Recombinant Enterococcus phage phiEF24C Virion protein 6

What is Enterococcus phage phiEF24C and why is it significant?

Enterococcus phage phiEF24C is a virulent bacteriophage that infects Enterococcus faecalis, including vancomycin-resistant strains (VRE), which have become a significant threat in nosocomial settings. The phage demonstrates remarkable therapeutic potential due to its broad host range, effective lytic activity, shorter latent period, and larger burst size compared to ordinary tailed phages .

Morphological and genomic analyses reveal that phiEF24C is a large myovirus (classified as family Myoviridae morphotype A1) with a linear double-stranded DNA genome of approximately 143 kbp . Its genomic features indicate that phiEF24C is a member of the SPO1-like phage genus with a close relationship to Listeria phage P100, which is already authorized for prophylactic use . This classification provides a rational basis for its potential therapeutic applications against E. faecalis infections.

What is the genomic structure of phiEF24C?

The complete genome of phiEF24C consists of 142,072 base pairs and is predicted to contain 221 open reading frames (ORFs) and five tRNA genes . Bioinformatic analyses have confirmed that the phage genome contains no undesirable elements for phage therapy, such as pathogenic or integration-related proteins . The genome shows noncompetitive directions of replication and transcription and host-adapted translation, features that are characteristic of therapeutic phages.

Several related phages share similar genomic characteristics:

This genomic similarity suggests evolutionary conservation among therapeutic Enterococcus phages .

How is Recombinant phiEF24C Virion protein 6 produced and purified?

Recombinant production of phiEF24C Virion protein 6 typically employs E. coli expression systems. The standard methodology involves:

• Gene cloning into an appropriate expression vector (often with a His6 tag for purification)

• Transformation into E. coli expression strains

• Induction of protein expression under controlled conditions

• Cell lysis and protein extraction

• Purification using affinity chromatography (leveraging the His-tag)

• Quality control assessment via SDS-PAGE (typically achieving >85% purity)

• Lyophilization for stability and storage

Commercial preparations may employ either N-terminal or C-terminal tags depending on protein stability requirements. The expression tag type (N-terminal vs. C-terminal) should be determined based on the specific experimental requirements and potential interference with protein function .

What are the optimal reconstitution and storage conditions for Recombinant phiEF24C Virion protein 6?

For optimal reconstitution and storage, researchers should follow these guidelines:

• Briefly centrifuge the lyophilized product before opening to ensure all material is at the bottom of the vial

• Reconstitute to a concentration of 0.1-1.0 mg/mL using deionized sterile water

• Add glycerol to a final concentration of 5-50% (commonly 50%) to prevent freeze-thaw damage

• Aliquot the reconstituted protein for long-term storage at -20°C/-80°C

• Avoid repeated freeze-thaw cycles which can compromise protein integrity

• Working aliquots can be stored at 4°C for up to one week

Storage stability varies based on formulation:

• Liquid form: approximately 6 months at -20°C/-80°C

• Lyophilized form: approximately 12 months at -20°C/-80°C

What experimental applications is Recombinant phiEF24C Virion protein 6 typically used for?

Recombinant phiEF24C Virion protein 6 is commonly employed in several research applications:

• Western Blotting (WB) - For detection and quantification of the protein in various experimental contexts

• Enzyme-Linked Immunosorbent Assay (ELISA) - For developing sensitive detection methods

• Structure-function studies - To understand the protein's role in phage assembly and infection

• Host-phage interaction analyses - To investigate mechanisms of bacterial targeting

• Antibody production - For developing detection reagents for phiEF24C in experimental or clinical samples

• Phage therapy research - As part of comprehensive studies evaluating phiEF24C as a therapeutic agent

The protein has also been used in phage display systems and protein-protein interaction studies to understand its binding partners and functional relationships within the phage structure.

How can researchers design experiments to evaluate phiEF24C therapeutic potential?

