predicting treatment response and prognosis. In this study, we found that combining the measurement of VEGF and soluble PDL-1 can be used for diagnosing and evaluating the progression of cervical cancer. To explore a more convenient approach for detecting and assessing cervical cancer, we designed and prepared an engineered fd bacteriophage, a human-safe viral nanofiber, equipped with two peptides targeting VEGF and PD-L1. The dual-display phage nanofiber specifically recognizes and binds to both proteins. Utilizing this nanofiber as a novel capture agent, we developed a new enzyme-linked immunosorbent assay (ELISA) method. This method shows significantly enhanced detection sensitivity compared to conventional ELISA methods, which use either anti-VEGF or anti-PD-L1 antibodies as capture agents. Therefore, the phage dual-display nanofiber presents significant potential in detecting cancer markers, evaluating medication efficacy, and advancing immunotherapy drug development. KEY POINTS: • The combined measurement of VEGF and soluble Programmed Death-Ligand 1(sPD-L1) demonstrates an additive effect in the diagnosis of cervical cancer. Fd phage nanofibers have been ingeniously engineered to display peptides that bind to VEGF and PD-L1, enabling the simultaneous detection of both proteins within a single assay • Genetically engineered phage nanofibers, adorned with two distinct peptides, can be utilized for the diagnosis and prognosis of cancer and can be mass-produced cost-effectively through bacterial infections • Employing dual-display fd phage nanofibers as capture probes, the phage ELISA method exhibited significantly enhanced detection sensitivity compared to traditional sandwich ELISA. Furthermore, phage ELISA facilitates the detection of a single protein or the simultaneous detection of multiple proteins, rendering them powerful tools for protein analysis and diagnosis across various fields, including cancer research.

Biophysical basis of filamentous phage tactoid-mediated antibiotic tolerance in P. aeruginosa.

Inoviruses are filamentous phages infecting numerous prokaryotic phyla. Inoviruses can self-assemble into mesoscale structures with liquid-crystalline order, termed tactoids, which protect bacterial cells in Pseudomonas aeruginosa biofilms from antibiotics. Here, we investigate the structural, biophysical, and protective properties of tactoids formed by the P. aeruginosa phage Pf4 and Escherichia coli phage fd. A cryo-EM structure of the capsid from fd revealed distinct biochemical properties compared to Pf4. Fd and Pf4 formed tactoids with different morphologies that arise from differing phage geometries and packing densities, which in turn gave rise to different tactoid emergent properties. Finally, we showed that tactoids formed by either phage protect rod-shaped bacteria from antibiotic treatment, and that direct association with a tactoid is required for protection, demonstrating the formation of a diffusion barrier by the tactoid. This study provides insights into how filamentous molecules protect bacteria from extraneous substances in biofilms and in host-associated infections.

Inoviruses.

Tropini and Burckhardt introduce inoviruses and discuss how they are unique amongst bacteriophages.

Analysis of Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis Revealed Novel Groups of Filamentous Bacteriophages.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in humans, especially in hospital patients with compromised host defence mechanisms, including patients with cystic fibrosis. Filamentous bacteriophages represent a group of single-stranded DNA viruses infecting different bacteria, including P. aeruginosa and other human and animal pathogens; many of them can replicate when integrated into the bacterial chromosome. Filamentous bacteriophages can contribute to the virulence of P. aeruginosa and influence the course of the disease. There are just a few isolated and officially classified filamentous bacteriophages infecting P. aeruginosa, but genomic studies indicated the frequent occurrence of integrated prophages in many P. aeruginosa genomes. An analysis of sequenced genomes of P. aeruginosa isolated from upper respiratory tract (throat and nasal swabs) and sputum specimens collected from Russian patients with cystic fibrosis indicated a higher diversity of filamentous bacteriophages than first thought. A detailed analysis of predicted bacterial proteins revealed prophage regions representing the filamentous phages known to be quite distantly related to known phages. Genomic comparisons and phylogenetic studies enabled the proposal of several new taxonomic groups of filamentous bacteriophages.

Cryo-EM structure of a bacteriophage M13 mini variant.

Filamentous bacteriophages package their circular, single stranded DNA genome with the major coat protein pVIII and the minor coat proteins pIII, pVII, pVI, and pIX. Here, we report the cryo-EM structure of a ~500 Å long bacteriophage M13 mini variant. The distal ends of the mini phage are sealed by two cap-like complexes composed of the minor coat proteins. The top cap complex consists of pVII and pIX, both exhibiting a single helix structure. Arg33 of pVII and Glu29 of pIX, located on the inner surface of the cap, play a key role in recognizing the genome packaging signal. The bottom cap complex is formed by the hook-like structures of pIII and pVI, arranged in helix barrels. Most of the inner ssDNA genome adopts a double helix structure with a similar pitch to that of the A-form double-stranded DNA. These findings provide insights into the assembly of filamentous bacteriophages.

