Recombinant Vibrio vulnificus UPF0133 protein VV2410 (VV2410)

What is currently known about the structural characteristics of VV2410?

VV2410 is classified as a nucleoid-associated protein belonging to the UPF0133 (Uncharacterized Protein Family 0133) family from Vibrio vulnificus. While specific structural data on VV2410 is limited, insights can be drawn from research on related UPF proteins. For example, the TTHA0281 protein from Thermus thermophilus HB8 (belonging to the UPF0150 family) adopts an α-β-β-β-α fold and forms a homotetramer in solution .

The structural analysis of UPF proteins typically requires multiple techniques. X-ray crystallography remains the gold standard for determining protein structure at atomic resolution, while nuclear magnetic resonance (NMR) spectroscopy can provide valuable information about protein dynamics in solution. Cryo-electron microscopy (cryo-EM) has also emerged as a powerful technique for structural determination of challenging proteins.

Based on other nucleoid-associated proteins, VV2410 likely contains DNA-binding domains that interact with bacterial chromosomes. Understanding its three-dimensional structure would provide critical insights into its function and potential role in V. vulnificus biology.

How might VV2410 contribute to Vibrio vulnificus pathogenesis?

While the specific role of VV2410 in virulence remains undetermined, nucleoid-associated proteins often function as global regulators of gene expression, potentially affecting virulence factor production. V. vulnificus possesses numerous virulence factors including pili, membrane proteins like OmpU, and flagella that contribute to attachment and invasion of host cells . Additionally, cytotoxins such as RtxA1, hemolysin (VvhA), and metalloproteases (VvpE and VvpM) cause cellular damage and cytotoxicity .

VV2410 could potentially regulate the expression of these virulence factors, similar to how ToxR functions as a virulence regulator in V. vulnificus. A study by Park et al. demonstrated that several proteins are differentially regulated in a toxR mutant of V. vulnificus, suggesting a network of virulence regulation .

The potential involvement of VV2410 in pathogenesis could be investigated through gene knockout studies, followed by virulence assessment in appropriate animal models. Comparative transcriptomic analyses between wild-type and VV2410 mutant strains would reveal genes under VV2410 regulation, particularly those involved in virulence.

What expression patterns does VV2410 exhibit under different environmental conditions?

The expression of VV2410 likely varies in response to environmental factors that V. vulnificus encounters in both marine environments and during host infection. Although specific data on VV2410 expression patterns is not available, research on stress responses in V. vulnificus provides insight into potential regulatory mechanisms.

Universal stress proteins in bacteria often show increased expression under stress conditions. For example, Rv2624c from M. tuberculosis, another bacterial stress protein, affects metabolic pathways in an ATP-dependent manner and enhances intracellular survival . Similar mechanisms might apply to VV2410, particularly given the challenging environments V. vulnificus must navigate during host infection.

Researchers investigating VV2410 expression should employ quantitative RT-PCR and RNA-seq analyses under various conditions including temperature shifts (representing transition from environment to host), varying salinity concentrations, nutrient limitation, and exposure to host immune factors. Proteomic approaches can further validate these findings at the protein level.

What are optimal methods for expression and purification of recombinant VV2410?

The production of recombinant VV2410 can be accomplished using several expression systems. Based on successful approaches with other V. vulnificus proteins, E. coli-based expression systems represent an effective starting point. The gene encoding VV2410 can be amplified by PCR and cloned into appropriate expression vectors such as pET21(b), which has been successfully used for other V. vulnificus proteins like VvpE .

A typical expression protocol would include:

• PCR amplification of the VV2410 gene from V. vulnificus genomic DNA

• Cloning into an expression vector with appropriate affinity tags (His6-tag is commonly used)

• Transformation into suitable E. coli expression strains (BL21(DE3) is frequently employed)

• Optimization of expression conditions including temperature, induction time, and IPTG concentration

• Cell lysis followed by purification steps

For purification, a multi-step approach is recommended:

• Initial capture using immobilized metal affinity chromatography (IMAC) for His-tagged protein

• Heat treatment (taking advantage of V. vulnificus proteins' relative thermostability)

• Ion exchange chromatography to remove charged contaminants

• Size exclusion chromatography for final polishing and oligomerization assessment

This approach has proven effective for other V. vulnificus proteins, including the recombinant VvpE metalloprotease that was successfully expressed and purified for vaccine studies .

How can researchers develop effective assays to characterize VV2410 function?

Developing functional assays for an uncharacterized protein like VV2410 requires a multi-faceted approach. Since VV2410 is annotated as a nucleoid-associated protein, DNA-binding assays should be prioritized. Electrophoretic mobility shift assays (EMSA) can determine if VV2410 binds DNA and identify sequence preferences. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) can map genome-wide binding sites in vivo.

For potential regulatory functions, reporter gene assays using promoters of suspected target genes fused to luciferase or fluorescent proteins can measure VV2410's effect on gene expression. These assays should be performed in both wild-type and VV2410 knockout strains.

