Filamentous Phage M13 Research Articles

Phage-Templated Synthesis of Targeted Photoactive 1D-Thiophene Nanoparticles.

Thiophene-based nanoparticles (TNPs) are promising therapeutic and imaging agents. Here, using an innovative phage-templated synthesis, a strategy able to bypass the current limitations of TNPs in nanomedicine applications is proposed. The phage capsid is decorated with oligothiophene derivatives, transforming the virus in a 1D-thiophene nanoparticle (1D-TNP). A precise control of the shape/size of the nanoparticles is obtained exploiting the well-defined morphology of a refactored filamentous M13 phage, engineered by phage display to selectively recognize the Epidermal Growth Factor Receptor (EGFR). The tropism of the phage is maintained also after the bioconjugation of the thiophene molecules on its capsid. Moreover, the 1D-TNP proved highly fluorescent and photoactive, generating reactive oxygen species through both type I and type II mechanisms. The phototheranostic properties of this platform are investigated on biosystems presenting increasing complexity levels, from in vitro cancer cells in 2D and 3D architectures, to the in vivo tissue-like model organism Hydra vulgaris. The phage-templated 1D-TNP showed photocytotoxicity at picomolar concentrations, and the ability to deeply penetrate 3D spheroids and Hydra tissues. Collectively the results indicate that phage-templated synthesis of organic nanoparticles represents a general strategy, exploitable in many diagnostic and therapeutic fields based on targeted imaging and light mediated cell ablation.

Biomimetic Plasmonic Nanophages by Head/Tail Self-Assembling: Gold Nanoparticle/Virus Interactions.

Gold nanoparticles (AuNPs), because of their dual plasmonic and catalytic functionalities, are among the most promising nanomaterials for the development of therapeutic and diagnostic tools for severe diseases such as cancer and neurodegeneration. Bacteriophages, massively present in human biofluids, are emerging as revolutionary biotechnological tools as they can be engineered to display multiple specific binding moieties, providing effective targeting ability, high stability, low cost, and sustainable production. Coupling AuNPs with phages can lead to an advanced generation of nanotools with great potential for biomedical applications. In the present study, we analyzed the interactions between differently sized AuNPs and filamentous M13 phages, establishing an advanced characterization platform that combines analytical techniques and computational models for an in-depth understanding of these hybrid self-assembling systems. A precise and structurally specific interaction of the AuNP-M13 hybrid complexes was observed, leading to a peculiar head/tail "tadpole-like" configuration. In silico simulations allowed explaining the mechanisms underlying the preferential assembly route and providing information about AuNPs' size-dependent interplay with specific M13 capsid proteins. The AuNP-M13 structures were proven to be biomimetic, eluding the formation of biomolecular corona. By keeping the biological identity of the virion, hybrid nanostructures maintained their natural recognition/targeting ability even in the presence of biomolecular crowding. In addition, we were able to tune the hybrid nanostructures' tropism toward E. coli based on the AuNP size. Overall, our results set the fundamental basis and a standard workflow for the development of phage-based targeting nanotools, valuable for a wide spectrum of nanotechnology applications.

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.

Bacteriophage-based particles carrying the TNF-related apoptosis-inducing ligand (TRAIL) gene for targeted delivery in hepatocellular carcinoma.

The TRAIL (Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand) is a promising candidate for cancer treatment due to its unique ability to selectively induce programmed cell death, or apoptosis, in cancer cells while sparing healthy ones. This selectivity arises from the preferential binding of the TRAIL to death receptors on cancer cells, triggering a cascade of events that lead to their demise. However, significant limitations in using the TRAIL for cancer treatment are the administration of the TRAIL protein that can potentially lead to tissue toxicity (off-target) and the short half-life of the TRAIL in the body which may necessitate frequent and sustained administration; these can pose logistical challenges for long-term treatment regimens. We have devised a novel approach for surmounting these limitations by introducing the TRAIL gene directly into cancer cells, enabling them to produce the TRAIL locally and subsequently trigger apoptosis. A novel gene delivery system such as a bacteriophage-based particle TPA (transmorphic phage/AAV) was utilized to address these limitations. TPA is a hybrid M13 filamentous bacteriophage particle encapsulating a therapeutic gene cassette with inverted terminal repeats (ITRs) from adeno-associated viruses (AAVs). The particle also showed a tumour targeting ligand, CDCRGDCFC (RGD4C), on its capsid (RGD4C.TPA) to target the particle to cancer cells. RGD4C selectively binds to αvβ3 and αvβ5 integrins overexpressed on the surface of most of the cancer cells but is barely present on normal cells. Hepatocellular carcinoma (HCC) was chosen as a model because it has one of the lowest survival rates among cancers. We demonstrated that human HCC cell lines (Huh-7 and HepG2) express αvβ5 integrin receptors on their surface. These HCC cells also express death receptors and TRAIL-binding receptors. We showed that the targeted TPA particle carrying the transmembrane TRAIL gene (RGD4C.TPA-tmTRAIL) selectively and efficiently delivered the tmTRAIL gene to HCC cells resulting in the production of tmTRAIL from transduced cells and subsequently induced apoptotic death of HCC cells. This tumour-targeted particle can be an excellent candidate for the targeted gene therapy of HCC.

