Filamentous Phage Research Articles

Crossing the blood-brain-barrier with nanoligand drug carriers self-assembled from a phage display peptide

The filamentous bacteriophage fd bind a cell target with exquisite specificity through its few copies of display peptides, whereas nanoparticles functionalized with hundreds to thousands of synthetically generated phage display peptides exhibit variable and often-weak target binding. We hypothesise that some phage peptides in a hierarchical structure rather than in monomeric form recognise and bind their target. Here we show hierarchial forms of a brain-specific phage-derived peptide (herein as NanoLigand Carriers, NLCs) target cerebral endothelial cells through transferrin receptor and the receptor for advanced glycation-end products, cross the blood-brain-barrier and reach neurons and microglial cells. Through intravenous delivery of NLC-β-secretase 1 (BACE1) siRNA complexes we show effective BACE1 down-regulation in the brain without toxicity and inflammation. Therefore, NLCs act as safe multifunctional nanocarriers, overcome efficacy and specificity limitations in active targeting with nanoparticles bearing phage display peptides or cell-penetrating peptides and expand the receptor repertoire of the display peptide.

Assessment of Non‐Uniform Sampling Schemes in Solid State NMR of Bacteriophage Viruses

AbstractThe set of tools offered to NMR spectroscopists has been greatly expanded in the last decades with the development of computer processing power. It has facilitated the use of non‐Fourier transform reconstruction algorithms, leading to the ability to acquire data using non‐uniform sampling. Its use in solid‐state NMR, especially for biological systems, emerges as the next leap toward the analysis and structure prediction of large proteins. Here we applied various non‐uniform sampling schemes to assess their performance towards a three‐dimensional N−C−C correlation experiment aimed to assign chemical shifts in the coat protein of an intact filamentous bacteriophage fd virus. We show that using the sparse multidimensional iterative lineshape‐enhanced (SMILE) reconstruction algorithm, biased sampling increases signal‐to‐noise without compromising linewidths and accuracy of chemical shift determination.

The Vibrio cholerae minor pilin TcpB mediates uptake of the cholera toxin phage CTXϕ

Virulent strains of the bacterial pathogen Vibrio cholerae cause the diarrheal disease cholera by releasing cholera toxin into the small intestine. V. cholerae acquired its cholera toxin genes by lysogenic infection with the filamentous bacteriophage CTXφ. CTXφ uses its minor coat protein pIII, located in multiple copies at the phage tip, to bind to the V. cholerae toxin-coregulated pilus (TCP). However, the molecular details of this interaction and the mechanism of phage internalization are not well-understood. The TCP filament is a polymer of major pilins, TcpA, and one or more minor pilin, TcpB. TCP are retractile, with both retraction and assembly initiated by TcpB. Consistent with these roles in pilus dynamics, we hypothesized that TcpB controls both binding and internalization of CTXφ. To test this hypothesis, we determined the crystal structure of the C-terminal half of TcpB and characterized its interactions with CTXφ pIII. We show that TcpB is a homotrimer in its crystallographic form as well as in solution and is present in multiple copies at the pilus tip, which likely facilitates polyvalent binding to pIII proteins at the phage tip. We further show that recombinant forms of TcpB and pIII interact in vitro, and both TcpB and anti-TcpB antibodies block CTXφ infection of V. cholerae Finally, we show that CTXφ uptake requires TcpB-mediated retraction. Our data support a model whereby CTXφ and TCP bind in a tip-to-tip orientation, allowing the phage to be drawn into the V. cholerae periplasm as an extension of the pilus filament.

Probing the dynamic stalk region of the ribosome using solution NMR

We describe an NMR approach based on the measurement of residual dipolar couplings (RDCs) to probe the structural and motional properties of the dynamic regions of the ribosome. Alignment of intact 70S ribosomes in filamentous bacteriophage enabled measurement of RDCs in the mobile C-terminal domain (CTD) of the stalk protein bL12. A structural refinement of this domain using the observed RDCs did not show large changes relative to the isolated protein in the absence of the ribosome, and we also found that alignment of the CTD was almost independent of the presence of the core ribosome particle, indicating that the inter-domain linker has significant flexibility. The nature of this linker was subsequently probed in more detail using a paramagnetic alignment strategy, which revealed partial propagation of alignment between neighbouring domains, providing direct experimental validation of a structural ensemble previously derived from SAXS and NMR relaxation measurements. Our results demonstrate the prospect of better characterising dynamical and functional regions of more challenging macromolecular machines and systems, for example ribosome–nascent chain complexes.