Additionally, since nucleoid-associated proteins often affect DNA topology, assays measuring DNA supercoiling, compaction, or protection from nuclease digestion would provide insights into VV2410's role in nucleoid organization. Protein-protein interaction assays including bacterial two-hybrid systems or co-immunoprecipitation can identify VV2410's interaction partners within the bacterial cell.

The data from these functional assays should be integrated with structural information and expression data to build a comprehensive model of VV2410's function in V. vulnificus.

What methods are most effective for generating and validating VV2410 mutants?

Creating VV2410 mutants is essential for understanding its function in V. vulnificus. Based on successful approaches with other V. vulnificus genes, several methods can be employed:

• Allelic exchange: This technique has been effectively used to create V. vulnificus mutants, as demonstrated in the construction of vvpE mutants . The approach involves:

  • Creating a construct with truncated or modified VV2410 gene

  • Cloning into a suicide vector (like pNQ705)

  • Conjugative transfer into V. vulnificus

  • Selection for recombinants

• CRISPR-Cas9 mutagenesis: This more modern approach allows precise genome editing and can be adapted for V. vulnificus using appropriate delivery vectors.

Validation of mutants should include:

• PCR verification of the mutation

• Southern blot analysis to confirm genomic integration

• Western blot using anti-VV2410 antibodies to verify protein absence

• Complementation studies to ensure phenotypes can be restored

Creating point mutations in specific functional domains (once identified through structural studies) can provide additional insights into structure-function relationships of VV2410.

What criteria should be used to evaluate VV2410 as a potential vaccine candidate?

Several V. vulnificus proteins have been investigated as vaccine candidates, providing a framework for evaluating VV2410's potential. The metalloprotease VvpE has shown 75% protective efficacy when used as a recombinant vaccine in mice , while the C-terminal region of RtxA1 induced protective immune responses against V. vulnificus infection .

To evaluate VV2410 as a vaccine candidate, researchers should assess:

• Conservation across clinically relevant V. vulnificus strains (essential for broad protection)

• Surface accessibility or secretion (for antibody recognition)

• Immunogenicity in animal models

• Ability to induce protective antibodies against challenge with virulent V. vulnificus

• Stability and safety profile

The evaluation process would typically include:

• Immunization of mice with purified recombinant VV2410 with appropriate adjuvants

• Measurement of specific antibody responses using ELISA

• Challenge studies with virulent V. vulnificus strains

• Quantification of bacterial load in blood and tissues

• Survival rate assessment

This approach mirrors successful studies with other V. vulnificus proteins such as OmpU, which has been evaluated as a potential vaccine antigen .

How might VV2410 be incorporated into multi-component vaccine formulations?

A multi-component vaccine approach may offer more comprehensive protection against V. vulnificus infection. Based on vaccine research with other V. vulnificus proteins, VV2410 could potentially be combined with established vaccine candidates like VvpE metalloprotease or the C-terminal region of RtxA1.

For effective multi-component formulation:

• Researchers should first establish non-interference between antigens by immunizing with individual components and combinations, then measuring antibody responses to each antigen.

• Adjuvant selection is critical - studies with V. vulnificus vaccines have typically used Freund's complete adjuvant for initial immunization followed by incomplete adjuvant for boosters .

• Different delivery strategies should be evaluated, including:

  • Co-administration of purified proteins

  • Combined DNA vaccine encoding multiple antigens

  • Recombinant bacterial vectors expressing multiple V. vulnificus antigens

A study by Guo et al. demonstrated the effectiveness of a bivalent vaccine using recombinant outer membrane proteins from V. vulnificus and Aeromonas hydrophila, indicating the feasibility of multi-component approaches .

What are the prospects for passive immunization using antibodies against VV2410?

Passive immunization represents an alternative approach to vaccine development, particularly for acute infections like those caused by V. vulnificus. Research has demonstrated that passive immunization with antibodies against the C-terminal region of RtxA1 provided significant protection in both prophylactic and therapeutic settings .

To evaluate the potential of anti-VV2410 antibodies for passive immunization:

• Polyclonal or monoclonal antibodies against VV2410 should be generated and characterized for specificity and affinity.

• Prophylactic efficacy should be tested by administering antibodies prior to V. vulnificus challenge.

• Therapeutic potential should be assessed by administering antibodies after infection has been established.

• Antibody half-life and tissue distribution studies would determine optimal dosing schedules.

• Potential synergistic effects with antibiotics should be investigated, as V. vulnificus has developed resistance to certain antibiotics .

The relatively rapid onset of V. vulnificus infections (often progressing to septicemia within 24 hours) makes passive immunization particularly relevant as a therapeutic intervention for high-risk individuals after potential exposure.

How can structural biology approaches advance our understanding of VV2410 function?