Characterisation of the Novel Filamentous Phage PMBT54 Infecting the Milk Spoilage Bacteria Pseudomonas carnis and Pseudomonas lactis.

Filamentous bacteriophages are lysogenic and pseudo-lysogenic viruses that do not lyse their host but are often continuously secreted from the infected cell. They belong to the order Tubulavirales, which encompasses three families, with the Inoviridae being the largest. While the number of identified inoviral sequences has greatly increased in recent years due to metagenomic studies, morphological and physiological characterisation is still restricted to only a few members of the filamentous phages. Here, we describe the novel filamentous phage PMBT54, which infects the spoilage-relevant Pseudomonas species P. carnis and P. lactis. Its genome is 7320 bp in size, has a mol% GC content of 48.37, and codes for 13 open-reading frames, two of which are located on the (-) strand. The virion exhibits a typical filamentous morphology and is secreted from the host cell at various lengths. The phage was shown to promote biofilm formation in both host strains and, therefore, has potential implications for milk spoilage, as biofilms are a major concern in the dairy industry.

Cryo-electron microscopy of the f1 filamentous phage reveals insights into viral infection and assembly.

Phages are viruses that infect bacteria and dominate every ecosystem on our planet. As well as impacting microbial ecology, physiology and evolution, phages are exploited as tools in molecular biology and biotechnology. This is particularly true for the Ff (f1, fd or M13) phages, which represent a widely distributed group of filamentous viruses. Over nearly five decades, Ffs have seen an extraordinary range of applications, yet the complete structure of the phage capsid and consequently the mechanisms of infection and assembly remain largely mysterious. In this work, we use cryo-electron microscopy and a highly efficient system for production of short Ff-derived nanorods to determine a structure of a filamentous virus including the tips. We show that structure combined with mutagenesis can identify phage domains that are important in bacterial attack and for release of new progeny, allowing new models to be proposed for the phage lifecycle.

Gut commensal Enterocloster species host inoviruses that are secreted in vitro and in vivo.

BACKGROUND: Bacteriophages in the family Inoviridae, or inoviruses, are under-characterized phages previously implicated in bacterial pathogenesis by contributing to biofilm formation, immune evasion, and toxin secretion. Unlike most bacteriophages, inoviruses do not lyse their host cells to release new progeny virions; rather, they encode a secretion system that actively pumps them out of the bacterial cell. To date, no inovirus associated with the human gut microbiome has been isolated or characterized. RESULTS: In this study, we utilized in silico, in vitro, and in vivo methods to detect inoviruses in bacterial members of the gut microbiota. By screening a representative genome library of gut commensals, we detected inovirus prophages in Enterocloster spp. (formerly Clostridium spp.). We confirmed the secretion of inovirus particles in in vitro cultures of these organisms using imaging and qPCR. To assess how the gut abiotic environment, bacterial physiology, and inovirus secretion may be linked, we deployed a tripartite in vitro assay that progressively evaluated bacterial growth dynamics, biofilm formation, and inovirus secretion in the presence of changing osmotic environments. Counter to other inovirus-producing bacteria, inovirus production was not correlated with biofilm formation in Enterocloster spp. Instead, the Enterocloster strains had heterogeneous responses to changing osmolality levels relevant to gut physiology. Notably, increasing osmolality induced inovirus secretion in a strain-dependent manner. We confirmed inovirus secretion in a gnotobiotic mouse model inoculated with individual Enterocloster strains in vivo in unperturbed conditions. Furthermore, consistent with our in vitro observations, inovirus secretion was regulated by a changed osmotic environment in the gut due to osmotic laxatives. CONCLUSION: In this study, we report on the detection and characterization of novel inoviruses from gut commensals in the Enterocloster genus. Together, our results demonstrate that human gut-associated bacteria can secrete inoviruses and begin to elucidate the environmental niche filled by inoviruses in commensal bacteria. Video Abstract.

Tripartite interactions between filamentous Pf4 bacteriophage, Pseudomonas aeruginosa, and bacterivorous nematodes.

The opportunistic pathogen Pseudomonas aeruginosa PAO1 is infected by the filamentous bacteriophage Pf4. Pf4 virions promote biofilm formation, protect bacteria from antibiotics, and modulate animal immune responses in ways that promote infection. Furthermore, strains cured of their Pf4 infection (ΔPf4) are less virulent in animal models of infection. Consistently, we find that strain ΔPf4 is less virulent in a Caenorhabditis elegans nematode infection model. However, our data indicate that PQS quorum sensing is activated and production of the pigment pyocyanin, a potent virulence factor, is enhanced in strain ΔPf4. The reduced virulence of ΔPf4 despite high levels of pyocyanin production may be explained by our finding that C. elegans mutants unable to sense bacterial pigments through the aryl hydrocarbon receptor are more susceptible to ΔPf4 infection compared to wild-type C. elegans. Collectively, our data support a model where suppression of quorum-regulated virulence factors by Pf4 allows P. aeruginosa to evade detection by innate host immune responses.

