Bacteriophage PP7 Research Articles

Removal of Pseudomonas type IV pili by a small RNA virus.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

RNA Aptamer-Mediated Gene Activation Systems for Inducible Transgene Expression in Animal Cells.

RNA expression analyses can be used to obtain various information from inside cells, such as physical conditions, the chemical environment, and endogenous signals. For detecting RNA, the system regulating intracellular gene expression has the potential for monitoring RNA expression levels in real time within living cells. Synthetic biology provides powerful tools for detecting and analyzing RNA inside cells. Here, we devised an RNA aptamer-mediated gene activation system, RAMGA, to induce RNA-triggered gene expression activation by employing an inducible complex formation strategy grounded in synthetic biology. This methodology connects DNA-binding domains and transactivators through target RNA using RNA-binding domains, including phage coat proteins. MS2 bacteriophage coat protein fused with a transcriptional activator and PP7 bacteriophage coat protein fused with the tetracycline repressor (tetR) can be bridged by target RNA encoding MS2 and PP7 stem-loops, resulting in transcriptional activation. We generated recombinant CHO cells containing an inducible GFP expression module governed by a minimal promoter with a tetR-responsive element. Cells carrying the trigger RNA exhibited robust reporter gene expression, whereas cells lacking it exhibited no expression. GFP expression was upregulated over 200-fold compared with that in cells without a target RNA expression vector. Moreover, this system can detect the expression of mRNA tagged with aptamer tags and modulate reporter gene expression based on the target mRNA level without affecting the expression of the original mRNA-encoding gene. The RNA-triggered gene expression systems developed in this study have potential as a new platform for establishing gene circuits, evaluating endogenous gene expression, and developing novel RNA detectors.

Virus breakthrough behavior during virus filtration: Effects of virus/protein concentration and operating conditions

Virus filtration in biopharmaceutical downstream process ensures viral safety with removal efficiency greater than 99.99%. Despite its robust performance, it has been reported that viruses can escape through the membrane under certain conditions. This study aimed to investigate virus breakthrough using Viresolve® Pro membrane with PP7 bacteriophage under high bacteriophage titer, high protein concentration, flow interruption, and low-flux operation. The results show high virus removal performance (LRV >6.8) up to 109 PFU/mL for 200 L/m2, with the first phage breakthrough observation over 1012 PFU/m2 of phage challenge in the membrane. As the protein concentration increased in the coexistence condition, a greater number of phages passed through the membrane. During the post-buffer flush and the operation at low filtrate flux, diffusion plays an important role in the escape of phages, which were detected as high as 1012 PFU/m2, but as low as 109 PFU/m2. These results suggest that the virus-retentive membrane may become susceptible to undesired virus breakthrough when exposed to phage challenges of > 1012 PFU/mL, providing important key factors to ensure viral safety during the downstream process of biopharmaceutical production.

Evaluation of low-cost viral concentration methods in wastewaters: Implications for SARS-CoV-2 pandemic surveillances

In the pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) many strategies have been performed in order to control viral spread in the population and known the real-time situation about the number of infected persons. In this sense, Wastewater Based Epidemiology (WBE) has been applied as an excellent tool to evaluate the virus circulation in a population. In order to obtain reliable results, three low-cost viral concentration methods were evaluated in this study, polyethylene glycol (PEG) precipitation, skimmed milk flocculation (SM) and Aluminum polychloride flocculation, for Pseudomonas aeruginosa bacteriophage PP7 as a surrogate for non-enveloped viruses and Bovine Coronavirus (BCoV) as a surrogate for enveloped virus, with emphasis for SARS- CoV-2. Our results suggest that PEG precipitation for viral concentration, for both enveloped and non-enveloped virus from wastewater is an appropriate approach since it was more sensitive compared to SM flocculation and Aluminum polychloride flocculation. This methodology can be used for WBE studies in order to follow the epidemiology of the SARS-CoV-2 pandemic, mainly in developing countries where the economic resources are frequently limited.

