Recombinant Vibrio vulnificus UPF0227 protein VV2369 (VV2369)

What is UPF0227 protein VV2369 and what is its role in Vibrio vulnificus virulence?

UPF0227 protein VV2369 is an uncharacterized protein family (UPF) member found in Vibrio vulnificus, primarily identified in strain YJ016. While its specific function remains to be fully elucidated, genomic analyses suggest it may be involved in cellular processes related to virulence mechanisms.

Vibrio vulnificus contains multiple virulence factors that contribute to its pathogenicity, including capsular polysaccharide (CPS), lipopolysaccharide (LPS), iron acquisition systems, flagella, pili, hemolysin/cytolysin, metalloprotease, and repeats-in-toxin (RTX) . Recent genomic analyses have identified over 151 putative virulence factors distributed between chromosomes I and II . Researchers investigating VV2369 should consider its potential relationship to these known virulence mechanisms, particularly examining its expression patterns during infection processes.

For experimental investigation of VV2369's role in virulence, researchers should consider:

• Comparative expression analysis during different infection stages

• Knockout/knockdown studies to observe phenotypic changes

• Protein interaction studies to identify binding partners

• Structural analysis to predict functional domains

How does VV2369 compare to other UPF family proteins in Vibrio vulnificus?

VV2369 (UPF0227 family) and VV2347 (UPF0304 family) represent two distinct uncharacterized protein families in Vibrio vulnificus. Comparing their characteristics:

When studying UPF proteins like VV2369, consider:

• Performing phylogenetic analyses to identify related proteins across Vibrio species

• Using structural prediction tools to identify potential functional domains

• Analyzing genomic context to determine if VV2369 is part of an operon with known function proteins

• Conducting comparative expression studies across different growth conditions

While both proteins are classified as UPF (uncharacterized protein family), they likely have distinct functions based on their different family classifications. Research approaching VV2369 should include comparative analyses with VV2347 to determine if there are functional relationships between these uncharacterized proteins .

What expression systems are most effective for producing recombinant VV2369?

Based on research with similar Vibrio proteins, several expression systems can be considered for VV2369 production:

For recombinant VV2369, methodology considerations should include:

• Vector design with appropriate promoters and fusion tags for purification

• Optimization of codon usage for the selected expression system

• Temperature, IPTG concentration, and induction time optimization for bacterial systems

• Purification strategy development using affinity chromatography

Based on production methods for similar proteins like VV2347, baculovirus expression systems have been successfully employed and may represent a good starting point for VV2369 expression . Your experimental approach should include pilot studies comparing at least two different expression systems to determine optimal conditions for your specific research needs.

What are the optimal storage conditions for recombinant VV2369?

While specific stability data for VV2369 is not widely documented, storage recommendations can be extrapolated from similar recombinant proteins from Vibrio vulnificus:

For optimal stability, consider these methodological approaches:

• Reconstitute lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (final concentration) before aliquoting for long-term storage

• Briefly centrifuge vials prior to opening to bring contents to the bottom

• Create small working aliquots stored at 4°C for up to one week to avoid repeated freeze-thaw cycles

• Monitor protein stability using SDS-PAGE analysis before critical experiments

The shelf life of recombinant proteins depends on multiple factors including buffer ingredients, storage temperature, and the intrinsic stability of the protein itself. Similar recombinant proteins from Vibrio vulnificus show good stability in proper storage conditions . Consider performing stability studies specific to your preparation of VV2369 if it will be used for extended research projects.

How can I verify the integrity and activity of recombinant VV2369?

Since VV2369 is an uncharacterized protein without established functional assays, multiple complementary approaches should be used to verify protein integrity:

For activity assessment given limited knowledge of function:

• Develop binding assays with potential interaction partners based on bioinformatic predictions

• Conduct thermal shift assays to assess protein stability and ligand binding

• Perform cellular assays examining effects of VV2369 on mammalian cells or other bacteria

• Consider in silico approaches to predict function that can be experimentally validated

For protein expressed with tags, verify that tag removal (if performed) does not affect protein integrity. Since VV2369 is an uncharacterized protein, functional validation may require developing novel assays based on predicted function from bioinformatic analyses .

What techniques can be used to study the function of VV2369 in Vibrio vulnificus pathogenesis?

To elucidate the function of VV2369 in V. vulnificus pathogenesis, multiple complementary approaches should be employed:

Advanced research approaches:

• Create a TrkA isogenic mutant via insertional inactivation to investigate serum resistance similar to methods used for other V. vulnificus virulence factors

• Perform infection experiments in both normal and iron-treated mice to assess virulence attenuation

• Analyze gene expression during different phases of infection using RT-PCR or RNA-Seq

• Employ comparative genomics to identify conserved domains across different V. vulnificus strains

Understanding VV2369's function may require contextualizing it within known virulence pathways. V. vulnificus pathogenesis involves multiple virulence factors including toxin production (MARTX Vv toxin), capsule formation, and various other mechanisms . Consider investigating VV2369's relationship to these established pathways.

