Recombinant Vibrio vulnificus Probable Fe (2+)-trafficking protein (VV2885)

What is Vibrio vulnificus and why is its Fe(2+)-trafficking protein significant?

Vibrio vulnificus is a food-borne bacterial pathogen associated with approximately 1% of all food-related deaths, primarily due to consumption of contaminated seafood . The Fe(2+)-trafficking protein (VV2885) is significant because iron acquisition and metabolism are critical for bacterial virulence and survival. The protein likely plays a role in iron homeostasis, which directly impacts the pathogen's ability to establish infection and produce toxins. Understanding this protein may provide insights into potential therapeutic targets and virulence mechanisms.

How does Fe(2+) regulate gene expression in Vibrio vulnificus?

Fe(2+) regulates gene expression in Vibrio vulnificus through interaction with RNA regulatory elements similar to the iron-responsive elements (IREs) found in many iron-regulated genes. The binding of Fe(2+) to these RNA structures can alter their conformation, particularly at terminal loops and bulged regions critical for protein interactions . This conformational change affects the binding of regulatory proteins such as iron regulatory proteins (IRPs), which can either enhance or inhibit the translation of specific mRNAs depending on the cellular iron status. In experimental settings, Fe(2+) has been shown to reverse IRP inhibition of protein synthesis, demonstrating its role as a positive regulator of iron-dependent gene expression .

What expression systems are most effective for producing recombinant VV2885?

For recombinant VV2885 expression, the following systems have demonstrated superior results based on protein yield and solubility:

Methodology notes: For optimal expression in E. coli systems, induction with 0.5 mM IPTG at OD600 0.6-0.8, followed by growth at 18°C for 16-18 hours has shown the best results for maintaining protein solubility. Addition of 0.1 mM FeSO4 to the growth medium can enhance proper folding of the iron-binding domains.

How should I design experiments to study the iron-binding properties of recombinant VV2885?

To effectively study the iron-binding properties of recombinant VV2885, a multi-methodological approach is recommended:

• Isothermal Titration Calorimetry (ITC): This provides direct measurement of binding affinity (Kd), stoichiometry, and thermodynamic parameters. Titrate Fe(2+) solutions into purified VV2885 under anaerobic conditions to prevent Fe(2+) oxidation.

• Spectroscopic Methods: UV-visible spectroscopy can detect changes in absorption spectra upon Fe(2+) binding. Monitor absorbance at 250-350 nm, where Fe(2+)-protein complexes typically show characteristic peaks.

• Fluorescence Quenching Assays: If VV2885 contains tryptophan residues near the binding site, intrinsic fluorescence will be quenched upon Fe(2+) binding. This can be quantified to determine binding constants.

• Circular Dichroism (CD): Use CD to detect conformational changes in the protein structure upon Fe(2+) binding.

Critical considerations: All experiments should be conducted in an oxygen-free environment using degassed buffers, as Fe(2+) rapidly oxidizes to Fe(3+) in the presence of oxygen. Include EDTA controls to verify specificity of iron binding versus general metal affinity. Compare results with other divalent cations (Mn(2+), Zn(2+)) to establish binding selectivity.

What purification strategy yields the highest purity and activity of recombinant VV2885?

The following optimized purification protocol has demonstrated superior results for obtaining high-purity, active VV2885:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using a His-tag on either N or C-terminus (C-terminal tag shows less interference with function).

• Secondary Purification: Ion exchange chromatography (IEX) - Based on the theoretical pI of VV2885 (~5.8), use anion exchange at pH 7.5.

• Polishing Step: Size exclusion chromatography (SEC) to remove aggregates and ensure monodispersity.

Purification efficiency data:

Critical considerations: Include 1-5 mM β-mercaptoethanol or DTT in all buffers to prevent oxidation of cysteine residues. Add 10% glycerol to enhance protein stability. For maximum activity retention, maintain a low concentration of Fe(2+) (5-10 μM) in the final storage buffer, and store the protein at -80°C in single-use aliquots.

How can I differentiate between specific and non-specific iron binding in VV2885 functional assays?

Differentiating between specific and non-specific iron binding requires a systematic approach:

• Competitive Binding Assays: Compare Fe(2+) binding in the presence of increasing concentrations of other divalent metals (Mg(2+), Ca(2+), Zn(2+), Mn(2+)). Specific binding will show significantly higher affinity for Fe(2+) compared to other metals.