Harnessing filamentous phages for enhanced stroke recovery.

Stroke poses a critical global health challenge, leading to substantial morbidity and mortality. Existing treatments often miss vital timeframes and encounter limitations due to adverse effects, prompting the pursuit of innovative approaches to restore compromised brain function. This review explores the potential of filamentous phages in enhancing stroke recovery. Initially antimicrobial-centric, bacteriophage therapy has evolved into a regenerative solution. We explore the diverse role of filamentous phages in post-stroke neurological restoration, emphasizing their ability to integrate peptides into phage coat proteins, thereby facilitating recovery. Experimental evidence supports their efficacy in alleviating post-stroke complications, immune modulation, and tissue regeneration. However, rigorous clinical validation is essential to address challenges like dosing and administration routes. Additionally, genetic modification enhances their potential as injectable biomaterials for complex brain tissue issues. This review emphasizes innovative strategies and the capacity of filamentous phages to contribute to enhanced stroke recovery, as opposed to serving as standalone treatment, particularly in addressing stroke-induced brain tissue damage.

A Novel Inovirus Reprograms Metabolism and Motility of Marine Alteromonas.

Members from the Inoviridae family with striking features are widespread, highly diverse, and ecologically pervasive across multiple hosts and environments. However, a small number of inoviruses have been isolated and studied. Here, a filamentous phage infecting Alteromonas abrolhosensis, designated ϕAFP1, was isolated from the South China Sea and represented a novel genus of Inoviridae. ϕAFP1 consisted of a single-stranded DNA genome (5986 bp), encoding eight putative ORFs. Comparative analyses revealed ϕAFP1 could be regarded as genetic mosaics having homologous sequences with Ralstonia and Stenotrophomonas phages. The temporal transcriptome analysis of A. abrolhosensis to ϕAFP1 infection revealed that 7.78% of the host genes were differentially expressed. The genes involved in translation processes, ribosome pathways, and degradation of multiple amino acid pathways at the plateau period were upregulated, while host material catabolic and bacterial motility-related genes were downregulated, indicating that ϕAFP1 might hijack the energy of the host for the synthesis of phage proteins. ϕAFP1 exerted step-by-step control on host genes through the appropriate level of utilizing host resources. Our study provided novel information for a better understanding of filamentous phage characteristics and phage-host interactions. IMPORTANCE Alteromonas is widely distributed and plays a vital role in biogeochemical in marine environments. However, little information about Alteromonas phages is available. Here, we isolated and characterized the biological characteristics and genome sequence of a novel inovirus infecting Alteromonas abrolhosensis, designated ϕAFP1, representing a novel viral genus of Inoviridae. We then presented a comprehensive view of the ϕAFP1 phage-Alteromonas abrolhosensis interactions, elucidating reprogramed host metabolism and motility. Our study provided novel information for better comprehension of filamentous phage characteristics and phage-host interactions.

Filamentous Pseudomonas Phage Pf4 in the Context of Therapy-Inducibility, Infectivity, Lysogenic Conversion, and Potential Application.

More than 20% of all Pseudomonas aeruginosa are infected with Pf4-related filamentous phage and although their role in virulence of P. aeruginosa strain PAO1 is well documented, its properties related to therapy are not elucidated in detail. The aim of this study was to determine how phage and antibiotic therapy induce Pf4, whether the released virions can infect other strains and how the phage influences the phenotype of new hosts. The subinhibitory concentrations of ciprofloxacin and mitomycin C increased Pf4 production for more than 50% during the first and sixth hour of exposure, respectively, while mutants appearing after infection with obligatory lytic phage at low MOI produced Pf4 more than four times after 12-24 h of treatment. This indicates that production of Pf4 is enhanced during therapy with these agents. The released virions can infect new P. aeruginosa strains, as confirmed for models UCBPP-PA14 (PA14) and LESB58, existing both episomally and in a form of a prophage, as confirmed by PCR, RFLP, and sequencing. The differences in properties of Pf4-infected, and uninfected PA14 and LESB58 strains were obvious, as infection with Pf4 significantly decreased cell autoaggregation, pyoverdine, and pyocyanin production, while significantly increased swimming motility and biofilm production in both strains. In addition, in strain PA14, Pf4 increased cell surface hydrophobicity and small colony variants' appearance, but also decreased twitching and swarming motility. This indicates that released Pf4 during therapy can infect new strains and cause lysogenic conversion. The infection with Pf4 increased LESB58 sensitivity to ciprofloxacin, gentamicin, ceftazidime, tetracycline, and streptomycin, and PA14 to ciprofloxacin and ceftazidime. Moreover, the Pf4-infected LESB58 was re-sensitized to ceftazidime and tetracycline, with changes from resistant to intermediate resistant and sensitive, respectively. The obtained results open a new field in phage therapy-treatment with selected filamentous phages in order to re-sensitize pathogenic bacteria to certain antibiotics. However, this approach should be considered with precautions, taking into account potential lysogenic conversion.

