Recombinant Vibrio vulnificus UPF0102 protein VV0603 (VV0603)

What is Recombinant Vibrio vulnificus UPF0102 protein VV0603?

Recombinant Vibrio vulnificus UPF0102 protein VV0603 is a bacterial protein belonging to the UPF (Uncharacterized Protein Family) 0102 group expressed in a heterologous system. Similar to other recombinant proteins from Vibrio vulnificus, it is typically produced with an affinity tag (such as His-tag) to facilitate purification and experimental manipulation. The protein is derived from the pathogenic Gram-negative bacterium Vibrio vulnificus, which is known to cause severe infections in humans. The recombinant form allows researchers to study the protein's structure, function, and potential role in bacterial pathogenicity under controlled laboratory conditions without needing to culture large amounts of the pathogenic organism itself .

How is recombinant VV0603 protein typically expressed and purified?

Recombinant VV0603 protein is typically expressed using prokaryotic expression systems, most commonly E. coli, similar to other Vibrio vulnificus recombinant proteins. The expression process typically involves:

• Amplification of the VV0603 gene from Vibrio vulnificus genomic DNA using specifically designed primers that incorporate restriction enzyme sites

• Cloning of the amplified gene into an expression vector containing an appropriate promoter and affinity tag (commonly His-tag)

• Transformation of the recombinant plasmid into a compatible E. coli expression strain

• Induction of protein expression using IPTG or other suitable inducers

• Cell lysis and purification using affinity chromatography, typically Ni-NTA for His-tagged proteins

This expression methodology is similar to that used for other Vibrio vulnificus proteins, such as the approach used for OmpU where specific primers were designed to amplify the gene with appropriate restriction sites for subsequent cloning .

What are optimal storage conditions for recombinant VV0603 protein?

Based on established protocols for similar Vibrio vulnificus recombinant proteins, the optimal storage conditions for VV0603 would include:

• Long-term storage: -20°C/-80°C in aliquots to avoid repeated freeze-thaw cycles

• Storage buffer: Tris/PBS-based buffer with stabilizers such as glycerol (typically 5-50%) and possibly trehalose (~6%)

• pH: Typically maintained around pH 8.0 for optimal stability

• Reconstitution: Deionized sterile water to a concentration of 0.1-1.0 mg/mL

Researchers should avoid repeated freeze-thaw cycles as this can lead to protein degradation and loss of activity. Working aliquots can be stored at 4°C for up to one week, but long-term storage requires -20°C or preferably -80°C temperatures .

What quality control methods should be used to verify recombinant VV0603 purity?

Quality control of recombinant VV0603 should involve multiple analytical techniques:

• SDS-PAGE analysis to confirm protein size and purity (aim for >90% purity)

• Western blot analysis using anti-His antibodies (if His-tagged) to confirm identity

• Mass spectrometry for accurate molecular weight determination and potential post-translational modifications

• Circular dichroism spectroscopy to assess proper protein folding

• Size exclusion chromatography to evaluate protein homogeneity and aggregation state

These methods collectively ensure that the purified protein meets the necessary quality standards for subsequent research applications. For Vibrio vulnificus proteins, SDS-PAGE analysis with a purity threshold of at least 90% is typically considered acceptable for research purposes .

What role might VV0603 play in Vibrio vulnificus pathogenicity compared to characterized virulence factors?

The potential role of VV0603 in Vibrio vulnificus pathogenicity should be examined in the context of known virulence factors. Vibrio vulnificus VvpM has been identified as a virulence factor that induces IL-1β production coupled with necrotic cell death in macrophages, promoting bacterial colonization. VvpM activates the NF-κB pathway and NLRP3 inflammasome through distinct mechanisms involving ANXA2 in different membrane compartments .

