Recombinant Vibrio vulnificus UPF0149 protein VV2847 (VV2847)

What is Vibrio vulnificus UPF0149 protein VV2847 and what is its significance?

VV2847 is a protein belonging to the UPF0149 protein family found in Vibrio vulnificus strain YJ016 . Vibrio vulnificus is a foodborne pathogen prevalent in coastal waters worldwide that causes septicemia with fatality rates exceeding 50% even with aggressive antibiotic therapy . The significance of VV2847 must be viewed in the context of V. vulnificus pathogenesis research, which has primarily focused on identifying virulence factors that could be targeted for therapeutic interventions. While the specific function of VV2847 is not yet fully characterized, proteins from the UPF0149 family are of interest as potential contributors to bacterial survival or virulence mechanisms.

How does VV2847 relate to other characterized virulence factors in Vibrio vulnificus?

While direct relationships between VV2847 and established virulence factors have not been definitively characterized in the available literature, V. vulnificus pathogenicity is known to involve multiple virulence factors. The multifunctional-autoprocessing RTX (MARTX) toxin, especially the RtxA1 variant, has been established as a significant virulence factor . Additionally, V. vulnificus hemolysin (VVH), a pore-forming cholesterol-dependent cytolysin, contributes to pathogenesis through various cellular outcomes . Research examining potential interactions between VV2847 and these established virulence factors would represent a valuable contribution to understanding V. vulnificus pathogenesis mechanisms.

What experimental approaches can be used to determine the function of VV2847 in Vibrio vulnificus?

Elucidating the function of VV2847 would require a multi-faceted experimental approach:

• Gene knockout studies: Creating VV2847-deficient mutants to assess changes in virulence, metabolism, or stress responses.

• Protein-protein interaction studies: Using pull-down assays, yeast two-hybrid systems, or co-immunoprecipitation to identify binding partners.

• Expression profiling: Analyzing VV2847 expression patterns under different environmental conditions and infection stages.

• Structural analysis: Determining the three-dimensional structure through X-ray crystallography or NMR spectroscopy.

• Comparative genomics: Analyzing homologs in related species to infer potential functions.

The availability of recombinant VV2847 with >85% purity (as determined by SDS-PAGE) enables many of these experimental approaches through in vitro studies.

How might VV2847 contribute to Vibrio vulnificus pathogenicity?

While the specific contribution of VV2847 to pathogenicity remains to be determined, several hypotheses could be tested:

• Role in bacterial survival: The protein might contribute to stress responses within hostile host environments.

• Host cell interactions: VV2847 could potentially interact with host cell receptors or intracellular components.

• Immunomodulation: The protein might affect host immune responses, similar to how other Vibrio proteins modulate host defenses.

• Regulatory functions: VV2847 could participate in regulatory networks that control expression of established virulence factors like MARTX toxins or VVH.

Testing these hypotheses would require comparisons to known virulence mechanisms, such as those of MARTX toxins which cause cytotoxic effects including actin depolymerization, apoptosis, necrosis, and induction of reactive oxygen species .

What are the most appropriate experimental conditions for studying VV2847 interactions with host proteins?

For studying potential interactions between VV2847 and host proteins, researchers should consider:

• Buffer composition: Phosphate-buffered saline with physiologically relevant pH (7.2-7.4) supplemented with appropriate stabilizing agents.

• Temperature control: Maintain 37°C for mammalian interactions or relevant environmental temperatures for ecological studies.

• Cell models: Human intestinal epithelial cells (like INT-407) have been successfully used in Vibrio protein studies .

• Protein labeling: Fluorescent or epitope tagging should be employed with caution to avoid disrupting native interactions.

• Validation techniques: Multiple complementary approaches (SPR, ELISA, microscopy) should be used to confirm any identified interactions.

When investigating potential cytotoxic effects, researchers can reference methodologies used with other Vibrio proteins, such as the study of VVH effects on human intestinal epithelial cells, which revealed that even low doses of recombinant protein can induce observable cellular responses .

What are the optimal storage and handling conditions for recombinant VV2847?

Based on manufacturer recommendations, researchers should adhere to the following storage and handling protocols:

• Storage temperature: -20°C to -80°C for long-term storage.

• Stability period: Liquid form maintains stability for approximately 6 months, while lyophilized preparations can remain stable for 12 months.

• Freeze-thaw cycles: Repeated freezing and thawing should be avoided; working aliquots can be stored at 4°C for up to one week.

• Reconstitution: The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Stabilization: Addition of glycerol to a final concentration of 5-50% (default: 50%) is recommended for long-term storage .

Proper storage and handling are critical for maintaining protein activity and ensuring experimental reproducibility.

What analytical methods are recommended for validating recombinant VV2847 quality and integrity?

Researchers should employ multiple analytical techniques to validate recombinant VV2847:

• SDS-PAGE: To confirm size (expected ~21 kDa) and initial purity assessment (>85% as indicated by manufacturer) .

• Western blotting: Using antibodies against the protein or any fusion tags to confirm identity.

• Mass spectrometry: For accurate mass determination and sequence confirmation.

• Circular dichroism: To assess secondary structure and proper folding.

• Size exclusion chromatography: To determine oligomerization state and detect aggregation.

How can researchers optimize expression systems for producing functional VV2847?