Arming Filamentous Bacteriophage, a Nature-Made Nanoparticle, for New Vaccine and Immunotherapeutic Strategies.

The pharmaceutical use of bacteriophages as safe and inexpensive therapeutic tools is collecting renewed interest. The use of lytic phages to fight antibiotic-resistant bacterial strains is pursued in academic and industrial projects and is the object of several clinical trials. On the other hand, filamentous bacteriophages used for the phage display technology can also have diagnostic and therapeutic applications. Filamentous bacteriophages are nature-made nanoparticles useful for their size, the capability to enter blood vessels, and the capacity of high-density antigen expression. In the last decades, our laboratory focused its efforts in the study of antigen delivery strategies based on the filamentous bacteriophage ‘fd’, able to trigger all arms of the immune response, with particular emphasis on the ability of the MHC class I restricted antigenic determinants displayed on phages to induce strong and protective cytotoxic responses. We showed that fd bacteriophages, engineered to target mouse dendritic cells (DCs), activate innate and adaptive responses without the need of exogenous adjuvants, and more recently, we described the display of immunologically active lipids. In this review, we will provide an overview of the reported applications of the bacteriophage carriers and describe the advantages of exploiting this technology for delivery strategies.

Genetically Engineered Fd Viruses for Site Specific Material Binding

Filamentous bacteriophages are viruses infecting only bacteria. In this study, filamentous fd viruses were genetically engineered to display specific gold- (V5) and platinum-binding (p7A) peptides on the head part (P9 minor coat protein). Gold-binding properties of recombinant p9V5, p9P7A, and wild-type fd viruses were investigated by Quartz Crystal Microbalance (QCM), scanning electron microscopy (SEM), atomic force microscopy (AFM), energy dispersive X-ray spectroscopy (EDX), and UV-Vis spectroscopy analyses. Displaying only 5 copies of V5 peptides on head part of viruses resulted in site-specific Au-binding. Surprisingly, p9p7A viruses, as well, showed enhanced gold-binding properties with respect to wild-type fd viruses. Filamentous fd viruses are good candidates of genetically programmable biotemplates for mineralization and metallization studies.

British Society for Gene and Cell Therapy Annual Conference Wednesday 19th June – Friday 21st June 2019 University of Sheffield, Sheffield, UK www.bsgct.org

British Society for Gene and Cell Therapy Annual Conference Wednesday 19th June – Friday 21st June 2019 University of Sheffield, Sheffield, UK www.bsgct.org

Hierarchically Structured, Self-Healing, Fluorescent, Bioactive Hydrogels with Self-Organizing Bundles of Phage Nanofilaments

Bacteriophages are essentially bionanoparticles with a protein coat, the composition of which can be controlled with atomic precision via genetic engineering, a property that makes them superior to synthetic nanoparticles as building blocks for bottom-up synthesis of multifunctional materials with advanced properties. We report hierarchically structured hydrogels of self-organized M13 bacteriophage bundles, composed of hundreds of M13 nanofilaments, which exhibit both long-range and micron-scale order, are visible in electron micrographs of the cross-linked state, and can adsorb up to 16× their weight in water. We further demonstrate that these hierarchical hydrogels of M13 exhibit advanced properties at room temperature, namely, self-healing under biological conditions, autofluorescence in three channels, which decays through biodegradation, potentiating non-destructive imaging capability, and bioactivity in the cross-linked state toward the host bacteria. The latter is, in particular, a powerful property, allowing the development of hydrogels with tunable bioactivity when combined with the phage display and/or recombinant DNA technology. Filamentous phage M13 has garnered significant attention in the past decade for the development of functional materials, ranging from tissue engineering scaffolds to batteries. Our investigation reveals the ability of these nanofilaments to self-organize into hierarchically structured soft matter, highlighting the power of self-organized M13 structures as building blocks for bottom-up synthesis.

