Recombinant Vibrio vulnificus UPF0234 protein VV1636 (VV1636)

What is the function of Vibrio vulnificus UPF0234 protein VV1636 in bacterial pathogenesis?

VV1636 belongs to the UPF0234 protein family, which remains largely uncharacterized in terms of specific biological function. Based on genomic analysis of V. vulnificus, VV1636 likely contributes to bacterial survival and potentially to virulence mechanisms. While not as well-studied as established virulence factors like VvhA (hemolysin) or MARTX toxins, researchers should consider investigating:

• Potential regulatory roles in metabolic pathways

• Interaction with known virulence factors

• Contribution to stress response mechanisms

• Possible involvement in post-translational modification networks

Methodological approach: Implement gene knockout studies using CRISPR-Cas9 to evaluate phenotypic changes in V. vulnificus pathogenicity, followed by complementation studies with recombinant VV1636 to confirm function. Comparative proteomic analysis between wild-type and VV1636-knockout strains can reveal affected pathways.

How does the genomic context of VV1636 compare across different Vibrio vulnificus strains?

The genomic organization surrounding VV1636 provides important clues about its biological role. Core genome analysis of multiple V. vulnificus strains has identified approximately 2,248 conserved genes across the species . To assess VV1636 conservation:

• Examine synteny of the genomic region containing VV1636

• Compare sequence conservation across biotype 1 and biotype 2 strains

• Analyze whether VV1636 is part of the core genome or accessory genome

• Investigate potential horizontal gene transfer events

Genomic comparison has revealed that V. vulnificus strains cluster into distinct clades based on core genome analysis , which may affect VV1636 expression patterns and function across clinical versus environmental isolates.

What expression systems are optimal for producing recombinant Vibrio vulnificus VV1636?

When expressing recombinant VV1636, consider the following expression systems based on research with similar V. vulnificus proteins:

Methodological recommendation: Start with E. coli BL21(DE3) using the pET expression system with a C-terminal 6×His tag. If solubility issues arise, try fusion partners like GST, MBP, or SUMO to enhance solubility. For functional studies requiring PTMs, consider yeast or insect cell expression systems.

How might VV1636 contribute to Vibrio vulnificus virulence compared to characterized toxins like VvhA?

VV1636 likely plays a different role than established cytotoxins such as VvhA and MARTX. While VvhA is a pore-forming cholesterol-dependent cytolysin that causes hemolysis, apoptosis, and necrosis in host cells , VV1636's UPF0234 family designation suggests potential regulatory functions.

Research strategies to differentiate VV1636's role from established toxins:

• Examine temporal expression patterns during infection using qRT-PCR

• Determine subcellular localization using fluorescent protein fusions

• Analyze protein-protein interactions with known virulence factors

• Investigate potential involvement in regulatory networks

Researchers should note that VvhA and MARTX toxins work additively to cause intestinal epithelial tissue damage and promote bacterial dissemination . VV1636 may function in complementary pathways or regulatory mechanisms rather than direct cytotoxicity.

What post-translational modifications might affect VV1636 function in Vibrio vulnificus?

Recent proteome-wide analysis of lysine acetylation in V. vulnificus identified 1,924 acetylated proteins (40.34% of all proteins) at 6,626 sites . While specific data on VV1636 acetylation is not directly reported, this high prevalence suggests VV1636 likely undergoes acetylation.

Consider investigating:

• Lysine acetylation sites using mass spectrometry

• Phosphorylation status under different growth conditions

• Protein methylation patterns that might affect activity

• Other modifications like SUMOylation or ubiquitination

Methodological approach: Perform immunoprecipitation of VV1636 followed by LC-MS/MS analysis under different environmental conditions (iron limitation, high/low osmolarity, different temperatures) to identify condition-specific PTM patterns. Site-directed mutagenesis of identified modification sites can confirm their functional significance.

How is VV1636 expression regulated in response to environmental signals?

V. vulnificus adapts to diverse environments through complex gene regulation. Given patterns observed with other virulence factors, VV1636 expression may be regulated by:

• Iron availability through Fur-dependent pathways

• Quorum sensing via LuxO/SmcR circuits

• Temperature changes through H-NS modulation

• Cyclic AMP and CRP during carbon source shifts

As demonstrated with VvhA regulation, V. vulnificus integrates environmental signals through global regulators like CRP, H-NS, and HlyU . Similar mechanisms likely control VV1636 expression.