VV0603, as a UPF0102 family protein, may potentially:

• Function in bacterial cell envelope maintenance or stress response

• Participate in host-pathogen interactions, possibly through adhesion mechanisms

• Contribute to bacterial survival in host environments

• Mediate cellular responses similar to those observed with other Vibrio proteins

Experimental approaches to determine its role in pathogenicity would include:

• Generating knockout mutants (ΔVVO603) and assessing virulence in infection models

• Evaluating inflammatory marker production (IL-1β, TNF-α) in response to purified VV0603

• Examining host cell death mechanisms (necrosis, pyroptosis) triggered by VV0603

• Investigating potential interactions with host factors using pull-down assays

Comparative analysis with known virulence factors like VvpM would provide valuable insights into the potential pathogenic role of VV0603 .

What experimental models are most suitable for studying VV0603 function?

Based on research approaches used for other Vibrio vulnificus proteins, the following experimental models would be most suitable for studying VV0603 function:

In vitro models:

• Macrophage cell lines (RAW264.7, THP-1) to assess inflammatory responses

• Intestinal epithelial cell lines to evaluate adhesion and invasion

• Human dermal fibroblasts to study wound infection mechanisms

• Reconstituted membrane systems for membrane protein functional studies

Ex vivo models:

• Primary human or mouse macrophages

• Human blood for serum resistance assays

• Tissue explants for colonization studies

In vivo models:

• Mouse infection models (intraperitoneal, subcutaneous, or oral infection routes)

• Zebrafish embryo infection model for real-time visualization

• Invertebrate models (Galleria mellonella, Caenorhabditis elegans)

The choice of model should align with the specific research question about VV0603 function. For instance, if investigating inflammatory responses, macrophage models would be appropriate, as demonstrated in studies with VvpM where these cells were used to assess IL-1β production and cell death mechanisms .

How can researchers investigate potential protein-protein interactions involving VV0603?

Several complementary approaches can be employed to investigate protein-protein interactions involving VV0603:

In vitro techniques:

• Pull-down assays using tagged VV0603 as bait

• Surface plasmon resonance (SPR) for real-time interaction kinetics

• Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Microscale thermophoresis for quantifying binding affinity

• Cross-linking mass spectrometry to identify interaction interfaces

Cellular techniques:

• Bimolecular fluorescence complementation (BiFC)

• Förster resonance energy transfer (FRET)

• Proximity labeling approaches (BioID, APEX)

• Co-immunoprecipitation from bacterial or host cells

• Yeast two-hybrid screening

Computational approaches:

• Molecular docking simulations

• Protein-protein interaction prediction algorithms

• Structural modeling to identify potential interaction domains

When investigating host-pathogen interactions, researchers should consider examining interactions with host proteins like annexin A2 (ANXA2), which has been identified as a target for VvpM in lipid and non-lipid raft compartments .

What techniques can reveal the structure-function relationship of VV0603?

Understanding the structure-function relationship of VV0603 requires a multi-faceted approach:

Structural analysis techniques:

Functional mapping techniques:

• Site-directed mutagenesis of conserved residues

• Truncation analysis to identify functional domains

• Chimeric protein construction with related UPF0102 family members

• Phage display for epitope mapping

• Hydrogen-deuterium exchange mass spectrometry to identify ligand-binding sites

Computational approaches:

• Homology modeling based on related proteins

• Molecular dynamics simulations to study conformational changes

• Sequence conservation analysis across bacterial species

• Functional residue prediction through evolutionary analysis

For membrane-associated proteins like VV0603, incorporating membrane mimetics (nanodiscs, liposomes) in structural studies would provide more physiologically relevant insights into structure-function relationships .

What are recommended reconstitution protocols for lyophilized VV0603?

For optimal reconstitution of lyophilized VV0603 protein, researchers should follow these methodological steps:

• Briefly centrifuge the vial containing lyophilized protein before opening to ensure all material is at the bottom of the vial

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

• Gently mix by slow inversion rather than vortexing to prevent protein denaturation

• Allow the protein to fully dissolve by incubating at room temperature for 5-10 minutes

• Add glycerol to a final concentration of 5-50% (with 50% being optimal for long-term storage) to prevent freeze-thaw damage

• Prepare small working aliquots to avoid repeated freeze-thaw cycles

• Flash-freeze aliquots in liquid nitrogen before transferring to -80°C for long-term storage

This approach minimizes protein denaturation and aggregation while maximizing stability and activity for subsequent experiments. For working with the reconstituted protein, aliquots can be stored at 4°C for up to one week, but for periods longer than this, storage at -20°C/-80°C is necessary .