Mass spectrometry enumeration of filamentous M13 bacteriophage

In the last decade, filamentous M13 bacteriophage has emerged into numerous biotechnological applications as a promising nontoxic and self-assembling biomaterial with specific binding properties. This raises a question about its upscale production that consequently requires an accurate phage enumeration during the various protocol developments. However, traditional methods of measuring phage concentration are mainly biological in nature and therefore time and labor intensive. These traditional methods also demonstrate poor reproducibility and are semi-quantitative at best. In the present work, we capitalized on mass spectrometry based absolute protein quantitation. We have optimized the quantitation conditions for a major coat protein, pVIII. Enumeration of M13 bacteriophage can be further performed using the determined molar concentration of pVIII, Avogadro's number, and known copy number of pVIII per phage. Since many different phages have well-defined copy number of capsid proteins, the proposed approach can be simply applied to any phage with known copy number of a specific capsid protein.

Remarkable Rigidity of the Single α-Helical Domain of Myosin-VI As Revealed by NMR Spectroscopy.

Although the α-helix has long been recognized as an all-important element of secondary structure, it generally requires stabilization by tertiary interactions with other parts of a protein’s structure. Highly charged single α-helical (SAH) domains, consisting of a high percentage (>75%) of Arg, Lys, and Glu residues, are exceptions to this rule but have been difficult to characterize structurally. Our study focuses on the 68-residue medial tail domain of myosin-VI, which is found to contain a highly ordered α-helical structure extending from Glu-6 to Lys-63. High hydrogen exchange protection factors (15–150), small (ca. 4 Hz) 3JHNHα couplings, and a near-perfect fit to an ideal model α-helix for its residual dipolar couplings (RDCs), measured in a filamentous phage medium, support the high regularity of this helix. Remarkably, the hydrogen exchange rates are far more homogeneous than the protection factors derived from them, suggesting that for these transiently broken helices the intrinsic exchange rates derived from the amino acid sequence are not appropriate reference values. 15N relaxation data indicate a very high degree of rotational diffusion anisotropy (D∥/D⊥ ≈ 7.6), consistent with the hydrodynamic behavior predicted for such a long, nearly straight α-helix. Alignment of the helix by a paramagnetic lanthanide ion attached to its N-terminal region shows a decrease in alignment as the distance from the tagging site increases. This decrease yields a precise measure for the persistence length of 224 ± 10 Å at 20 °C, supporting the idea that the role of the SAH helix is to act as an extension of the myosin-VI lever arm.

Immuno-Based Molecular Scaffolding of Glucose Dehydrogenase and Ferrocene Mediator on fd Viral Particles Yields Enhanced Bioelectrocatalysis

A virus-based nanostructuring strategy is proposed for improving the catalytic performance of integrated redox enzyme electrodes. Random arrays of adsorbed filamentous fd bacteriophage particles, used as scaffolds, are assembled onto gold electrode surfaces. The viral particles are endowed with functionally coupled enzymatic and redox properties, by the sequential immunological assembly of quinoprotein glucose dehydrogenase conjugated antibodies and ferrocene PEGylated antibodies on their protein shell. The resulting virus-scaffolded enzyme/redox mediator integrated system displays a large enhancement in the catalytic current generated per enzyme molecule (i.e., in enzymatic turnover) as compared with nonscaffolded integrated glucose oxidizing enzyme electrodes. The mechanism underlying the observed scaffolding-induced catalytic enhancement is deciphered. Confinement of the mediator on the viral scaffold enables fast electron transport rate and shifts the enzyme behavior into its most effective cooperative kinetic mode.

Immunological properties of the SLLTEVET epitope of Influenza A virus in multiple display on filamentous M13 phage

Small peptides require large carriers to stimulate the humoral immune system. The filamentous phages, such as M13, have been proposed as vectors for expressing and carrying these peptides on their capsid surface. M2e 2-9 (SLLTEVET) residues of the transmembrane protein M2 of Influenza A virus are conserved and considered as a suitable target for immunization against a wide range of Influenza A virus strains. Here, M2e (2-9) sequence of Influenza A virus was fused to the N-terminus of the major coat protein gpVIII of M13 phage and was used to immunize broiler chickens. The results showed that the SLLTEVET peptide on the surface of M13 phage was expressed, the hybrid phage was immunogenic and produced specific antibodies against M2e (2-9) in broiler chickens.