While the commercially available recombinant VV2847 is produced in yeast , researchers developing their own expression systems should consider:

• Expression host selection:

  • E. coli: For high yield but potential folding issues

  • Yeast: For better eukaryotic post-translational modifications

  • Mammalian cells: For highest authenticity but lower yield

• Vector design:

  • Fusion tags selection (His, GST, MBP) for improved solubility and purification

  • Codon optimization for the selected expression host

  • Inducible promoters for controlled expression

• Culture conditions:

  • Induction timing and temperature (lower temperatures often improve folding)

  • Media composition and supplementation

  • Harvest timing to maximize yield while minimizing degradation

Successful expression would be evidenced by high yield of soluble protein with correct folding and biological activity.

What computational approaches can predict potential functions of VV2847?

Several bioinformatic approaches can provide insights into VV2847 function:

• Sequence homology analysis: Identifying functionally characterized homologs in other bacterial species.

• Domain prediction: Tools like PFAM, SMART, or InterPro can identify conserved domains.

• Structural modeling: Using AlphaFold or similar tools to predict tertiary structure based on amino acid sequence.

• Molecular docking: Predicting potential interactions with host proteins or small molecules.

• Genomic context analysis: Examining the genes surrounding VV2847 for functional clues.

This approach could reveal connections to known virulence mechanisms in V. vulnificus, such as the MARTX toxin system or VVH, which have established roles in pathogenesis .

How can researchers determine if VV2847 plays a role in antibiotic resistance or stress responses?

To investigate potential roles in antibiotic resistance or stress responses, researchers should:

• Generate VV2847 knockout strains and measure:

  • Minimum inhibitory concentrations (MICs) for various antibiotics

  • Growth curves under oxidative, osmotic, or acid stress conditions

  • Survival rates in serum or during phagocytosis

• Perform complementation studies:

  • Re-introducing wild-type VV2847 to confirm phenotype restoration

  • Introducing modified variants to identify critical residues

• Measure expression changes:

  • qRT-PCR or RNA-seq to quantify VV2847 expression changes under stress

  • Promoter-reporter fusion assays to visualize expression patterns

Since V. vulnificus infections have high mortality rates even with aggressive antibiotic therapy , understanding potential contributions to antibiotic resistance would be particularly valuable.

What approaches can be used to study potential immunogenic properties of VV2847?

To investigate the immunogenic properties of VV2847, researchers could:

• Epitope mapping:

  • In silico prediction of B and T cell epitopes

  • Peptide array analysis to identify immunodominant regions

  • ELISA with overlapping peptides to confirm predictions

• Immunological assays:

  • Measuring cytokine production by immune cells exposed to VV2847

  • Flow cytometry to assess immune cell activation markers

  • Western blotting with sera from infected individuals to detect antibody recognition

• Animal models:

  • Immunization studies to assess protective capacity

  • Challenge studies to evaluate vaccine potential

  • Passive immunization with anti-VV2847 antibodies

This approach would complement existing vaccine research, such as studies on the C-terminal region of MARTX toxin (RtxA1-C), which has shown promise as a protective antigen .

How might VV2847 be incorporated into diagnostic assays for Vibrio vulnificus?

VV2847 could potentially contribute to improved diagnostic methods:

• Antibody-based detection:

  • Development of anti-VV2847 antibodies for immunoassays

  • Implementation in lateral flow devices for rapid field testing

  • Sandwich ELISA systems for laboratory diagnostics

• Nucleic acid detection:

  • PCR primers targeting the VV2847 gene as a species marker

  • LAMP assays for point-of-care detection

  • Multiplexed detection systems incorporating multiple genetic markers

• Biosensor development:

  • Antibody-coated surfaces for surface plasmon resonance

  • Electrochemical detection systems

  • Aptamer-based biosensors

Improved diagnostics would address the critical need for rapid identification, given the high mortality rate of V. vulnificus infections, which exceeds 50% even with antibiotic treatment .

What role could VV2847 play in the development of vaccines against Vibrio vulnificus?

While not yet established as a vaccine target, VV2847 could be investigated in vaccine development:

• As a potential antigen:

  • Evaluating immunogenicity in animal models

  • Testing protective efficacy against challenge

  • Combining with established antigens for broader protection

• As a carrier protein:

  • Fusion with weakly immunogenic epitopes

  • Presentation platform for critical protective antigens

• For comparative studies:

  • Understanding immune responses across different V. vulnificus proteins

  • Identifying optimal antigen combinations

This approach would build upon established vaccine research, such as studies with recombinant RtxA1-C protein, which has shown promise in eliciting protective immune responses and significant protection against lethal challenge with V. vulnificus .

How can VV2847 be used to study genetic variation among Vibrio vulnificus strains?

VV2847 could serve as a marker for studying genetic diversity:

• Sequence analysis across strains:

  • PCR amplification and sequencing of the VV2847 gene

  • Identification of single nucleotide polymorphisms or structural variants

  • Phylogenetic analysis to establish evolutionary relationships

• Expression analysis:

  • qRT-PCR to measure differential expression across isolates

  • Western blotting to assess protein production levels

  • Transcriptomic comparisons under standardized conditions

• Functional comparisons:

  • Activity assays with recombinant variants from different strains

  • Host interaction studies to identify strain-specific differences

This approach would complement existing research on genetic variation in V. vulnificus virulence factors, such as the studies on rtxA1 gene variants, which have revealed distinct toxin arrangements across different strains .