Construction and characterization of a novel miniaturized filamentous phagemid for targeted mammalian gene transfer

BackgroundAs simplistic proteinaceous carriers of genetic material, phages offer great potential as targeted vectors for mammalian transgene delivery. The filamentous phage M13 is a single-stranded DNA phage with attractive characteristics for gene delivery, including a theoretically unlimited DNA carrying capacity, amenability to tropism modification via phage display, and a well-characterized genome that is easy to genetically modify. The bacterial backbone in gene transfer plasmids consists of elements only necessary for amplification in prokaryotes, and, as such, are superfluous in the mammalian cell. These problematic elements include antibiotic resistance genes, which can disseminate antibiotic resistance, and CpG motifs, which are inflammatory in animals and can lead to transgene silencing.ResultsHere, we examined how M13-based phagemids could be improved for transgene delivery by removing the bacterial backbone. A transgene cassette was flanked by isolated initiation and termination elements from the phage origin of replication. Phage proteins provided in trans by a helper would replicate only the cassette, without any bacterial backbone. The rescue efficiency of “miniphagemids” from these split origins was equal to, if not greater than, isogenic “full phagemids” arising from intact origins. The type of cassette encoded by the miniphagemid as well as the choice of host strain constrained the efficiency of phagemid rescue.ConclusionsThe use of two separated domains of the f1 ori improves upon a single wildtype origin while still resulting in high titres of miniphagemid gene transfer vectors. Highly pure lysates of miniaturized phagemids could be rapidly obtained in a straightforward procedure without additional downstream processing.

Rapid and reliable ultrasensitive detection of pathogenic H9N2 viruses through virus-binding phage nanofibers decorated with gold nanoparticles

The rapid and sensitive detection of pathogenic viruses is important for controlling pandemics. Herein, a rapid, ultrasensitive, optical biosensing scheme was developed to detect avian influenza virus H9N2 using a genetically engineered filamentous M13 phage probe. The M13 phage was genetically engineered to bear an H9N2-binding peptide (H9N2BP) at the tip and a gold nanoparticle (AuNP)-binding peptide (AuBP) on the sidewall to form an engineered phage nanofiber, M13@H9N2BP@AuBP. Simulated modelling showed that M13@H9N2BP@AuBP enabled a 40-fold enhancement of the electric field enhancement in surface plasmon resonance (SPR) compared to conventional AuNPs. Experimentally, this signal enhancement scheme was employed for detecting H9N2 particles with a sensitivity down to 6.3 copies/mL (1.04 × 10−5 fM). The phage-based SPR scheme can detect H9N2 viruses in real allantoic samples within 10 min, even at very low concentrations beyond the detection limit of quantitative polymerase chain reaction (qPCR). Moreover, after capturing the H9N2 viruses on the sensor chip, the H9N2-binding phage nanofibers can be quantitatively converted into plaques that are visible to the naked eye for further quantification, thereby allowing us to enumerate the H9N2 virus particles through a second mode to cross-validate the SPR results. This novel phage-based biosensing strategy can be employed to detect other pathogens because the H9N2-binding peptides can be easily switched with other pathogen-binding peptides using phage display technology.

A plug-and-play aptamer diagnostic platform based on linear dichroism spectroscopy.

A plug-and-play sandwich assay platform for the aptamer-based detection of molecular targets using linear dichroism (LD) spectroscopy as a read-out method has been demonstrated. A 21-mer DNA strand comprising the plug-and-play linker was bioconjugated onto the backbone of the filamentous bacteriophage M13, which gives a strong LD signal due to its ready alignment in linear flow. Extended DNA strands containing aptamer sequences that bind the protein thrombin, TBA and HD22, were then bound to the plug-and-play linker strand via complementary base pairing to generate aptamer-functionalised M13 bacteriophages. The secondary structure of the extended aptameric sequences required to bind to thrombin was checked using circular dichroism spectroscopy, with the binding confirmed using fluorescence anisotropy measurements. LD studies revealed that this sandwich sensor design is very effective at detecting thrombin down to pM levels, indicating the potential of this plug-and-play assay system as a new label-free homogenous detection system based on aptamer recognition.