When designing experiments to evaluate phiEF24C's therapeutic potential, researchers should consider:

• In vitro efficacy studies:

  • Host range determination using the streak test against clinical E. faecalis isolates

  • Measurement of phage adsorption rate (90% adsorption can occur within one minute for effective phages)

  • One-step growth curve analysis to determine latent period and burst size

  • Killing assays under various multiplicity of infection (MOI) values

• Animal model validation:

  • Mouse sepsis models have demonstrated efficacy at low concentrations

  • Endophthalmitis mouse models show reduced bacterial load and neutrophil infiltration after phage treatment

  • Monitoring of host response to single and repeated phage exposures

• Physiological parameters optimization:

  • Evaluation of Ca²⁺ and Mg²⁺ effects on lytic activity

  • pH, temperature, and storage stability assessments

  • Formulation development for specific administration routes

• Safety assessments:

  • In silico genomic screening for undesirable features (toxins, antibiotic resistance genes)

  • Observation of animal health during repeated exposure experiments

  • Evaluation of immune responses to phage components

How should researchers approach host range determination for phiEF24C and related phages?

Host range determination is critical for therapeutic applications and should include:

• Comprehensive methodology:

  • Primary screening using the streak test on double-layered agar plates

  • Record whether bacteria are lysed, show "lysis from without," or remain unlysed

  • Define lysis from without as a short transparent line without plaque formation

• Strain selection strategy:

  • Include diverse clinical isolates, particularly vancomycin-resistant strains

  • Incorporate temporally and geographically diverse strain collections

  • Include biofilm-forming strains that may exhibit different susceptibility

• Quantitative assessment:

  • Determine efficiency of plating (EOP) across different strains

  • Measure adsorption rates on different host strains

  • Evaluate phage multiplication kinetics on various hosts

• Molecular basis investigation:

  • Identify receptor molecules through resistance development studies

  • Sequence analysis of resistant mutants to identify resistance mechanisms

  • Correlation of Virion protein 6 sequence variants with host range alterations

What approaches can resolve contradictory findings in phage protein characterization?

When faced with contradictory findings about phiEF24C Virion protein 6, researchers should implement:

• Standardization approaches:

  • Define consistent buffer compositions, pH, and temperature for experiments

  • Standardize protein production, purification, and storage protocols

  • Establish reference materials and positive controls across laboratories

• Complementary structural analyses:

  • Combine multiple techniques (X-ray crystallography, NMR, cryo-EM)

  • Use the SWISS-MODEL Repository data (entry P85230) as a starting point

  • Validate structural models through functional assays

• Methodological variations assessment:

  • Evaluate the impact of different tags (N-terminal vs. C-terminal)

  • Test various expression systems to rule out host-specific effects

  • Analyze the influence of protein concentration on observed properties

• Collaborative validation:

  • Conduct inter-laboratory comparisons with standardized protocols

  • Organize blind testing of protein samples

  • Perform meta-analysis of published data with statistical evaluation

How can researchers isolate and characterize new phiEF24C-like phages for comparative studies?

To isolate and characterize new phiEF24C-like phages, researchers should:

• Isolation protocol:

  • Filter environmental water samples through 0.45 μm pore surfactant-free cellulose acetate membranes

  • Mix filtrate with appropriate medium (e.g., HIB, BHI, or TSB)

  • Inoculate with E. faecalis culture and incubate overnight

  • Filter culture and perform single plaque isolation at least three times

• Purification methodology:

  • Layer crude phage suspension on a discontinuous gradient of iodixanol (30-40%) or CsCl

  • Ultracentrifuge (50,000-100,000×g, 1-2 hours, 4°C)

  • Collect phage band and store at 4°C

• Genomic characterization:

  • Extract DNA using standard phage DNA preparation methods

  • Sequence using modern platforms (Illumina, 454, etc.)

  • Assemble reads using appropriate software (SPAdes, Newbler)

  • Annotate genomes using prokaryotic genome annotation pipelines like DFAST

• Functional comparison:

  • Compare adsorption rates, latent periods, and burst sizes

  • Evaluate host ranges using standardized strain panels

  • Assess therapeutic efficacy in appropriate animal models

  • Compare specific proteins (like Virion protein 6) for sequence and functional conservation

What are the optimal approaches for studying phage-bacterium interactions in complex environments?

To study phage-bacterium interactions in complex settings, researchers should consider:

• Mixed culture systems:

  • Design experiments with both target and non-target bacteria

  • Evaluate phage impact on microbial community structure

  • Assess potential collateral effects on bystander bacteria

• Biofilm models:

  • Establish single-species and multi-species biofilm systems

  • Evaluate phage penetration and efficacy in biofilm structures

  • Measure biofilm disruption and reformation after phage treatment

• Host response dynamics:

  • Monitor bacterial transcriptional responses to phage infection

  • Investigate potential induction of bacterial defense mechanisms

  • Assess development of resistance during extended exposure

• Physiological relevance:

  • Incorporate host factors (serum proteins, immune cells) in experimental designs

  • Model relevant infection sites (e.g., endophthalmitis model for eye infections)

  • Utilize ex vivo tissue models to bridge in vitro and in vivo studies

• Measurement approaches:

  • Use fluorescent reporter systems for real-time monitoring

  • Employ confocal microscopy for spatial distribution analysis

  • Implement metagenomics and transcriptomics for community-level effects

How might structural insights into phiEF24C Virion protein 6 inform engineered phage development?