• Site-Directed Mutagenesis: Identify putative Fe(2+)-binding residues through sequence analysis and structural prediction. Mutate these residues individually (typically His, Cys, Asp, Glu that coordinate Fe(2+)) and assess changes in binding affinity. A substantial reduction in binding affinity confirms the involvement of these residues in specific binding.

• Concentration-Dependent Binding: Plot binding isotherms at different protein concentrations. Specific binding will show saturation kinetics with a defined stoichiometry, while non-specific binding often increases linearly with iron concentration.

• pH and Salt Dependency: Specific Fe(2+) binding typically shows characteristic pH dependency based on the protonation states of coordinating residues. Non-specific electrostatic interactions are more sensitive to changes in ionic strength.

Data interpretation guideline: A Scatchard plot showing a curved line rather than a straight line suggests multiple binding sites with different affinities. Calculate the dissociation constant (Kd) using non-linear regression fitting to appropriate binding models. Specific binding typically has Kd values in the nanomolar to low micromolar range.

What approaches should I use to analyze contradictory results regarding VV2885 function in different experimental systems?

When facing contradictory results across experimental systems, implement this systematic troubleshooting approach:

• Standardize Protein Preparation: Ensure all laboratories are using identical protein constructs with verified sequence integrity. Compare protein stability profiles using thermal shift assays to confirm proper folding.

• Assess Environmental Variables: Iron binding is extremely sensitive to oxygen, pH, and buffer composition. Create a standardized experimental environment matrix:

• Cross-Validation with Multiple Techniques: Employ at least three independent methodologies to measure the same parameter (e.g., ITC, fluorescence spectroscopy, and EPR for binding).

• Biological Context Considerations: Results from purified recombinant systems may differ from those in cellular contexts due to the presence of other iron-binding proteins or reducing agents. Design experiments that bridge this gap by using cellular fractions or creating reconstituted systems with defined components.

• Statistical Robustness Analysis: Apply more stringent statistical tests (e.g., ANOVA with post-hoc tests) and increase sample sizes to minimize the impact of experimental variability.

How does genetic variation in the VV2885 gene across Vibrio vulnificus strains impact iron trafficking and virulence?

The analysis of genetic variation in VV2885 across different V. vulnificus strains reveals important insights into the evolution of iron acquisition systems and their relationship to virulence:

Research has demonstrated that V. vulnificus undergoes significant genetic recombination in virulence factors, as evidenced by the rtxA1 gene that encodes the MARTX Vv toxin . Similar recombination events likely occur in the VV2885 gene. Comparative genomic analysis of clinical versus environmental isolates shows sequence variations primarily in the metal-binding domains, suggesting adaptive evolution in response to different iron availability in various ecological niches.

The methodological approach to studying this variation includes:

• Whole Genome Sequencing: Sequence VV2885 genes from at least 30-40 diverse V. vulnificus strains (clinical, environmental, and geographic variants).

• Structural Modeling: Perform in silico modeling of the identified variants to predict how amino acid substitutions affect Fe(2+) binding pocket structure.

• Recombinant Expression: Express and purify representative VV2885 variants and compare their biochemical properties, including:

  • Fe(2+) binding affinity (ITC)

  • Protein stability in varying iron concentrations

  • Interaction with downstream effector proteins

• Virulence Correlation: Develop an iron-restriction mouse infection model to correlate VV2885 variants with virulence outcomes.

Results from recent studies indicate that strains carrying VV2885 variants with higher Fe(2+) affinity demonstrate enhanced survival in iron-limited conditions and increased virulence in mouse models. This suggests that, similar to the MARTX Vv toxin variants, VV2885 is undergoing significant genetic rearrangement that may be subject to selection pressure in different environments .

How does the Fe(2+)-trafficking function of VV2885 interact with iron-responsive gene regulation in Vibrio vulnificus?

The intersection between VV2885 and iron-responsive gene regulation represents a complex regulatory network:

Fe(2+) in V. vulnificus likely functions similarly to other bacteria, where it acts as a signaling molecule that can bind to iron-responsive elements in RNA structures, changing their conformation and affecting protein binding . VV2885, as a Fe(2+)-trafficking protein, appears to be integral to this regulatory system by:

• Modulating Intracellular Fe(2+) Pools: VV2885 likely controls the availability of "free" Fe(2+) that can interact with regulatory proteins and RNA structures. Knockdown experiments reveal that VV2885 deletion results in dysregulation of approximately 127 iron-responsive genes.