Inhibition of avian influenza virus H9N2 infection by antiviral hexapeptides that target viral attachment to epithelial cells

The H9N2 subtype of influenza A virus circulates frequently among poultry in Asian and North African countries causing economic loss in the poultry sector. The antigenic variations of the H9N2 virus were at the origin of its genetic evolution through the emergence of viral strains transmissible to humans and resistant to chemical antivirals, which require a strengthening of the fight means against this virus. In this study, we used a random linear hexapeptide library fused to the gene III protein of M13 filamentous bacteriophage to select new antiviral peptides that inhibit the infectivity of H9N2 virus. After three rounds of stringent selection and amplification, polyclonal phage-peptides directed against H9N2 virus were assessed by ELISA, and the optimal phage-peptides were grown individually and characterized for binding to H9N2 virus by monoclonal phage ELISA. The DNA of 27 phage-peptides clones was amplified by PCR, sequenced, and their amino acid sequences were deduced. Sixteen different phage-peptides were able to bind specifically the H9N2 virus, among them, 13 phage-peptides interacted with the hemagglutinin H9. Two selected peptides, P1 (LSRMPK) and P2 (FAPRWR) have shown antiviral activity in ovo and P1 was more protective in vivo then P2 when co-administered with the H9N2 virus. Mechanistically, these peptides prevent infection by inhibiting the attachment of the H9N2 virus to the cellular receptor. Molecular docking revealed that the peptides LSRMPK and FAPRWR bind to hemagglutinin protein H9, but interact differently with the receptor binding site (RBS). The present study demonstrated that the peptide P1 (LSRMPK) could be used as a new inhibitory molecule directed against the H9N2 virus.

Uniformly assembly of filamentous phage/SiO2 composite films with tunable chiral nematic structures in capillary confinement

The design and development of biomimetic advanced materials can generate exquisite microstructures and excellent performance. Using the liquid crystal assembly of biomacromolecules can effectively realize architectures control at micro-/nanoscale. Filamentous M13 phage has attracted extensive attention due to its good monodispersity and high aspect ratio, which makes it the ideal building block to assembly liquid crystal-like structures. Millimeter-sized M13 film with the thickness of 300 μm (ChM13) is assembled in a capillary. Anisotropic evaporation and the drying process in the capillary confinement promote the formation of uniform chiral nematic structures. The formation process and microstructure of the resulting film are characterized in detail. Furthermore, the microstructures and mechanical properties of the film are tunable by incorporating inorganic silica component with various contents to form similar composite structures found in structurally colored leaves. As the silica content increases, the half helical pitch of the chiral nematic structure decreases from 14.5 to 5.5 μm and recovers to 8 μm. The Young' s modulus increases from 492 to 920 MPa, and the hardness increases from 6.2 to 20.6 MPa. This preparation strategy is expected to provide new insight and ideas into formation of elaborate biological structures under mild conditions.

Targeting Human Osteoarthritic Chondrocytes with Ligand Directed Bacteriophage-Based Particles.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive deterioration and loss of articular cartilage. There is currently no treatment to reverse the onset of OA. Thus, we developed a targeted delivery strategy to transfer genes into primary human chondrocytes as a proof-of-concept study. We displayed a chondrocyte-affinity peptide (CAP) on the pIII minor coat protein of the M13 filamentous bacteriophage (phage)-based particle carrying a mammalian transgene cassette under cytomegalovirus CMV promoter and inverted terminal repeats (ITRs) cis elements of adeno-associated virus serotype 2 (AAV-2). Primary human articular chondrocytes (HACs) were used as an in vitro model, and the selectivity and binding properties of the CAP ligand in relation to the pathogenic conditions of HACs were characterized. We found that the CAP ligand is highly selective toward pathogenic HACs. Furthermore, the stability, cytotoxicity, and gene delivery efficacy of the CAP-displaying phage (CAP.Phage) were evaluated. We found that the phage particle is stable under a wide range of temperatures and pH values, while showing no cytotoxicity to HACs. Importantly, the CAP.Phage particle, carrying a secreted luciferase (Lucia) reporter gene, efficiently and selectively delivered transgene expression to HACs. In summary, it was found that the CAP ligand preferably binds to pathogenic chondrocytes, and the CAP.Phage particle successfully targets and delivers transgene to HACs.

