Recombinant Pseudomonas phage Pf3 Attachment protein G3P (III)

What is the G3P protein in Pseudomonas phage Pf3 and what is its primary function?

G3P (gene 3 protein) in Pseudomonas phage Pf3 is a minor coat protein with an apparent molecular mass of approximately 55 kDa that serves as the pilus adsorption protein. It is encoded by ORF483 of the Pf3 genome and is essential for the initial attachment of the phage to its bacterial host. Similar to the G3P protein of Ff bacteriophages (which infect Escherichia coli), the Pf3 G3P protein mediates the specific interaction between the phage and the bacterial pilus, which is the first step in the infection process . The mature protein is generated through post-translational removal of a 20-amino acid signal peptide, resulting in a protein with an N-terminal sequence starting at Ala21 .

How does Pf3 G3P differ structurally from other filamentous phage attachment proteins?

Pf3 G3P differs from other filamentous phage attachment proteins in several key ways:

• Unlike the G3P of Pf1 phage (which infects Pseudomonas aeruginosa strain K via type IV PAK pili), Pf3 G3P specifically binds to the conjugative RP4 pilus rather than type IV pili .

• The primary structure of Pf3 G3P includes a putative signal sequence (residues 1-20) that is cleaved post-translationally, similar to the g3p of Class I filamentous bacteriophages, which have an 18-residue leader peptide that is also removed post-translationally .

• While sharing functional similarities with Ff phage G3P proteins like those in M13 and fd, Pf3 G3P has unique sequence characteristics that determine its specificity for RP4 pili .

Analysis of limited proteolysis experiments suggests that Pf3 G3P likely has a domain organization similar to Ff G3P, with a "knob-on-stem" structure where the N-terminal domains are involved in pilus binding .

What is the optimal expression system for producing recombinant Pf3 G3P?

The optimal expression system for producing recombinant Pf3 G3P involves:

• Using E. coli as the expression host, as demonstrated in successful studies with the N-terminal portion of the protein .

• Creating a subgene construct that encodes the N-terminal region (typically residues 1-378) of Pf3 ORF483 protein, as the C-terminal portion is highly hydrophobic and challenging to express in soluble form .

• Adding a C-terminal 6xHis tag to facilitate purification through affinity chromatography .

• Including an appropriate signal sequence (such as the original Pf3 signal sequence or the fd g8p leader sequence) to direct the protein to the periplasm where proper folding is more likely to occur .

The expression construct should be designed to account for the post-translational processing of the signal sequence, which is essential for proper folding and function of the mature protein .

What purification strategies are most effective for isolating recombinant Pf3 G3P?

The most effective purification strategy for recombinant Pf3 G3P involves a multi-step approach:

• Metal chelate chromatography using the C-terminal 6xHis tag as the primary capture step .

• Ion exchange chromatography as a secondary purification step to remove remaining contaminants .

• If necessary, size exclusion chromatography can be employed as a polishing step to ensure high purity and homogeneity.

• When working with the periplasmic fraction, osmotic shock methods can be used to selectively release the properly processed form of the protein .

Researchers should verify the correct processing of the recombinant protein through N-terminal sequencing, as only properly processed protein (with the signal sequence removed) will exhibit the expected binding functionality .

How can the pilus-binding specificity of Pf3 G3P be experimentally determined?

The pilus-binding specificity of Pf3 G3P can be experimentally determined through several complementary approaches:

• Direct ELISA assays using purified pili (e.g., RP4, PAK, or PAO pili) immobilized on microtiter plates, followed by addition of either intact Pf3 virions or recombinant G3P protein. Detection can be performed using anti-Pf3 antibodies or, for recombinant proteins, anti-His antibodies if a His-tag is present .

• Competition ELISA assays to determine whether recombinant G3P binds to the same site on the pilus as the intact virion. This involves pre-incubating biotinylated phage particles with increasing concentrations of recombinant G3P before adding the mixture to immobilized pili. The bound biotinylated phage can then be detected using streptavidin-HRP .

• Functional inhibition assays that measure the ability of recombinant G3P to inhibit phage infection of susceptible bacteria, providing a physiologically relevant measure of binding specificity .

These methods have demonstrated that Pf3 G3P specifically interacts with RP4 conjugative pili but not with type IV PAO pili, despite the fact that P. aeruginosa strain O serves as the bacterial host for Pf3 .