Interparticle effects in the cotransport of viruses and engineered nanoparticles in saturated porous media

The effects of P25 TiO2 nanoparticles on the transport in saturated porous media of the bacteriophage PP7 was investigated. Two levels of ionic strength were considered, given by monovalent and divalent cations, Na+ and Ca2+. The viruses and the P25 particles were characterized with respect to size and surface charge, by dynamic light scattering and electron microscopy. The breakthrough curves were obtained for suspensions of each particle and both combined, using UV absorbance to quantify P25 and RT-qPCR for PP7. In the single transport experiments, P25 was retained, while the viruses mostly eluted the sand bed. However, in the cotransport experiments, retention increased for the virus and decreased for the TiO2 nanoparticles under high viral content, due to heteroaggregation among the colloids in the incoming suspension, electrostatic interactions, and ripening. The interparticle interactions were modeled by DLVO theory, resulting in agreement with the experimental observations, which highlights the electrostatic nature.

Virus filter scalability: Demonstration of consistent viral clearance across laboratory and manufacturing scales

Virus removal filtration processes in biopharmaceutical manufacturing are developed, optimized and validated for viral clearance using laboratory scale filters. Thus, the scalability of these filters is critical for accurately extrapolating filtration performance and reliably extending viral clearance to manufacturing scale. Virus removal filter manufacturers generally validate scalability of filtration performance based on various filtration parameters, and virus removal capability is extended to manufacturing scale filters using inert, size-appropriate particles such as gold nanoparticles to avoid the risks associated with using mammalian viruses in large feed volumes. In this study, we use bacteriophage PP7 as a parvovirus model to directly demonstrate viral clearance on Planova™ BioEX virus removal filters across all scales, including manufacturing scale. Filters with hollow fibers from three spinning series with filter sizes ranging from 0.0003 to 4.0 m2 were tested for virus removal, flux, and protein recovery performance using BSA spiked with PP7. Complete viral clearance was observed across all filter sizes with PP7 LRV of ≥4.7 or higher. Flux and protein recovery were also consistent. These results demonstrate the scalability of filtration performance and consistent virus removal at all sizes, supporting the use of laboratory scale filters to validate viral clearance at manufacturing scales.

CDNA-Derived RNA Phage Assembly Reveals Critical Residues in the Maturation Protein of the Pseudomonas aeruginosa Leviphage PP7.

PP7 is a leviphage, with a single-stranded RNA genome, that infects Pseudomonas aeruginosa PAO1. A reverse genetic system for PP7 was previously created by using reverse-transcribed cDNA (PP7O) from a virion-derived RNA genome. Here, we have found that the PP7O cDNA contained 20 nucleotide differences from the PP7 genome sequence deposited in the database. We created another reverse genetic system exploiting chemically synthesized cDNA (PP7S) based on the database sequence. Unlike PP7O, which yielded infectious PP7 virions, PP7S-derived particles were incapable of plaque formation on PAO1 cells, which was restored in the PAO1 cells expressing the maturation protein (MP) from PP7O Using this reverse genetic system, we revealed two amino acid residues involved in the known roles of MP (i.e., adsorption and genome replication), fortuitously providing a lesson that the viral RNA genome sequencing needs functional verification, possibly by a reverse genetic system.IMPORTANCE The biological significance of RNA phages has been largely ignored, ironically, because few studies have focused on RNA phages. As an initial attempt to properly represent RNA phages in the phageome, we previously created, by using reverse-transcribed cDNA, a reverse genetic system for the small RNA phage PP7, which infects the opportunistic human pathogen Pseudomonas aeruginosa We report another system by using chemically synthesized cDNA based on the database genome that has 20 nucleotide differences from the previous cDNA. Investigation of those cDNA-derived phage virions revealed that two amino acids of the maturation protein are crucial for the normal phage lifecycle at different steps. Our study provides insight into the molecular basis for the RNA phage lifecycle and a lesson that the RNA genome sequencing needs to be carefully validated by cDNA-based phage assembly systems.