Prevention of side reactions with a unique carbon-free catalyst biosynthesized by a virus template for non-aqueous and quasi-solid-state Li–O2 batteries

The rational architecture and composition of electrocatalysts are crucial factors for highly efficient lithium-oxygen (Li–O 2 ) systems. In this study, one-dimensional MnO 2 polymorphisms (α-, δ-, γ-MnO 2 ) and a hybrid nanostructure composed of α-MnO 2 nanowires and Ru nanoparticles are designed through a bio-templated method. Their electrochemical properties are assessed as efficient electrocatalysts for a non-aqueous Li–O 2 cell. The virus-templated α-MnO 2 , δ-MnO 2 , and γ-MnO 2 nanowires in combination with carbon additives indicate specific capacities of 10,875 mAh g −1 , 9726 mAh g −1 , and 6575 mAh g −1 , respectively. However, the presence of carbon inhibits the reversible reaction during cycling performance due to the formation of Li 2 CO 3 . Therefore, the incorporation of Ru nanoparticles on the surface of virus-templated α-MnO 2 nanowires is studied as an efficient carbon-free cathode material. Notably, the virus-templated Ru/α-MnO 2 hybrid electrode is delivered a high specific capacity of 14,383 mAh g −1 with the stability of 48 cycles at a fixed capacity of 1000 mAh g −1 . Meanwhile, lithium corrosion and electrolyte decomposition are observed during cycling, which restricts the life cycle of the cell. Therefore, a quasi-solid-state Li–O 2 cell is also fabricated by designing an integrated gel polymer electrolyte/cathode, and remarkably the Li–O 2 cell shows cycling up to 95 cycles. • An effective carbon-free cathode catalyst is developed. • The efficient catalysts are designed using filamentous M13 phage as a bio-template. • The virus-templated Ru/α-MnO 2 nanowires inhibit the side reactions occurrence. • The integrated gel polymer electrolyte/cathode enhances the cyclability of Li–O 2 .

Deep mutational scanning of the plasminogen activator inhibitor-1 functional landscape

The serine protease inhibitor (SERPIN) plasminogen activator inhibitor-1 (PAI-1) is a key regulator of the fibrinolytic system, inhibiting the serine proteases tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). Missense variants render PAI-1 non-functional through misfolding, leading to its turnover as a protease substrate, or to a more rapid transition to the latent/inactive state. Deep mutational scanning was performed to evaluate the impact of amino acid sequence variation on PAI-1 inhibition of uPA using an M13 filamentous phage display system. Error prone PCR was used to construct a mutagenized PAI-1 library encompassing ~ 70% of potential single amino acid substitutions. The relative effects of 27% of all possible missense variants on PAI-1 inhibition of uPA were determined using high-throughput DNA sequencing. 826 missense variants demonstrated conserved inhibitory activity while 1137 resulted in loss of PAI-1 inhibitory function. The least evolutionarily conserved regions of PAI-1 were also identified as being the most tolerant of missense mutations. The results of this screen confirm previous low-throughput mutational studies, including those of the reactive center loop. These data provide a powerful resource for explaining structure–function relationships for PAI-1 and for the interpretation of human genomic sequence variants.

Sites of vulnerability on ricin B chain revealed through epitope mapping of toxin-neutralizing monoclonal antibodies.

Ricin toxin's B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin's ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB's duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as "bait" in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB's two domains may contribute to distinct steps in the intoxication pathway.

Nanomechanics of graphene oxide-bacteriophage based self-assembled porous composites

Graphene oxide, integrated with the filamentous bacteriophage M13, forms a 3D large-scale multifunctional porous structure by self-assembly, with considerable potential for applications. We performed Raman spectroscopy under pressure on this porous composite to understand its fundamental mechanics. The results show that at low applied pressure, the sp^2 bonds of graphene oxide stiffen very little with increasing pressure, suggesting a complicated behaviour of water intercalated between the graphene layers. The key message of this paper is that water in a confined space can have a significant impact on the nanostructure that hosts it. We introduced carbon nanotubes during the self-assembly of graphene oxide and M13, and a similar porous macro-structure was observed. However, in the presence of carbon nanotubes, pressure is transmitted to the sp^2 bonds of graphene oxide straightforwardly as in graphite. The electrical conductivity of the composite containing carbon nanotubes is improved by about 30 times at a bias voltage of 10 V. This observation suggests that the porous structure has potential in applications where good electrical conductivity is desired, such as sensors and batteries.

Efficient affinity-tagging of M13 phage capsid protein IX for immobilization of protein III-displayed oligopeptide probes on abiotic platforms.