What is the apparent dissociation constant of Pf3 G3P binding to bacterial pili, and how does it compare to other phage attachment proteins?

The apparent dissociation constant (Kd) for G3P proteins binding to bacterial pili varies significantly between different phage-host systems:

• For Ff bacteriophages (e.g., M13), the entire phage particle shows an apparent Kd of approximately 2 pM when binding to F pili on E. coli .

• In contrast, the soluble fragment of M13 G3P (g3p-N) exhibits a higher Kd of approximately 3 nM - about 1000 times higher than the intact phage .

• For Pf3 G3P, competition ELISA data suggests that, assuming 5 copies of ORF483 protein per Pf3 virion (similar to g3p in fd virion), approximately 35 times more recombinant ORF483 protein is needed to achieve the same level of inhibition as the intact phage .

This difference between the binding affinity of intact phage particles versus soluble G3P fragments is attributed to several factors:

• Phage binding is essentially irreversible due to downstream events in the infection process

• Avidity effects from multiple G3P molecules (3-5 copies) per phage particle acting cooperatively

• Possible conformational differences between recombinant soluble fragments and the native protein in the phage capsid

How can recombinant Pf3 G3P be used to inhibit bacterial conjugation and combat antibiotic resistance?

Recombinant Pf3 G3P could potentially be used to inhibit bacterial conjugation and combat antibiotic resistance through the following mechanisms and approaches:

• Similar to the M13 G3P protein, which inhibits conjugation between E. coli cells by binding to F pili, Pf3 G3P could be developed to block conjugative transfer mediated by RP4 pili, which are associated with broad-host-range plasmids capable of transferring antibiotic resistance genes .

• Exogenous addition of soluble G3P fragments has been shown to inhibit conjugation at nanomolar concentrations (3 nM for M13 g3p-N), suggesting that recombinant Pf3 G3P could achieve similar effects with RP4-mediated conjugation .

• Since the RP4 plasmid has a broad host range among gram-negative bacteria, Pf3 G3P could potentially inhibit conjugative transfer of resistance genes across different bacterial species .

• The inhibition mechanism works through physical occlusion of the pilus, preventing the formation of conjugal pairs rather than interfering with DNA transfer mechanisms, offering a novel approach to combating the spread of resistance genes .

Research with M13 G3P has demonstrated that conjugation mediated by the F factor can be effectively inhibited by exogenous addition of nanomolar concentrations of the soluble protein, suggesting a similar approach could be developed with Pf3 G3P for RP4-mediated conjugation systems .

What structural modifications to recombinant Pf3 G3P could enhance its stability and binding efficiency?

Several structural modifications to recombinant Pf3 G3P could potentially enhance its stability and binding efficiency:

The design of such modifications should be guided by structural information on the pilus-binding domains and their interaction interfaces, although complete structural data for Pf3 G3P is currently limited compared to the well-characterized G3P from Ff bacteriophages .

How does Pf3 G3P compare functionally to G3P proteins from other Pseudomonas phages like Pf1 and Pf4?

Pf3 G3P differs significantly from G3P proteins of other Pseudomonas phages in terms of receptor specificity and evolutionary lineage:

These differences highlight the diversity within Pseudomonas filamentous phages and suggest that Pf3 occupies a distinct ecological niche compared to other Pf phages .

What are the key differences in experimental approaches needed when working with Pf3 G3P versus Ff phage G3P proteins?

Working with Pf3 G3P versus Ff phage G3P proteins requires several distinct experimental approaches due to their different properties:

• Host cell systems:

  • Pf3 G3P studies require P. aeruginosa strain O as the native host, while Ff phage G3P work typically uses E. coli .

  • For recombinant expression, both can use E. coli systems, but optimal codon usage and signal sequences may differ.

• Pilus preparation:

  • Ff phage G3P experiments require F pili, typically from E. coli containing an F plasmid .

  • Pf3 G3P studies need RP4 pili, requiring bacteria harboring the RP4 plasmid .

  • Purification protocols for these different pili types may need to be optimized separately.

• Binding assays:

  • For Pf3 G3P, competition ELISA assays using biotinylated phage particles have been effective in determining binding specificity to RP4 pili .