Filamentous bacteriophage delays healing of Pseudomonas-infected wounds.

Chronic wounds infected by Pseudomonas aeruginosa (Pa) are characterized by disease progression and increased mortality. We reveal Pf, a bacteriophage produced by Pa that delays healing of chronically infected wounds in human subjects and animal models of disease. Interestingly, impairment of wound closure by Pf is independent of its effects on Pa pathogenesis. Rather, Pf impedes keratinocyte migration, which is essential for wound healing, through direct inhibition of CXCL1 signaling. In support of these findings, a prospective cohort study of 36 human patients with chronic Pa wound infections reveals that wounds infected with Pf-positive strains of Pa are more likely to progress in size compared with wounds infected with Pf-negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian cells. These findings suggest Pf may have potential as a biomarker and therapeutic target in chronic wounds.

Modelling of filamentous phage-induced antibiotic tolerance of P. aeruginosa.

Filamentous molecules tend to spontaneously assemble into liquid crystalline droplets with a tactoid morphology in environments with high concentration on non-adsorbing molecules. Tactoids of filamentous Pf bacteriophage, such as those produced by Pseudomonas aeruginosa, have been linked to increased antibiotic tolerance. We modelled this system and show that tactoids composed of filamentous Pf virions can lead to antibiotic tolerance by acting as an adsorptive diffusion barrier. The continuum model, reminiscent of descriptions of reactive diffusion in porous media, has been solved numerically and good agreement was found with the analytical results, obtained using a homogenisation approach. We find that the formation of tactoids significantly increases antibiotic diffusion times which may lead to stronger antibiotic resistance.

Filamentous bacteriophages, natural nanoparticles, for viral vaccine strategies.

Filamentous bacteriophages are natural nanoparticles formed by the self-assembly of structural proteins that have the capability of replication and infection. They are used as a highly efficient vaccine platform to enhance immunogenicity and effectively stimulate the innate and adaptive immune response. Compared with traditional vaccines, phage-based vaccines offer thermodynamic stability, biocompatibility, homogeneity, high carrying capacity, self-assembly, scalability, and low toxicity. This review summarizes recent research on phage-based vaccines in virus prevention. In addition, the expression systems of filamentous phage-based virus vaccines and their application principles are discussed. Moreover, the prospect of the prevention of emerging infectious diseases, such as coronavirus 2019 (COVID-19), is also discussed.

A Filamentous Bacteriophage Protein Inhibits Type IV Pili To Prevent Superinfection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections in a variety of settings. Many P. aeruginosa isolates are infected by filamentous Pf bacteriophage integrated into the bacterial chromosome as a prophage. Pf virions can be produced without lysing P. aeruginosa. However, cell lysis can occur during superinfection, which occurs when Pf virions successfully infect a host lysogenized by a Pf prophage. Temperate phages typically encode superinfection exclusion mechanisms to prevent host lysis by virions of the same or similar species. In this study, we sought to elucidate the superinfection exclusion mechanism of Pf phage. Initially, we observed that P. aeruginosa that survive Pf superinfection are transiently resistant to Pf-induced plaquing and are deficient in twitching motility, which is mediated by type IV pili (T4P). Pf utilize T4P as a cell surface receptor, suggesting that T4P are suppressed in bacteria that survive superinfection. We tested the hypothesis that a Pf-encoded protein suppresses T4P to mediate superinfection exclusion by expressing Pf proteins in P. aeruginosa and measuring plaquing and twitching motility. We found that the Pf protein PA0721, which we termed Pf superinfection exclusion (PfsE), promoted resistance to Pf infection and suppressed twitching motility by binding the T4P protein PilC. Because T4P play key roles in biofilm formation and virulence, the ability of Pf phage to modulate T4P via PfsE has implications in the ability of P. aeruginosa to persist at sites of infection. IMPORTANCE Pf bacteriophage (phage) are filamentous viruses that infect Pseudomonas aeruginosa and enhance its virulence potential. Pf virions can lyse and kill P. aeruginosa through superinfection, which occurs when an already infected cell is infected by the same or similar phage. Here, we show that a small, highly conserved Pf phage protein (PA0721, PfsE) provides resistance to superinfection by phages that use the type IV pilus as a cell surface receptor. PfsE does this by inhibiting assembly of the type IV pilus via an interaction with PilC. As the type IV pilus plays important roles in virulence, the ability of Pf phage to modulate its assembly has implications for P. aeruginosa pathogenesis.