Supramolecular Encapsulation of Small-Ultrared Fluorescent Proteins in Virus-Like Nanoparticles for Noninvasive In Vivo Imaging Agents.

Icosahedral virus-like particles (VLPs) derived from bacteriophages Qβ and PP7 encapsulating small-ultrared fluorescent protein (smURFP) were produced using a versatile supramolecular capsid disassemble-reassemble approach. The generated fluorescent VLPs display identical structural properties to their nonfluorescent analogs. Encapsulated smURFP shows indistinguishable photochemical properties to its unencapsulated counterpart, exhibits outstanding stability toward pH, and produces bright in vitro images following phagocytosis by macrophages. In vivo imaging allows the biodistribution to be imaged at different time points. Ex vivo imaging of intravenously administered encapsulated smURFP reveals a localization in the liver and kidneys after 2 h blood circulation and substantial elimination after 16 h of imaging, highlighting the potential application of these constructs as noninvasive in vivo imaging agents.

Development of small-scale models to understand the impact of continuous downstream bioprocessing on integrated virus filtration.

We designed small‐scale virus filtration models to investigate the impact of the extended process times and dynamic product streams present in continuous manufacturing. Our data show that the Planova 20N and BioEX virus filters are capable of effectively removing bacteriophage PP7 (>4 log) when run continuously for up to 4 days. Additionally, both Planova 20N and BioEX filters were able to successfully process a mock elution peak of increased protein, salt, and bacteriophage concentrations with only an increase in filtration pressure observed during the higher protein concentration peak. These experiments demonstrated that small‐scale viral clearance studies can be designed to model a continuous virus filtration step with specific process parameters.

A Stable Pep2-proapoptotic Peptide Inducing Apoptosis of Acute Myeloid Leukemia Cells by Down-Regulating EZH2.

Proapoptotic peptide, (KLAKLAK)2, exhibits strong anti-tumor effect with the help of cell-penetrating peptides such as Pep2, targeting TLR2 with high expression in acute myeloid leukemia (AML). However, the applications are limited due to the peptide's instability and high cost of synthesis. Recombinant PP7 bacteriophage-like particles (VLPs) can protect the peptides from degradation by proteases, based on their ability to display foreign peptides. Here, we evaluated the feasibility of PP7 VLPs carrying Pep2 and (KLAKLAK)2 (2PP7-Pep2-KLAK VLPs) expressed in E. coli. We further investigated the characteristics including size, toxicity, thermal stability, penetrating ability, anti-tumor activity, and potential anti-tumor mechanism of 2PP7-Pep2-KLAK VLPs. 2PP7-Pep2-KLAK VLPs was expressed in E. coli BL21(DE3) successfully with high yield and thermal stability. They penetrated the AML cells THP-1 rapidly after 30min of incubation. Moreover, 2PP7-Pep2-KLAK VLPs were non-replicative, non-infectious, and non-toxic against normal cells, but inhibited the proliferation of THP-1 cells by inducing cell apoptosis after 24h of exposure. This effect extends through 120h of exposure, indicating their anti-proliferation effect was superior to that of synthetic peptides. In addition to the mitochondrial apoptotic pathway, the anti-tumor activity of 2PP7-Pep2-KLAK VLPs was also correlated with down-regulation of expression of enhancer of zeste homolog 2 (EZH2) and trimethylation of histone H3K27. We identified the feasibility to prepare the stable, active Pep2-KLAK peptide by using PP7 bacteriophage as the vehicle. We revealed this peptide was an inhibitor of EZH2. 2PP7-Pep2-KLAK VLPs may have significant clinical implications in the treatment of MLL-AF9 AML as an epigenetic modulator.