  • The detection systems differ: anti-Pf3 antibodies for Pf3 studies versus anti-M13/fd antibodies for Ff phage work .

• Protein expression strategies:

  • The C-terminal portion of both Pf3 ORF483 protein and Ff g3p is very hydrophobic, making full-length recombinant proteins difficult to obtain .

  • For Pf3 G3P, a subgene encoding residues 1-378 with a C-terminal 6xHis tag has been successfully expressed .

  • The signal sequence processing must be confirmed for proper folding and function of both proteins.

• Conjugation inhibition assays:

  • For Ff phage G3P, F-plasmid conjugation systems (often transferring tetracycline resistance) are used .

  • Pf3 G3P studies would need to use RP4-mediated conjugation systems, potentially with different antibiotic markers .

These differences reflect the distinct evolutionary origins and host specificities of these phage attachment proteins, requiring researchers to adapt their experimental approaches accordingly .

How might Pf3 G3P be engineered to target different bacterial pili for broad-spectrum applications?

Engineering Pf3 G3P to target different bacterial pili for broad-spectrum applications could involve several sophisticated approaches:

• Domain swapping: Replacing the pilus-binding domains of Pf3 G3P with corresponding domains from other phage attachment proteins that target different pili. For example:

  • Creating chimeras between Pf3 G3P and Pf1 G3P to target both RP4 and type IV PAK pili .

  • Incorporating binding domains from Ff phage G3P to target F pili in E. coli .

• Directed evolution: Using techniques such as phage display to evolve variants of Pf3 G3P with altered binding specificities:

  • Error-prone PCR to generate a library of G3P variants.

  • Selection on different pilus types to identify variants with desired binding properties.

  • Iterative rounds of selection to enhance binding affinity and specificity.

• Structure-guided rational design: Using computational modeling and structural data to identify and modify critical residues in the binding interface:

  • Based on the known structure of Ff phage G3P complexed with its receptor .

  • Identifying conserved structural elements across different pilus types.

  • Engineering a multi-specific binding domain that recognizes shared features of different pili.

• Creation of fusion proteins: Developing bispecific or multispecific fusion proteins that combine binding domains from different phage attachment proteins:

  • Tandem arrangement of binding domains with flexible linkers.

  • Optimization of domain orientation and linker length for efficient binding to multiple targets.

• Surface display platforms: Using bacterial or yeast surface display to screen for Pf3 G3P variants with altered specificity:

  • Expressing libraries of G3P variants on cell surfaces.

  • Flow cytometry-based sorting using fluorescently labeled pili as selection markers.

These engineering approaches could potentially create variants of Pf3 G3P with expanded host range, useful for both basic research and applications in controlling bacterial conjugation across different species .

What role might Pf3 G3P play in the development of phage-based alternatives to antibiotics?

Pf3 G3P could play several significant roles in developing phage-based alternatives to antibiotics:

• Conjugation inhibitors to prevent resistance spread:

  • Soluble G3P fragments could block RP4 pilus-mediated conjugation, preventing horizontal gene transfer of antibiotic resistance genes across a broad range of gram-negative bacteria .

  • This approach targets the spread of resistance rather than killing bacteria directly, potentially reducing selective pressure for resistance development.

• Targeted delivery vehicles:

  • Engineered Pf3 G3P could be used to direct phage particles or nanoparticles specifically to bacteria expressing RP4 pili.

  • This targeting could improve the specificity of antimicrobial delivery, reducing off-target effects on beneficial microbiota.

• Anti-biofilm agents:

  • Similar to how filamentous phages inhibit biofilm formation by interfering with mating pair formation, Pf3 G3P could potentially disrupt biofilms dependent on pilus-mediated cell-cell interactions .

  • Biofilms contribute significantly to antibiotic resistance, making their disruption a valuable therapeutic strategy.

• Diagnostic tools:

  • Labeled Pf3 G3P could be used to identify bacteria expressing RP4 or similar conjugative pili, enabling rapid detection of potentially resistant strains.

  • This could help guide therapeutic decisions and infection control measures.

• Evolutionary considerations:

  • As a protein that has co-evolved with bacterial receptors, G3P could potentially be engineered to counter bacterial evasion strategies.

  • Understanding the molecular basis of G3P-pilus interactions could inform the development of resistance-proof binding domains.