Advanced structural biology techniques can provide crucial insights into VV2410's function. X-ray crystallography has been successfully applied to other UPF proteins, such as TTHA0281 from T. thermophilus, revealing an α-β-β-β-α fold and homotetramer formation . Similar approaches could elucidate VV2410's structure.

For comprehensive structural characterization of VV2410, researchers should consider:

• Protein crystallization trials with both full-length VV2410 and functional domains

• Co-crystallization with potential binding partners or DNA fragments

• Cryo-EM for visualizing larger complexes

• NMR spectroscopy for dynamic regions and ligand interactions

• Small-angle X-ray scattering (SAXS) for solution structure and conformational changes

The structural data can guide site-directed mutagenesis experiments to identify critical residues for VV2410 function. For example, mutations in ATP-binding residues of the universal stress protein Rv2624c from M. tuberculosis abrogated its growth advantage in macrophage cells . Similar approaches could reveal functional mechanisms of VV2410.

What omics approaches can best elucidate VV2410's role in Vibrio vulnificus biology?

Integrated omics approaches offer powerful tools for understanding VV2410's role in V. vulnificus biology. A comprehensive research strategy should include:

• Transcriptomics: RNA-seq comparing wild-type and VV2410 mutant strains under various conditions. This approach successfully identified genes affected by universal stress protein Rv2624c in M. tuberculosis, revealing effects on histidine, arginine, and proline metabolism .

• Proteomics: Mass spectrometry-based proteome analysis to identify proteins with altered abundance in VV2410 mutants. Similar approaches with ToxR mutants revealed several differentially regulated proteins in V. vulnificus .

• Metabolomics: Liquid chromatography-mass spectrometry (LC-MS/MS) analysis to detect metabolic changes in VV2410 mutants, potentially revealing affected biochemical pathways.

• ChIP-seq: To map VV2410 binding sites across the V. vulnificus genome, providing direct evidence of genes under VV2410 regulation.

Integration of these multi-omics data can create a comprehensive model of VV2410's function and its position within regulatory networks governing V. vulnificus physiology and virulence.

How might genome engineering approaches contribute to understanding VV2410's role in virulence?

Advanced genome engineering approaches can precisely dissect VV2410's role in V. vulnificus virulence. CRISPR-Cas9 technology offers several advantages over traditional mutagenesis methods:

• Creation of clean deletions without polar effects on downstream genes

• Introduction of specific point mutations to test structure-function hypotheses

• Development of conditional expression systems to study essential genes

• Multiplexed targeting to investigate potential redundant functions

Beyond creating knockout mutants, researchers should consider:

• Domain swapping experiments with homologous proteins from other bacteria to identify functional regions

• Creation of reporter fusions to monitor VV2410 expression in real-time during infection

• Inducible expression systems to control VV2410 levels at different infection stages

• Tagging VV2410 with fluorescent proteins to track localization during cell cycle and stress responses

These approaches can establish causal relationships between VV2410 and specific virulence phenotypes, moving beyond correlative observations to mechanistic understanding.

How does VV2410 compare with UPF proteins from other pathogens?

UPF (Uncharacterized Protein Family) proteins are found across diverse bacterial species, including many pathogens. Comparative analysis of VV2410 with other UPF proteins can provide functional insights based on evolutionary relationships and conservation patterns.

The UPF0150 protein family includes proteins like TTHA0281 from T. thermophilus, which forms a homotetramer with each monomer adopting an α-β-β-β-α fold . Although VV2410 belongs to the UPF0133 family, structural comparison with characterized UPF proteins can reveal shared architectural features.

Many UPF proteins are involved in stress responses, similar to universal stress protein Rv2624c from M. tuberculosis, which enhances intracellular survival in monocytes through ATP binding . Functional comparisons should include:

• Sequence alignment with UPF proteins from other pathogens

• Structural modeling based on solved structures

• Expression pattern comparison under similar stress conditions

• Phylogenetic analysis to identify closely related functional homologs

These comparative approaches can accelerate functional characterization by leveraging insights from better-studied UPF proteins in other bacterial systems.

What is the relationship between VV2410 and other virulence factors in Vibrio vulnificus?

Understanding how VV2410 integrates with established virulence mechanisms in V. vulnificus requires systematic comparative analysis. V. vulnificus possesses several well-characterized virulence factors:

• Metalloprotease (VvpE) - contributes to tissue damage and hemorrhagic effects

• RTX toxins (RtxA1) - causes cell rounding and cytoskeletal rearrangements

• Outer membrane proteins (OmpU) - involved in adhesion and fibronectin binding

• Capsular polysaccharide (CPS) - provides resistance to complement-mediated lysis

The relationship between VV2410 and these factors can be investigated through:

• Expression correlation analysis under various conditions

• Protein-protein interaction studies using co-immunoprecipitation or bacterial two-hybrid systems

• Epistasis analysis with double mutants to identify genetic interactions

• Comparative virulence studies in animal models

This integrated approach can position VV2410 within the virulence regulatory network of V. vulnificus and identify potential synergistic effects with established virulence factors.