Investigation of microbial cell deformability by filter cake compressibility using ultrafiltration membranes

This study presents the investigation of deformability of various microbial cells in terms of filter cake compressibility during cake filtration using ultrafiltration membranes in dead-end mode. The examined microbial cells include mycoplasma, Gram-positive and Gram-negative bacteria, and Pseudomonas aeruginosa phage PP7. Polystyrene particles were used as an incompressible reference. The compressibility results were correlated to the deformability of a microbial cell, induced by its cell envelope.To determine the deformability of the different microbial cells under different process conditions, their cake resistance was measured under varying pressures from 10 to 250 kPa and temperatures from 2 to 35 °C. In addition, the influence of different culture media on the cell properties of Acholeplasma laidlawii and its behavior under different pressure and temperature was determined.The results of the pressure and temperature experiments revealed that Gram-positive S. epidermidis was found to be relatively stiff due to the thickness of the peptidoglycan layer, under different pressure and temperature conditions. No significant increase of the specific cake resistance of S. epidermidis could be determined. B. diminuta however showed a high deformation tendency when the pressure was increased indicating relatively soft cells. Mycoplasma A. laidlawii cells cultivated in three different media showed a different, but significant, effect of pressure and temperature.

Virus Size Analysis by Gas-Phase Mobility Measurements: Resolution Limits.

Electrospraying (ES) dissolved viral particles, followed by charge reduction and size analysis with a differential mobility analyzer (DMA), offers a flexible size-analysis tool for small particles in solution. The technique relies on pioneering work by Kaufman and colleagues, commercialized by TSI, and often referred to as GEMMA. However, viral studies with TSI's GEMMA have suffered from limited resolving power, possibly because of imperfections in either the instrument (DMA or charge reduction) or the sample solution preparation. Here, we explore the limits of the resolution achievable by GEMMA, taking advantage of (i) cleaner charge reduction methods and (ii) DMAs of higher resolving power. Analysis of the literature provides indications that mobility peak widths (fwhm) of 2% or less may be achieved by combining careful sample preparation with improved instrumentation. Working with purified PP7 bacteriophage particles small enough to be classifiable by existing high-resolution DMAs, we confirm that fairly narrow viral mobility peaks may be obtained (relative full width at half-maximum fwhm <5%). Comparison of spectra of a given apian virus sample obtained with TSI's GEMMA and our improved instrumentation confirms that one critical limitation is the DMA. This is further verified by narrow peaks from murine parvovirus, norovirus, and encephalomyelitis virus samples, obtained in our improved GEMMA with little sample preparation, directly from infected cell cultures. Classification of purified large (60 nm) coliphage PR772 particles leads to broad peaks, due to both viral degradation and limited intrinsic resolution of the DMAs used to cover the range of such large particles. We conclude that improved DMAs suitable for high-resolution analysis of particles larger than 30 nm need to be developed to determine the intrinsic mobility width of viral particles.

Investigation of virus retention by size exclusion membranes under different flow regimes.

Virus removal by filter membranes is regarded as a robust and efficient unit operation, which is frequently applied in the downstream processing of biopharmaceuticals. The retention of viruses by virus filtration membranes is predominantly based on size exclusion. However, recent results using model membranes and bacteriophage PP7 point to the fact that virus retention can also significantly be influenced by adsorptive interactions between virus, product molecules, and membranes. Furthermore, the impact of flow rate and flow interruptions on virus retention have been studied and responsible mechanisms discussed. The aim of this investigation was to gain a holistic understanding of the underlying mechanisms for virus retention in size exclusion membranes as a function of membrane structure and membrane surface properties, as well as flow and solution conditions. The results of this study contribute to the differentiation between size exclusion and adsorptive effects during virus filtration and broaden the current understanding of mechanisms related to virus breakthroughs after temporary flow interruptions. Within the frame of a Design of Experiments approach it was found that the level of retention of virus filtration membranes was mostly influenced by the membrane structure during typical process-related flow conditions. The retention performance after a flow interruption was also significantly influenced by membrane surface properties and solution conditions. While size exclusion was confirmed as main retention mechanism, the analysis of all results suggests that especially after a flow interruption virus retention can be influenced by adsorptive effects between the virus and the membrane surface. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2747, 2019.