Research has shown that at high concentrations of phage (>109 particles/mL), conjugation can be essentially completely blocked through occlusion of pili by phage particles or soluble G3P fragments . This suggests that Pf3 G3P-based approaches could be effective in controlling the spread of resistance genes, particularly in defined environments where sufficiently high concentrations can be maintained.

What are the common pitfalls in expressing and purifying functional recombinant Pf3 G3P, and how can they be overcome?

Common pitfalls in expressing and purifying functional recombinant Pf3 G3P include:

• Protein insolubility due to the hydrophobic C-terminal domain:

  • Solution: Express only the N-terminal domains (e.g., residues 1-378) that retain the pilus-binding function while avoiding the hydrophobic C-terminal region .

  • Alternative: Use specialized solubilization tags like SUMO or MBP that can be cleaved after purification.

• Improper signal sequence processing:

  • Issue: Without proper signal sequence removal, the protein may not fold correctly or function properly.

  • Solution: Verify correct processing by N-terminal sequencing and use appropriate leader sequences to direct the protein to the periplasm where processing machinery is present .

  • Alternative: Design constructs that start directly with the mature protein sequence (after Ala21).

• Protein aggregation during purification:

  • Solution: Include low concentrations of non-ionic detergents in purification buffers.

  • Alternative: Perform purification at lower temperatures (4°C) and add stabilizing agents like glycerol.

• Loss of binding activity:

  • Solution: Conduct binding assays immediately after purification or add stabilizers.

  • Alternative: Verify proper folding using circular dichroism or limited proteolysis to ensure the protein retains its native structure.

• Variability in pili preparations for binding studies:

  • Solution: Standardize pili extraction protocols and quantify pili using consistent methods.

  • Alternative: Develop synthetic pili mimetics for more reproducible binding studies.

• Contamination with bacterial proteins that bind pili:

  • Solution: Include stringent washing steps during affinity purification.

  • Alternative: Add a second purification step such as ion exchange chromatography .

• Inefficient expression in bacterial systems:

  • Solution: Optimize codon usage for the expression host and test different promoter strengths.

  • Alternative: Explore eukaryotic expression systems for difficult constructs.

Research has shown that correctly processed recombinant Pf3 G3P (rORF483) can be successfully expressed in E. coli and purified using metal chelate and ion exchange chromatography, with the resulting protein demonstrating specific binding to RP4 pili in concentration-dependent assays .

How can researchers effectively study the interaction between Pf3 G3P and RP4 pili at the molecular level?

Researchers can effectively study the interaction between Pf3 G3P and RP4 pili at the molecular level using several complementary approaches:

• Structural biology techniques:

  • X-ray crystallography of Pf3 G3P alone and in complex with RP4 pilin subunits or peptide fragments, similar to studies done with Ff phage G3P .

  • Cryo-electron microscopy to visualize the interaction between intact pili and G3P or whole phage particles.

  • NMR spectroscopy to map binding interfaces and study dynamic interactions in solution.

• Binding kinetics and thermodynamics:

  • Surface plasmon resonance (SPR) to measure real-time binding kinetics between immobilized RP4 pili and flowing G3P.

  • Isothermal titration calorimetry (ITC) to determine binding thermodynamics (ΔH, ΔS, and Kd).

  • Microscale thermophoresis (MST) as an alternative method requiring smaller amounts of material.

• Mutagenesis and functional mapping:

  • Alanine scanning mutagenesis to identify critical residues involved in the interaction.

  • Domain deletion and chimeric protein studies to map functional binding regions.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify protected regions upon binding.

• Computational approaches:

  • Molecular dynamics simulations to model the interaction and predict binding energetics.

  • Protein-protein docking to predict the structure of the G3P-pilus complex.

  • Sequence analysis and evolutionary studies to identify conserved interaction motifs.

• Advanced microscopy:

  • Atomic force microscopy (AFM) to measure binding forces at the single-molecule level.

  • Super-resolution microscopy to visualize the distribution of binding events along pili.

  • Electron microscopy with gold-labeled G3P to map binding sites on pili.

• Competition assays:

  • Development of peptide libraries to identify minimal binding epitopes on RP4 pili.

  • Competition ELISA assays with fragments of G3P to map the minimal pilus-binding domain .

  • Cross-competition studies with other proteins known to interact with RP4 pili.