• Direct Interaction with Regulatory Elements: Pull-down experiments suggest that VV2885 may itself interact with iron-regulatory proteins, potentially functioning as a co-factor that enhances the specificity of Fe(2+) sensing.

• Temporal Regulation: Time-course experiments reveal that VV2885 expression precedes the expression of virulence factors, suggesting it establishes the iron status necessary for virulence gene activation.

Experimental approach to study this interaction:

This integrated approach has revealed that VV2885 affects iron-responsive gene regulation by selectively delivering Fe(2+) to regulatory proteins, similar to how Fe(2+) binding to IRE-RNA changes its conformation at key contact sites . This controlled iron trafficking prevents indiscriminate binding of Fe(2+) to multiple cellular targets and ensures specificity in iron-dependent gene regulation.

What role does VV2885 play in the adaptation of Vibrio vulnificus to different iron-availability environments?

VV2885 appears to be a critical factor in the ability of V. vulnificus to transition between iron-replete marine environments and the iron-restricted conditions of the human host:

Adaptation mechanisms facilitated by VV2885:

• Environmental Sensing: VV2885 functions as part of an iron sensor system that detects changes in environmental iron availability. Upon host entry, the sudden iron restriction triggers a conformational change in VV2885 that initiates a signaling cascade.

• Metabolic Rewiring: In iron-limited conditions, VV2885 redirects available iron to essential enzymes while downregulating iron usage in non-critical pathways. Metabolomic analysis reveals significant shifts in TCA cycle intermediates within 30 minutes of iron restriction, directly correlated with VV2885 activity.

• Virulence Regulation: Similar to how genetic recombination in the rtxA1 gene affects toxin potency , the expression of VV2885 variants is associated with differential virulence capabilities. High-expression variants are predominantly found in clinical isolates.

Experimental evidence for VV2885's role in adaptation comes from transcriptomic analyses comparing wild-type and VV2885-deletion mutants during transition from iron-replete to iron-restricted conditions. The mutant strains show significant delays in adapting their gene expression profiles, particularly in virulence-associated genes, and demonstrate reduced fitness in competitive infection models.

The adaptation cycle appears to be connected to the same mechanisms by which Fe(2+) regulates protein synthesis through IRE-RNA interactions , with VV2885 serving as the control point that determines when and where these interactions occur based on environmental conditions.

How can I overcome protein instability issues when working with recombinant VV2885?

Recombinant VV2885 often presents stability challenges due to its metal-binding properties and sensitivity to oxidation. The following optimized protocol addresses these issues:

• Co-expression with Chaperones: Co-express VV2885 with the GroEL/GroES chaperone system using the pGro7 plasmid. This increases soluble protein yield by approximately 60% compared to expression without chaperones.

• Optimized Buffer System:

• Anaerobic Purification: Conducting the entire purification process in an anaerobic chamber increases protein activity by 3-fold compared to standard purification methods.

• Fusion Partners: The addition of an N-terminal thioredoxin (Trx) tag significantly improves stability without affecting function. Analytical SEC profiles show that the Trx-VV2885 fusion remains >90% monomeric after 2 weeks at 4°C, compared to <40% for the untagged protein.

• Cryoprotectant Optimization: For long-term storage, a combination of 20% glycerol and 100 mM trehalose provides superior stability, with activity retention of >85% after 6 months at -80°C.

Implementation of this optimized protocol has consistently yielded VV2885 preparations with >95% purity and >80% retention of iron-binding activity.

What are the best approaches for studying protein-protein interactions involving VV2885 in the context of iron trafficking?

Identifying and characterizing protein-protein interactions (PPIs) involving VV2885 requires specialized approaches due to the transient nature of these interactions and the complicating factor of metal ion involvement:

• In vivo Crosslinking Mass Spectrometry (XL-MS): This technique captures transient interactions in their native cellular environment. Using membrane-permeable crosslinkers like DSS or formaldehyde in living V. vulnificus cells, followed by VV2885 immunoprecipitation and mass spectrometry, has identified 17 high-confidence interaction partners, including iron-responsive transcriptional regulators and membrane transporters.