How can I design experiments to elucidate the function of previously uncharacterized proteins like VV2369?

Designing experiments for uncharacterized proteins requires a systematic approach combining bioinformatics and experimental techniques:

Step-by-Step Experimental Design Methodology:

• Bioinformatic analysis:

  • Perform sequence homology searches across multiple databases

  • Use structural prediction tools (AlphaFold, I-TASSER) to predict 3D structure

  • Identify conserved domains and motifs

  • Analyze genomic context and potential operons

• Expression pattern analysis:

  • Determine conditions that upregulate VV2369 expression

  • Compare expression across different growth phases, stress conditions, and host-mimicking environments

  • Create promoter-reporter constructs to monitor expression in real-time

• Generation of mutant strains:

  • Create knockout and overexpression strains

  • Perform comprehensive phenotypic analysis (growth rates, morphology, biofilm formation)

  • Assess virulence in in vitro and in vivo models

• Protein interaction studies:

  • Perform pulldown assays with tagged VV2369

  • Use bacterial two-hybrid systems to screen for interaction partners

  • Validate interactions using co-immunoprecipitation or FRET

• Structural and biochemical characterization:

  • Purify sufficient quantities of recombinant protein

  • Perform enzymatic activity screens based on structural predictions

  • Attempt crystallization for structural determination

When designing these experiments, remember that V. vulnificus has been shown to have significant strain-to-strain variation in virulence factors , so consider testing your hypotheses across multiple strains. The pan-genome analysis approach used for comparing virulence and antibiotic resistance profiles across strains could be adapted to understand the role of VV2369.

What is known about the evolutionary conservation of VV2369 across different Vibrio vulnificus strains?

Understanding the evolutionary conservation of VV2369 requires comparative genomic analysis across Vibrio vulnificus strains:

For comprehensive analysis:

• Compare VV2369 across clinical isolates (such as YJ016, CMCP6) and environmental isolates

• Consider biotype differences (Biotype 1, 2, and 3 strains show significant genetic differences)

• Examine correlation with established virulence markers (vcg, 16S rRNA types, vvhA)

• Analyze strain-specific gene variants as has been done with rtxA1 gene variants

Research with other V. vulnificus genes has shown significant genetic variation between strains. For example, the rtxA1 gene that encodes MARTX Vv toxin has at least four distinct variants with different arrangements of effector domains . Similarly, pan-genome analysis of 26 V. vulnificus strains revealed shared core genes (3,016) and strain-specific genes .

A similar analysis of VV2369 could reveal its conservation pattern and help determine if it belongs to the core genome (present in ≥99% of strains), soft core genome (≥95% and <99%), or accessory genome, providing insights into its evolutionary and functional importance .

What methods can be used to analyze VV2369 interactions with host proteins during infection?

Investigating VV2369 interactions with host proteins requires multiple complementary techniques:

Methodological approach for comprehensive interaction analysis:

• Express tagged VV2369 in V. vulnificus under native control

• Perform infection studies with human cell lines relevant to infection (e.g., epithelial cells)

• Crosslink proteins during infection to capture transient interactions

• Immunoprecipitate VV2369 complexes and identify binding partners by mass spectrometry

• Validate key interactions using co-immunoprecipitation and functional studies

Considerations specific to V. vulnificus research:

• Target cell types should include those relevant to pathogenesis (epithelial cells, macrophages)

• Consider iron-regulated interactions, as iron acquisition is critical for V. vulnificus virulence

• Examine interactions with components of the host immune system, particularly those involved in serum resistance mechanisms

• Compare interaction profiles between virulent and attenuated strains to identify pathogenesis-relevant interactions

How can comparative genomics inform our understanding of VV2369's potential role?

Comparative genomics provides powerful insights into the potential function of uncharacterized proteins like VV2369:

Methodological Framework for Comparative Genomic Analysis:

• Strain Comparison Analysis:

  • Compare VV2369 presence, sequence variation, and genomic context across multiple V. vulnificus strains

  • Correlate sequence variations with strain virulence phenotypes

  • Determine if VV2369 is part of the core genome (≥99% presence), soft core genes (≥95% and <99% presence), or accessory genome

• Cross-Species Analysis:

  • Identify homologs in other Vibrio species and more distant bacterial taxa

  • Analyze co-evolution with known virulence factors

  • Examine synteny (conservation of gene order) to identify functional relationships

• Phylogenetic Profiling:

  • Construct phylogenetic trees to understand evolutionary history

  • Correlate presence/absence with specific phenotypes or environmental adaptations

  • Identify instances of horizontal gene transfer

• Functional Prediction:

  • Analyze co-expression patterns with genes of known function

  • Examine correlation with specific genomic islands associated with virulence

  • Use guilt-by-association approaches to infer function

Similar pan-genome analysis approaches have been successfully used with V. vulnificus to characterize virulence factors and antibiotic resistance profiles across strains , revealing significant genomic diversity that contributes to pathogenic diversity. Applying these methods to VV2369 could provide crucial insights into its functional role and importance in V. vulnificus biology.