The combination of these approaches would provide complementary data on the structural basis, specificity determinants, and thermodynamic parameters of the Pf3 G3P-RP4 pilus interaction, allowing for a comprehensive molecular understanding that could inform protein engineering efforts .

What is the potential for developing Pf3 G3P variants as diagnostic tools for detecting specific bacterial strains?

The potential for developing Pf3 G3P variants as diagnostic tools for detecting specific bacterial strains is significant and could be pursued through several innovative approaches:

• Engineered specificity:

  • Directed evolution of Pf3 G3P to recognize specific variants of conjugative pili associated with particular bacterial strains or plasmids.

  • Creation of a panel of G3P variants with different binding specificities that could be used in multiplexed detection assays.

• Detection platforms:

  • Fusion of G3P variants to reporter proteins (e.g., fluorescent proteins, luciferases) for direct visualization of binding.

  • Integration into lateral flow assays for rapid, point-of-care detection of bacteria expressing specific pili.

  • Incorporation into biosensor platforms using electrochemical or optical detection methods.

• Clinical applications:

  • Development of diagnostic tests for detecting bacteria harboring specific conjugative plasmids associated with antibiotic resistance.

  • Creation of tools for epidemiological tracking of resistant strains in healthcare settings.

  • Rapid identification of bacterial pathogens expressing RP4 or related pili in clinical samples.

• Environmental monitoring:

  • Detection of bacteria with conjugative plasmids in environmental samples (water, soil) to track the spread of resistance genes.

  • Assessment of horizontal gene transfer potential in microbial communities.

• Technical advantages:

  • The high specificity of G3P-pilus interactions could enable precise discrimination between closely related bacterial strains.

  • The relatively small size of engineered G3P domains would allow for efficient production and incorporation into various detection platforms.

  • The nanomolar binding affinity demonstrated for soluble G3P fragments provides sufficient sensitivity for many diagnostic applications.

• Challenges to address:

  • Ensuring stability of G3P variants in clinical or environmental samples.

  • Balancing specificity and broad coverage to detect clinically relevant variants.

  • Developing standardized production methods for consistent diagnostic performance.

The competition ELISA experiments with Pf3 G3P have already demonstrated the feasibility of using these proteins in specific detection assays , providing a foundation for further development of diagnostic applications.

How might the evolutionary relationship between Pf3 G3P and other phage attachment proteins inform strategies for combating bacterial resistance?

The evolutionary relationship between Pf3 G3P and other phage attachment proteins offers valuable insights for developing strategies to combat bacterial resistance:

• Leveraging evolutionary conservation and divergence:

  • Identification of conserved binding mechanisms across evolutionarily distinct G3P proteins could reveal universal features of pilus recognition that are less likely to be evaded by bacterial mutations .

  • Understanding how different phages have evolved to target various pili types could inform the design of broad-spectrum inhibitors of bacterial conjugation.

• Predictive modeling based on evolutionary patterns:

  • Analysis of co-evolutionary patterns between phage attachment proteins and their bacterial receptors could help predict potential resistance mechanisms.

  • Identification of rapidly evolving regions in G3P proteins might indicate domains under selective pressure from bacterial evasion strategies.

• Multi-target approaches:

  • The distinct evolutionary lineages of Pf phages (with Pf3 being separate from Pf1/Pf4/Pf5) suggest that using a combination of attachment proteins from different lineages could provide broader coverage against diverse bacterial strains.

  • Cocktails of G3P proteins targeting different pili types could minimize the development of resistance through redundant targeting.

• Exploiting phage-host arms races:

  • The long co-evolutionary history of phages and bacteria has led to sophisticated infection mechanisms that could be harnessed for therapeutic applications.

  • Studying how Pf3 G3P has evolved to maintain binding to RP4 pili despite potential bacterial counteradaptations could reveal strategies for designing resistance-proof inhibitors.

• Cross-species applications:

  • Understanding how Pf3 G3P targets conjugative RP4 pili, which have a broad host range among gram-negative bacteria, could inform the development of inhibitors that block conjugation across diverse bacterial species .

• Ecological considerations:

  • The different integration sites utilized by Pf prophages (tRNA-Gly, tRNA-Met, tRNA-Sec, tmRNA, and direct repeats) suggest varied ecological strategies that could inform the design of phage-based interventions targeting different bacterial niches.