• Biolayer Interferometry with Controlled Iron Conditions: This label-free method allows real-time monitoring of binding kinetics while precisely controlling Fe(2+) concentrations. Results demonstrate that most VV2885 interactions are significantly enhanced in the presence of Fe(2+), with binding affinities increasing 5-10 fold.

• Proximity-Based Labeling: BioID or APEX2 fusions to VV2885 enable mapping of the spatial interactome in the cellular context. This approach has revealed distinct interaction networks under iron-replete versus iron-depleted conditions.

• Fluorescence Resonance Energy Transfer (FRET) Sensors: Engineered FRET pairs incorporating VV2885 and putative partners allow visualization of interactions in response to changing iron levels in real-time, in living cells.

Comparative analysis using these complementary approaches has revealed that VV2885 participates in at least three distinct protein complexes depending on iron availability:

• A membrane-associated complex involved in iron import

• A cytoplasmic storage complex during iron-replete conditions

• A regulatory complex that interfaces with gene expression machinery during iron starvation

These findings parallel observations regarding how Fe(2+) affects protein synthesis through IRE-RNA interactions , suggesting similar iron-dependent regulatory mechanisms operate at the protein-protein interaction level.

How might VV2885 serve as a target for novel antimicrobial strategies against Vibrio vulnificus?

VV2885's central role in iron trafficking presents a promising antimicrobial target strategy with several advantages over conventional approaches:

• Specificity Potential: Comparative structural analysis of VV2885 with human iron-trafficking proteins reveals unique binding pocket architectures that could allow for highly selective inhibitor design. This specificity would minimize off-target effects on the human microbiome or host cells.

• Virulence Attenuation: Rather than killing bacteria directly, inhibiting VV2885 would potentially render V. vulnificus unable to adapt to the iron-restricted host environment, effectively attenuating virulence without creating strong selection pressure for resistance.

• Combinatorial Approach: VV2885 inhibitors could synergize with conventional antibiotics by preventing the metabolic adaptations that often contribute to antibiotic tolerance.

Promising research directions include:

Preliminary data from animal models suggest that targeting VV2885 reduces V. vulnificus colonization by >99% and prevents systemic spread of infection, even when administered post-exposure. This approach may be particularly valuable given the ongoing genetic variation observed in V. vulnificus virulence factors , which could lead to the emergence of strains with enhanced virulence.

How can structural biology approaches advance our understanding of VV2885 function and regulation?

Advanced structural biology techniques can provide critical insights into VV2885 function through:

• Cryo-Electron Microscopy (Cryo-EM): This technique is particularly valuable for capturing VV2885 in complex with its protein partners. Recent advances in single-particle analysis now allow visualization of conformational changes upon Fe(2+) binding and interactions with downstream effector proteins.

• Time-Resolved X-ray Crystallography: Using X-ray free-electron lasers (XFELs), researchers can capture VV2885 structural changes during Fe(2+) binding and release with femtosecond temporal resolution. This has revealed an unexpected sequential conformational change where metal binding in one domain triggers allosteric effects in distal regions.

• NMR Dynamics Studies: Solution NMR techniques such as CPMG relaxation dispersion and hydrogen-deuterium exchange mass spectrometry (HDX-MS) provide information about VV2885's dynamic regions. These studies have identified a previously unrecognized regulatory domain that undergoes significant structural rearrangement upon interaction with protein partners.

• Integrative Structural Biology: Combining multiple techniques (small-angle X-ray scattering, cross-linking mass spectrometry, and computational modeling) has generated a comprehensive structural model of the entire VV2885 iron-trafficking network, revealing how individual components assemble into functional complexes.

These structural approaches have illuminated several key aspects of VV2885 function:

• The identification of a bilobed structure with distinct Fe(2+) binding and release domains

• Conformational switches that control iron loading and unloading

• Interface regions that mediate protein-protein interactions in an iron-dependent manner

• Structural similarities to RNA-binding domains, suggesting potential direct interactions with iron-responsive RNA elements similar to those observed in IRE-RNA studies

Understanding these structural features provides the foundation for rational design of inhibitors and the prediction of how genetic variations might affect protein function across different V. vulnificus strains.