An in Vivo Binding Assay for RNA-Binding Proteins Based on Repression of a Reporter Gene

We study translation repression in bacteria by engineering a regulatory circuit that functions as a binding assay for RNA binding proteins (RBP) in vivo. We do so by inducing expression of a fluorescent protein-RBP chimera, together with encoding its binding site at various positions within the ribosomal initiation region (+11-13 nt from the AUG) of a reporter module. We show that when bound by their cognate RBPs, the phage coat proteins for PP7 (PCP) and Qβ (QCP), strong repression is observed for all hairpin positions within the initiation region. Yet, a sharp transition to no-effect is observed when positioned in the elongation region, at a single-nucleotide resolution. Employing in vivo Selective 2'-hydroxyl acylation analyzed by primer extension followed by sequencing (SHAPE-seq) for a representative construct, established that in the translationally active state the mRNA molecule is nonstructured, while in the repressed state a structured signature was detected. We then utilize this regulatory phenomena to quantify the binding affinity of the coat proteins of phages MS2, PP7, GA, and Qβ to 14 cognate and noncognate binding sites in vivo. Using our circuit, we demonstrate qualitative differences between in vitro to in vivo binding characteristics for various variants when comparing to past studies. Furthermore, by introducing a simple mutation to the loop region for the Qβ-wt site, MCP binding is abolished, creating the first high-affinity QCP site that is completely orthogonal to MCP. Consequently, we demonstrate that our hybrid transcriptional-post-transcriptional circuit can be utilized as a binding assay to quantify RNA-RBP interactions in vivo.

Induced Protein-RNA Interactions in the 5’ UTR Lead to Conformational Changes that Alter Translation

We study translational regulation by a 5’ UTR sequence encoding the binding site of an RNA-binding protein (RBP) in bacteria, using a reporter assay and selective 2′-hydroxyl acylation analysed by primer extension sequencing (SHAPE-Seq). We tested constructs containing a single hairpin, based on the binding sites of the coat RBPs of bacteriophages GA, MS2, PP7, and Qβ, positioned in the 5’ UTR of a reporter gene. With specifically-bound RBP present, either weak repression or up-regulation is observed, depending on the binding site and its flanking sequence. SHAPE-Seq data for a representative construct exhibiting up-regulation, indicates a partially-folded hairpin and non-reactive upstream and downstream flanking region, which we attribute to intermediate structures that apparently block translation. RBP binding stabilizes the fully-folded hairpin state facilitating translation, suggesting that the up-regulating constructs function as protein-sensing regulatory elements. This finding is further supported by lengthening the binding-site stem, which in turn destabilizes the translationally-inactive state, and abolishes the up-regulating behavior. Finally, we find that the combination of two binding sites, positioned in the 5’ UTR and the ribosomal initiation region within the CDS of the same transcript, can yield a cooperative regulatory response. Together, we show that the interaction of an RBP with its RNA target facilitates structural changes in the RNA, which is reflected by a controllable range of binding affinities and dose response behaviors. Thus, demonstrating that RNA-RBP interactions can provide a platform for constructing gene regulatory networks that are based on translational, rather than transcriptional, regulation.

Adenovirus and rotavirus recovery from a treated effluent through an optimized skimmed-milk flocculation method.

Sewage treatment may be insufficient for the complete removal of enteric viruses, such as human adenoviruses (HAdV) and group A rotavirus (RVA). The differences in the efficiency of the treatment methodologies used may interfere with the detection of these viruses. The objective of this study was to optimize a skimmed-milk flocculation technique for the recovery of HAdV and RVA in the samples of treated effluent. The treated effluent collected at the wastewater treatment plant (WWTP) was processed via four protocols including modifications in the initial centrifugation step and the final concentration of skimmed-milk. The viral load and recovery rate were determined by quantitative PCR TaqMan® System. The highest recovery rates of HAdV, RVA, and bacteriophage PP7 (internal control process) were obtained when the concentration of skimmed-milk was doubled and no centrifugation step was used for the sample clarification. The optimized protocol was assessed in a field study conducted with 24 treated effluent samples collected bi-monthly during 2015. HAdV and RVA were detected in 50.0% (12/24) and 33.3% (08/24) of the samples tested, respectively, throughout the year, without seasonal variation (p > 0.05). This study corroborates the use of the organic flocculation method for virus recovery in environmental samples with the adaptation of the protocols to different aquatic matrices.