Research approach: Construct a VV1636 promoter-reporter fusion and monitor expression under various environmental conditions. Follow with chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the promoter region.

What purification strategies yield optimal recovery of functional recombinant VV1636?

Based on experience with similar V. vulnificus proteins, a multi-step purification strategy is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged VV1636

• Intermediate purification: Ion exchange chromatography (IEX)

• Polishing step: Size exclusion chromatography (SEC)

Critical considerations:

• Add protease inhibitors throughout purification to prevent degradation

• Include reducing agents (DTT or β-mercaptoethanol) if cysteine residues are present

• Determine optimal buffer conditions through thermal shift assays

• Consider detergent addition if hydrophobic regions are identified in sequence analysis

For enhanced solubility, fusion tags like MBP or SUMO often outperform standard His-tags alone. If inclusion bodies form, evaluate refolding protocols using stepwise dialysis with decreasing concentrations of urea or guanidine hydrochloride.

How can researchers design experiments to identify potential VV1636 interaction partners?

Understanding protein-protein interactions is crucial for elucidating VV1636 function:

• Yeast two-hybrid screening:

  • Use VV1636 as bait against a prey library of V. vulnificus proteins

  • Confirm interactions using reciprocal bait-prey configurations

  • Validate with controls for auto-activation

• Pull-down assays with co-immunoprecipitation:

  • Express tagged VV1636 in V. vulnificus

  • Capture protein complexes under native conditions

  • Identify partners using mass spectrometry

• Proximity-based labeling:

  • Fuse VV1636 to BioID or APEX2

  • Express in V. vulnificus and allow proximity labeling

  • Purify biotinylated proteins and identify by MS

• Surface plasmon resonance:

  • Immobilize purified VV1636 on a sensor chip

  • Flow candidate interacting proteins

  • Determine binding kinetics and affinity constants

Consider focusing on potential interactions with known virulence regulators like HlyU, SmcR, and CRP, which modulate expression of other virulence factors in V. vulnificus .

What animal models are appropriate for studying VV1636's role in Vibrio vulnificus pathogenesis?

When investigating VV1636's contribution to pathogenesis, consider these animal models based on established V. vulnificus research:

Research has shown that V. vulnificus strain MCCC 1A08743 causes liver lesions in C57BL/6J mice, with more severe phenotypes in non-alcoholic fatty liver disease models . This suggests that liver disease models are particularly relevant for studying V. vulnificus virulence factors.

Experimental design recommendations:

• Use subcutaneous injection (10^8 CFU) for initial virulence assessment

• Analyze tissue distribution at 12-24 hour intervals

• Compare wild-type versus VV1636 knockout strains

• Evaluate complementation with purified recombinant VV1636

How can researchers apply transcriptomic approaches to understand VV1636's role in Vibrio vulnificus biology?

Modern transcriptomic approaches offer powerful insights into gene function:

• RNA-Seq analysis:

  • Compare wild-type vs. VV1636 knockout expression profiles

  • Analyze different growth conditions (iron limitation, temperature shifts)

  • Identify co-regulated genes suggesting functional relationships

• Independent Component Analysis (ICA):

  • Apply machine learning to identify co-regulated gene sets (iModulons)

  • Recently identified novel biofilm-related genes in V. vulnificus

  • May reveal VV1636's role in previously uncharacterized pathways

• Single-cell RNA-Seq:

  • Capture expression heterogeneity across bacterial populations

  • Identify subpopulations with distinct VV1636 expression patterns

  • Understand potential bet-hedging strategies

• Dual RNA-Seq:

  • Simultaneously profile host and pathogen transcriptomes during infection

  • Correlate VV1636 expression with host response patterns

  • Identify condition-specific regulation

Independent Component Analysis has proven particularly valuable for identifying novel gene functions in V. vulnificus beyond what differential expression analysis alone can detect . This approach could reveal whether VV1636 belongs to specific regulatory networks not previously associated with UPF0234 family proteins.