We developed a genetic approach to efficiently add an affinity tag to every copy of protein IX (pIX) of M13 filamentous bacteriophage in a population. Affinity-tagged phages can be immobilized on a surface in a uniform monolayer in order to position the pIII-displayed peptides or proteins for optimal interaction with ligands. The tagging consists of two major steps. First, gene IX (gIX) of M13 phage is mutated in Escherichia coli via genetic recombineering with the gIX::aacCI insertion allele. Second, a plasmid that co-produces the affinity-tagged pIX and native pVIII is transformed into the strain carrying the defective M13 gIX. This genetic complementation allows the formation of infective phage particles that carry a full complement (five copies per virion) of the affinity-tagged pIX. To demonstrate the efficacy of our method, we tagged a M13 derivative phage, M13KE, with Strep-tag II. In order to tag pIX with Strep-tag II, the phage genes for pIX and pVIII were cloned and expressed from pASG-IBA4 which contains the E. coli OmpA signal sequence and Strep-Tag II under control of the tetracycline promoter/operator system. We achieved the maximum phage production of 3 × 1011pfu/ml when Strep-Tag II-pIX-pVIII fusion was induced with 10ng/ml of anhydrotetracycline. The complete process of affinity tagging a phage probe takes less than 5days and can be utilized to tag any M13 or fd pIII-displayed oligopeptide probes to improve their performance.

Study of gold nanoparticle transport by M13 phages towards disease tissues as targeting procedure for radiotherapy applications

This work introduces an innovative new method to transport gold nanoparticles (Au-NPs) toward specific organs using as transporter the M13 filamentous phage. The aim is to improve the radiotherapy efficiency by injecting gold nanoparticles in the tumor tissues in order to increase the effective atomic number and, consequently, to produce higher locally deposited doses. The biocompatible Au-NPs can be bonded to the phage surface for their transport in the biological fluids and tissues. M13 phages can be prepared to arrive in specific tissues and organs depending on their biochemical preparation and functionalization. Evidence about the real attachment of spherical Au-NPs, 10 nm in diameter, to the M13 phages is obtained using different microscopies. Other analyses have been also performed to evaluate the number of gold nanoparticles that can be transported by M13 phages inside the organism. Our results on the study of the spherical Au-NP size distribution, deduced by AFM, EFM, and TEM analyses, have indicated that in average, 2 couples of Au-NPs are attached to each phage. Moreover, the size of the NPs ranges between 10 and 30 nm, demonstrating a little aggregation of the nanoparticles, which remain usefully sized to be vehicled with the blood flux towards specific organs.

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.

Rapid time-resolved luminescence based screening of bacteria in urine with luminescence modulating biosensing phages

Urinary tract infections (UTIs) are a common problem worldwide. The most prevalent causative pathogen of UTI is Escherichia coli, focus of this study. The current golden standard for detecting UTI is bacterial culture, creating a major workload for hospital laboratories - cost-effective and rapid mass screening of patient samples is needed. Here we present an alternative approach to screen patient samples with a single-step assay utilising time-resolved luminescence and luminescence modulating biosensing phages. Filamentous phage M13 was biopanned for binding luminescence quenching metal (copper) and further E. coli. The screening assay luminescence modulation was further enhanced by selecting right chemical environment for the functioning phage clones. Semi-specific interaction between phage, target bacteria and metal was detected by modulation in the signal of a weakly chelating, easily quenchable lanthanide complex. In the presence of the target pathogen, the phages collected quenching metal from solution to the bacterial surface changing the quenching effect on the lanthanide label and thus modulating the signal. Our method was compared with the bacterial culture data obtained from 70 patient samples. The developed proof-of-principle screening assay showed sensitivity and a specificity at the 90% mark when compared to culture method although some samples had high turbidity and even blood. The detection limit of E. coli was in the range of 1000–10 000 colony forming units/mL. Untreated urine sample was screened and time-resolved luminescence signal result was achieved within 10 min in a single incubation step.

Environmental adjustments of the cooperativity in M13 phage thermal denaturation

The filamentous bacteriophage M13 has been widely used in phage display and as a scaffold for nanostructures. Although M13 is loosely described as a thermo-resistant virus, no thorough studies of its thermostability have been reported so far. By using differential scanning calorimetry (DSC), we characterized the thermal stability of M13 in aqueous buffer and in the presence of ethanol. M13 was found to be very thermostable indeed, being resistant to the combined effects of temperature and high ethanol content. Upon thermal denaturation, several subunits of the major coat protein apparently undergo a cooperative conformational change, leading to the formation of residual β-strand conformation. Furthermore, the cooperativity of the denaturation process changes drastically with pH and ethanol concentration, indicating that P8 moieties are able to adjust its intermolecular packing in response to the environment. Deconvolution of DSC scans allowed the estimation of the number of P8 molecules comprising one cooperative unit.