Filamentous bacteriophages affect pathogenicity of Pseudomonas infections in cystic fibrosis.

This study shows that the presence of filamentous bacteriophages affects the pathogenicity of Pseudomonas infections in patients with cystic fibrosis.

Infection-boosting phage

Cystic Fibrosis Chronic Pseudomonas aeruginosa infection is common in patients with cystic fibrosis (CF). Filamentous bacteriophage (Pf phage) can infect P. aeruginosa and contribute to bacterial virulence in animal models. Whether Pf phage plays a role in the pathogenicity of P. aeruginosa in CF is unknown. Burgener et al. found that Pf phage was abundant in sputum samples from three cohorts of patients with CF. Pf phage presence was associated with increased antibiotic resistance and reduced lung function, suggesting that it may play a role in the pathogenicity of P. aeruginosa infection in CF. Sci. Transl. Med. 11 , eaau9748 (2019).

Filamentous bacteriophages are associated with chronic Pseudomonas lung infections and antibiotic resistance in cystic fibrosis.

Filamentous bacteriophage (Pf phage) contribute to the virulence of Pseudomonas aeruginosa infections in animal models, but their relevance to human disease is unclear. We sought to interrogate the prevalence and clinical relevance of Pf phage in patients with cystic fibrosis (CF) using sputum samples from two well-characterized patient cohorts. Bacterial genomic analysis in a Danish longitudinal cohort of 34 patients with CF revealed that 26.5% (n = 9) were consistently Pf phage positive. In the second cohort, a prospective cross-sectional cohort of 58 patients with CF at Stanford, sputum qPCR analysis showed that 36.2% (n = 21) of patients were Pf phage positive. In both cohorts, patients positive for Pf phage were older, and in the Stanford CF cohort, patients positive for Pf phage were more likely to have chronic P. aeruginosa infection and had greater declines in pulmonary function during exacerbations than patients negative for Pf phage presence in the sputum. Last, P. aeruginosa strains carrying Pf phage exhibited increased resistance to antipseudomonal antibiotics. Mechanistically, in vitro analysis showed that Pf phage sequesters these same antibiotics, suggesting that this mechanism may thereby contribute to the selection of antibiotic resistance over time. These data provide evidence that Pf phage may contribute to clinical outcomes in P. aeruginosa infection in CF.

Filamentous phages: masters of a microbial sharing economy.

Bacteriophage ("bacteria eaters") or phage is the collective term for viruses that infect bacteria. While most phages are pathogens that kill their bacterial hosts, the filamentous phages of the sub-class Inoviridae live in cooperative relationships with their bacterial hosts, akin to the principal behaviours found in the modern-day sharing economy: peer-to-peer support, to offset any burden. Filamentous phages impose very little burden on bacteria and offset this by providing service to help build better biofilms, or provision of toxins and other factors that increase virulence, or modified behaviours that provide novel motile activity to their bacterial hosts. Past, present and future biotechnology applications have been built on this phage-host cooperativity, including DNA sequencing technology, tools for genetic engineering andmolecular analysis of gene expression and protein production, and phage-display technologies for screening protein-ligand and protein-protein interactions. With the explosion of genome and metagenome sequencing surveys around the world, we are coming to realize that our knowledge of filamentous phage diversity remains at a tip-of-the-iceberg stage, promising that new biology and biotechnology are soon to come.

Phage Display Selection and Identification of Novel Pancreatic Cancer Targeting Peptides