Retention of Acholeplasma laidlawii by Sterile Filtration Membranes: Effect of Cultivation Medium and Filtration Temperature.

ALCM: A. laidlawii culture medium; ASTM: American Society for Testing and Materials; ATCC: American Type Culture Collection; CFU/mL: colony-forming units per milliliter; DLS: Dynamic light scattering; LRV: Log reduction value; PES: Polyethersulfone; PFU/mL: Plaque-forming units per milliliter; PSD: Particle size distribution; PVP: Polyvinylpyrrolidone; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SLB: Saline lactose broth; TMP: Transmembrane pressure; TSB: Tryptic soy broth.

Evaluating the effect of in-process material on the binding mechanisms of surrogate viral particles to a multi-modal anion exchange resin

Bacteriophage binding mechanisms to multi-modal anion exchange resin may include both anion exchange and hydrophobic interactions, or the mechanism can be dominated by a single moiety. However, previous studies have reported binding mechanisms defined for simple solutions containing only buffer and a surrogate viral spike (i.e. bacteriophage ΦX174, PR772, and PP7). We employed phage spiked in-process monoclonal antibody (mAb) pools to model binding under bioprocessing conditions. These experiments allow the individual contributions of the mAb, in-process impurities, and buffer composition on mechanistic removal of phages to be studied. PP7 and PR772 use synergetic binding by the positively charged quaternary amine and the hydrophobic aromatic phenyl group to bind multi-modal resin. ΦX174′s binding mechanism remains inconclusive due to operating conditions.

Electrostatic interactions in virus removal by ultrafiltration membranes

Ultrafiltration membranes are increasingly used in potabilization to remove viral particles. This removal is controlled by electrostatic repulsion, attachment and size exclusion. The effect of electrostatic interaction in virus filtration was investigated. Our work included characterization of bacteriophage PP7 and polyethersulfone membrane with respect to size and surface charge; the removal of this bacteriophage at laboratory scale by ultrafiltration membrane process and the mechanism and limitations were analyzed and discussed under DLVO and XDLVO theories. A partial removal of the bacteriophage was achieved; however, enhanced separation may be achieved considering that the process is affected by the aqueous matrix. The presence of divalent cations diminished the effectiveness of the procedure as opposed to monovalent cations and species with amphoteric behavior such as bicarbonate. DLVO and XDLVO predicted the interactions studied between bacteriophage PP7 and polyethersulfone membrane.

Determination of pore size distributions of virus filtration membranes using gold nanoparticles and their correlation with virus retention

Virus filtration membranes contribute substantially to the virus safety of biopharmaceutical drugs due to their capability to retain viral particles mainly based on the size-exclusion mechanisms. In this work, virus filtration membranes were challenged with gold nanoparticles (GNPs) in order to determine pore size distributions (PSDs) for a wide range of different commercial and non-commercial parvovirus retentive membranes differing in structure, material and surface chemistry. The retention mechanism of GNPs was investigated and effectively shifted towards size-exclusion by using an anionic surfactant to suppress particle adsorption to the membrane surface. This allowed insights into the relevance of particle retention based on size-exclusion mechanisms of the respective membranes. Membrane PSDs investigated through GNP challenges were for some membranes compared with PSDs investigated by liquid-liquid displacement porometry (LLDP). In addition, virus retention performance using Pseudomonas aeruginosa bacteriophage PP7 as accepted model virus was determined for the entire set of membranes and correlated with the cut-off pore size obtained from experiments using GNPs. Exemplarily, retention was examined for one membrane type using a set of different sized viruses or phages (PCV-2, PP7, MVM, HAV) ranging from 18 to 28nm and compared to GNP retention.