Structural understanding of Virion protein 6 could advance engineered phages through:

• Structure-guided modifications:

  • Identification of critical domains for targeted mutagenesis

  • Design of chimeric proteins combining elements from different phages

  • Engineering of stabilizing modifications to enhance shelf-life

• Host range alterations:

  • If involved in host recognition, modifications could alter specificity

  • Creation of phage variants with expanded or narrowed host ranges

  • Development of phages targeting specific antibiotic-resistant strains

• Therapeutic enhancement:

  • Addition of biofilm-degrading domains for improved efficacy

  • Modification of immunogenic epitopes to reduce immune clearance

  • Engineering for improved stability under physiological conditions

• Diagnostic applications:

  • Development of reporter phage systems based on structural knowledge

  • Creation of phage-based biosensors for E. faecalis detection

  • Design of imaging agents for infection visualization

What research is needed to better understand the immune response to phiEF24C during therapeutic applications?

Future research on immune responses to phiEF24C should focus on:

• Immunogenicity characterization:

  • Mapping of immunogenic epitopes on Virion protein 6 and other structural components

  • Evaluation of antibody development after single and repeated exposures

  • Assessment of neutralizing vs. non-neutralizing antibody responses

• Impact on therapeutic efficacy:

  • Determination of how pre-existing immunity affects subsequent treatments

  • Investigation of dosing strategies to overcome immune clearance

  • Development of formulations to reduce immunogenicity

• Beneficial immune modulation:

  • Exploration of phage components that positively modulate host immune response

  • Investigation of synergies between phage therapy and conventional immune responses

  • Potential for phage-mediated immunomodulation in polymicrobial infections

• Individualized approaches:

  • Development of patient screening for pre-existing anti-phage antibodies

  • Personalized phage cocktail formulation based on immune status

  • Investigation of adjunctive immunomodulatory strategies

How can phage endolysins from phiEF24C be utilized alongside the complete phage for enhanced antimicrobial strategies?

Phage endolysins represent a complementary therapeutic approach:

• Endolysin characterization:

  • Two endolysins from the related iF6 phage demonstrated activity against enterococci in both logarithmic and stationary growth phases

  • The HU-Gp84 endolysin showed activity against 77% of tested enterococci strains and remained active after 1 hour incubation at 60°C

• Combination strategies:

  • Sequential treatment with phages followed by endolysins

  • Co-administration protocols for synergistic effects

  • Engineering of phages to overexpress endolysins upon infection

• Resistance management:

  • Evaluation of resistance development to phages vs. endolysins

  • Development of alternating treatment protocols

  • Assessment of fitness costs associated with resistance to each component

• Formulation challenges:

  • Development of vehicles that maintain activity of both components

  • Investigation of controlled release systems for sequential delivery

  • Stability testing under various environmental and physiological conditions

What methodological advances are needed to optimize high-throughput screening for improved phiEF24C variants?

Advancing high-throughput screening for phiEF24C improvement requires:

• Library generation approaches:

  • Error-prone PCR for random mutagenesis of Virion protein 6 and other structural proteins

  • Site-directed mutagenesis based on structural insights

  • DNA shuffling with related phage proteins

  • CRISPR-based genome editing of whole phage genomes

• Screening system design:

  • Development of reporter systems for phage infection

  • High-throughput host range determination methods

  • Automated plaque analysis systems

  • Microfluidic devices for rapid phenotypic characterization

• Selection parameter optimization:

  • Screens for enhanced thermal or pH stability

  • Selection for improved host range or specific targeting

  • Assays for reduced immunogenicity

  • Systems to identify variants with enhanced tissue penetration

• Data analysis infrastructure:

  • Machine learning approaches for pattern recognition

  • Predictive modeling of structure-function relationships

  • Systems biology integration of phage-host interactions

  • Computational approaches to predict successful variant combinations