Pancreatic ductal adenocarcinoma (PDA) is currently the fourth leading cause of cancer deaths in the U.S. It has been recently estimated that the disease causes approximately 30,000 deaths per year in the U.S., and that pancreatic cancer patients exhibit a 5‐year survival rate of only 3%. The high lethality of pancreatic cancer is due to the fact that most cases are diagnosed at advanced metastatic stages. While there are diagnostic methods in use clinically, most are inadequate, potentially leading to delayed diagnosis and further spreading of the cancer prior to treatment. Due to insufficient diagnostic techniques, it is imperative to develop new methods, which may facilitate early diagnosis. One such modality employs the use of peptides with high binding affinity and specificity to a pancreatic cancer biomarker.Bacteriophage (phage) display technology is a high throughput method for discovering peptides that confer specific binding properties to a single molecular target. The peptides are typically displayed on the distal end of coat protein III of the Fd filamentous phage. Here we report using phage display technology to select peptides with specific binding to human pancreatic cancer cells. To do so, a naïve fUSE5 15‐mer library (kind gift from Dr. George Smith) was negatively selected against normal pancreatic ductal cells (hTERT‐hPNE). Unbound cells were collected, amplified by infection of E. coli K91BK overnight, and isolated using polyethylene glycol (PEG), NaCl precipitation and centrifugation. Amplified phage were then subjected to four rounds of positive selection against pancreatic cancer cells (Mia‐Paca‐2) grown to 80–90% confluency. Between each round of selection, phage were collected, amplified in E. coli K91BK and isolated as described above. Stringency was increased in the fourth round of selection by performing the selection against cells grown to 60% confluency. After the final round of selection, phage DNA was extracted and polymerase chain reaction (PCR) amplicons were identified by paired‐end (2×150bp) next generation sequencing (MiSeq) followed by translation and sorting of the sequences (Genewiz, NJ). The “top” 10 peptides identified by this method will next be screened in cell binding assays to determine individual binding constants. Peptides that exhibit high binding affinity and specificity for Mia‐Paca‐2 cells may subsequently be employed as probes in the detection of pancreatic cancer.Support or Funding InformationWestern Illinois University Research Council GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A qPCR Targeted Against the Viral Replication Origin Designed to Quantify Total Amount of Filamentous Phages and Phagemids.

Filamentous bacteriophages are widely used in phage display technology. The most common quantification method is lysis plaque formation test (PFT). This technique has several disadvantages, and only quantifies infective phages and is not effective when phagemids are used. We developed a qPCR method directed against the M13 replication origin, which detects between 3.3 × 103 and 3.3 × 108 viral genome copies with a linearity of R 2 = 0.9998. Using this method we were able to observe a difference of approximately ten more phages than with the PFT. This difference was not due to the presence of a free genome, which suggests the presence of non-infective particles. Using a DNaseI treatment, we observed the presence of 30% to 40% of unpackaged genome in recombinant phage modified in PIII or PVIII. The qPCR method with a DNase I treatment is an efficient method to quantify the total amount of filamentous phages.

Bacteriophage trigger antiviral immunity and prevent clearance of bacterial infection.

Bacteriophage are abundant at sites of bacterial infection, but their effects on mammalian hosts are unclear. We have identified pathogenic roles for filamentous Pf bacteriophage produced by Pseudomonas aeruginosa (Pa) in suppression of immunity against bacterial infection. Pf promote Pa wound infection in mice and are associated with chronic human Pa wound infections. Murine and human leukocytes endocytose Pf, and internalization of this single-stranded DNA virus results in phage RNA production. This triggers Toll-like receptor 3 (TLR3)- and TIR domain-containing adapter-inducing interferon-β (TRIF)-dependent type I interferon production, inhibition of tumor necrosis factor (TNF), and the suppression of phagocytosis. Conversely, immunization of mice against Pf prevents Pa wound infection. Thus, Pf triggers maladaptive innate viral pattern-recognition responses, which impair bacterial clearance. Vaccination against phage virions represents a potential strategy to prevent bacterial infection.

Phage subverts immune response

Immunology Pseudomonas aeruginosa ( Pa ) is a multidrug-resistant Gramnegative bacterium commonly found in health care settings. Pa infections frequently result in considerable morbidity and mortality. Sweere et al. found that a type of temperate filamentous bacteriophage that infects and integrates into Pa is associated with chronic human wound infections. Likewise, wounds in mice colonized with phage-infected Pa were more severe and longer-lasting than those colonized by Pa alone. Immune cell uptake of phage-infected Pa resulted in phage RNA production and inappropriate antiviral immune responses, impeding bacterial clearance. Both phage vaccination and transfer of antiphage antibodies were protective against Pa infection. Science , this issue p. [eaat9691][1] [1]: /lookup/doi/10.1126/science.aat9691