What strategies can optimize experimental design when studying VV0603 and host cell interactions?

When designing experiments to study VV0603 interactions with host cells, consider the following optimization strategies:

Experimental controls:

• Include protein buffer-only treatment to control for buffer effects

• Use heat-inactivated VV0603 as a negative control for structure-dependent functions

• Include a known Vibrio vulnificus protein (e.g., VvpM) as a positive control for host responses

• Incorporate an irrelevant recombinant protein with similar tag as specificity control

Dose and time considerations:

• Perform dose-response experiments (0.1-10 μg/mL range typically appropriate)

• Establish detailed time-course studies (early responses: 30 min-4 hrs; late responses: 6-72 hrs)

• Consider pulsed exposure vs. continuous exposure paradigms

Cell culture optimization:

• Test multiple cell types relevant to infection (macrophages, epithelial cells, fibroblasts)

• Standardize cell density and passage number to reduce variability

• Optimize serum conditions (serum-free vs. serum-containing) based on experimental goals

• Consider 3D culture systems for more physiologically relevant interactions

Analytical endpoints:

• Combine multiple readouts (e.g., cytokine production, cell death, signaling pathway activation)

• Include both early (NF-κB activation, ROS production) and late (IL-1β production) endpoints

• Consider single-cell approaches to account for population heterogeneity

For studying inflammatory responses, researchers should measure markers similar to those examined in VvpM studies, including IL-1β production, ROS generation, autophagy induction (LC3 puncta formation), and NLRP3 inflammasome activation .

How can researchers differentiate between direct and indirect effects of VV0603 on host cells?

Differentiating between direct and indirect effects of VV0603 on host cells requires carefully designed experiments:

Approaches to identify direct effects:

• Use purified recombinant protein at defined concentrations

• Employ fluorescently labeled VV0603 to track cellular localization

• Utilize protein immobilization techniques to restrict protein to cell surface

• Perform binding assays with isolated cell membrane fractions

• Use cell-free systems to reconstitute potential signaling pathways

Strategies to distinguish indirect effects:

• Apply specific inhibitors of key signaling molecules to block potential mediators

• Use genetically modified cells lacking specific receptors or signaling components

• Perform conditioned media experiments to identify secreted mediators

• Employ time-course studies to establish sequential events

• Use single-cell approaches to identify heterogeneous responses within cell populations

Analytical techniques:

• Cell fractionation followed by Western blotting to determine subcellular localization

• Proximity labeling to identify direct interaction partners

• Real-time biosensors to monitor immediate cellular responses

• Cytokine neutralization experiments to block potential paracrine effects

• Transcriptomics to distinguish primary response genes from secondary response genes

This methodological approach has been successfully applied to VvpM, where researchers identified direct ANXA2 binding and subsequent differential effects on lipid raft and non-lipid raft compartments, leading to both ROS production and autophagy induction .

What special considerations apply when expressing membrane-associated proteins like VV0603?

Expressing membrane-associated proteins from Vibrio vulnificus requires specialized approaches:

Expression system selection:

• E. coli strains optimized for membrane protein expression (C41(DE3), C43(DE3), Lemo21)

• Cell-free expression systems with membrane mimetics

• Eukaryotic expression systems for complex membrane proteins

• Consideration of codon optimization for the expression host

Vector design considerations:

• Inclusion of appropriate signal sequences if applicable

• Selection of solubilizing fusion partners (MBP, SUMO, Mistic)

• Carefully positioned affinity tags to avoid interference with membrane insertion

• Inducible promoters with fine control over expression levels

Expression conditions:

• Lower induction temperatures (16-20°C) to slow folding and prevent aggregation

• Reduced inducer concentrations to prevent overwhelming the membrane insertion machinery

• Addition of specific lipids to culture media to support proper folding

• Extended expression times with minimal inducer concentration

Extraction and purification strategies:

• Careful selection of detergents based on protein characteristics

• Detergent screening panel (mild to harsh) to optimize extraction

• Inclusion of stabilizers like glycerol and specific lipids in all buffers

• Consideration of native nanodiscs or styrene maleic acid lipid particles (SMALPs) for detergent-free extraction

For functional studies, reconstitution into liposomes or nanodiscs may be necessary to maintain native activity, particularly if VV0603 functions depend on the membrane environment .

How can VV0603 be utilized in studies of host immune responses to Vibrio vulnificus?

VV0603 can serve as a valuable tool in studying host immune responses to Vibrio vulnificus through several experimental applications:

Inflammatory response characterization:

• Measuring pro-inflammatory cytokine production (IL-1β, TNF-α, IL-6) in response to purified VV0603

• Assessing inflammasome activation by monitoring caspase-1 cleavage and pyroptosis

• Evaluating NF-κB pathway activation through reporter assays and phosphorylation studies

• Comparing responses in different immune cell populations (macrophages, dendritic cells, neutrophils)

Cell death mechanism analysis:

• Distinguishing between pyroptosis, necrosis, and apoptosis triggered by VV0603

• Measuring membrane permeabilization and inflammatory marker release

• Assessing cellular morphology changes through microscopy

• Evaluating the role of ROS production in cell death pathways

Specific protein marker analysis:
Table 1: Potential immune markers for assessment in VV0603 studies

Based on studies with VvpM, researchers should investigate whether VV0603 similarly recruits NOX enzymes coupled with ANXA2 to facilitate ROS production, potentially influencing epigenetic and transcriptional regulation of NF-κB in the IL-1β promoter .

What approaches can identify potential binding partners or receptors for VV0603?

To identify potential binding partners or receptors for VV0603, researchers should employ a comprehensive discovery workflow:

Unbiased screening approaches:

• Affinity purification-mass spectrometry (AP-MS) using tagged VV0603 as bait

• Yeast two-hybrid screening against host cDNA libraries

• Protein microarray screening against host proteome arrays

• Phage display selection against VV0603

• BioID or APEX2 proximity labeling in relevant host cells

Candidate-based approaches:

• Direct binding assays with predicted partners based on homology to known interactions

• Co-immunoprecipitation with suspected binding partners

• ELISA-based binding assays with recombinant proteins

• Surface plasmon resonance (SPR) with purified candidate receptors

• Liposome recruitment assays for membrane-associated interactions

Validation strategies:

• Knockdown/knockout of identified partners to confirm functional relevance

• Competition assays with blocking antibodies or peptides

• Mutagenesis of key binding residues in both VV0603 and potential partners

• Co-localization studies using fluorescence microscopy

• Functional assays to demonstrate biological significance of the interaction

From studies with other Vibrio proteins, membrane-associated proteins like annexin A2 (ANXA2) represent promising candidates for investigation, as these proteins can mediate interactions in both lipid raft and non-lipid raft compartments, potentially initiating distinct signaling pathways .

How can researchers assess the contribution of VV0603 to bacterial fitness and virulence?

Assessing VV0603's contribution to bacterial fitness and virulence requires a multifaceted experimental approach:

Genetic manipulation strategies:

• Generation of clean deletion mutants (ΔVV0603) using allelic exchange

• Complementation studies using plasmid-based expression systems

• Construction of point mutants to identify critical functional residues

• Conditional expression systems to study essentiality

• Reporter fusions to study expression patterns under different conditions

In vitro phenotypic characterization:

• Growth kinetics under various stress conditions (pH, temperature, osmolarity)

• Biofilm formation assays

• Motility assessments (swimming, swarming)

• Antibiotic susceptibility testing

• Competitive growth assays with wild-type strains

Virulence assessment:

• Cell culture infection models measuring adhesion, invasion, and cytotoxicity

• Ex vivo survival in human serum or whole blood

• Mouse infection models with various routes (oral, intraperitoneal, wound)

• Measurement of bacterial burden in tissues

• Survival curve analysis in animal models

Molecular mechanisms:

• Transcriptomic analysis comparing wild-type and mutant strains

• Proteomic profiling to identify compensatory changes

• Metabolomic analysis to identify altered metabolic pathways

• Secretome analysis to identify altered protein secretion

This approach mirrors successful strategies used to characterize other Vibrio virulence factors, such as VvpM, where mutation of the vvpM gene demonstrated its role in IL-1β production during infection and bacterial colonization in animal models .

What are the challenges in translating in vitro findings with VV0603 to in vivo infection models?

Translating in vitro findings with VV0603 to in vivo infection models presents several methodological challenges:

Physiological relevance challenges:

• Protein concentration discrepancies between in vitro studies and in vivo expression levels

• Timing differences between acute protein exposure and progressive infection

• Microenvironment complexity not replicated in cell culture systems

• Host factor interactions that may modify protein activity in vivo

• Tissue-specific responses that vary from cell culture models

Technical challenges:

Experimental design considerations:

• Use multiple animal models to address species-specific differences

• Employ tissue-specific conditional knockouts to isolate effects

• Develop reporter systems to track protein expression in vivo

• Utilize ex vivo organ culture systems as intermediate complexity models

• Consider multiple infection routes to capture different disease manifestations

Validation approaches:

• Correlate findings with clinical samples from human infections

• Use complementary in vivo approaches (genetic deletion, protein administration)

• Employ systems biology approaches to integrate multi-level data

• Develop mathematical models to predict in vivo outcomes based on in vitro parameters

These challenges and approaches reflect the complexity of studying bacterial proteins in the context of infection, as demonstrated in studies with VvpM, where both in vitro mechanistic studies and in vivo mouse infection models were required to fully characterize its pathogenic role .

What emerging technologies could advance understanding of VV0603 function?

Several cutting-edge technologies show promise for advancing VV0603 research:

Advanced structural biology approaches:

• Cryo-electron tomography for visualizing protein localization in near-native contexts

• Microcrystal electron diffraction (MicroED) for structural determination with minimal material

• Integrative structural biology combining multiple data types (NMR, SAXS, crosslinking MS)

• AlphaFold2 and related AI tools for predictive structural modeling

• Time-resolved structural methods to capture conformational changes

Single-cell technologies:

• Single-cell transcriptomics to identify heterogeneous responses to VV0603

• Mass cytometry (CyTOF) for high-dimensional analysis of cellular responses

• Live-cell biosensors to monitor real-time signaling dynamics

• Digital spatial profiling for in situ protein localization and pathway activation

• Microfluidic cell culture systems for controlled exposure studies

Genome-wide screening approaches:

• CRISPR-Cas9 screens to identify host factors involved in VV0603 responses

• Bacterial transposon sequencing (Tn-Seq) to identify genetic interactions

• CRISPR interference/activation (CRISPRi/a) for functional genomics

• Genome-wide association studies in clinical isolates to correlate VV0603 variants with virulence

• Saturation mutagenesis to comprehensively map functional domains

Advanced imaging methods:

• Super-resolution microscopy for nanoscale localization

• Expansion microscopy for improved spatial resolution

• Label-free imaging techniques to study native proteins

• Correlative light and electron microscopy for structural context

• Intravital microscopy for in vivo visualization

These technologies could reveal unprecedented insights into VV0603 function, similar to how detailed mechanistic studies elucidated the dual pathways of VvpM in stimulating NF-κB-dependent IL-1β production and autophagy-mediated NLRP3 inflammasome activation .

How might comparative analysis of VV0603 with homologs from other bacterial species inform function?

Comparative analysis of VV0603 with homologs from other bacterial species represents a powerful approach to infer function:

Phylogenetic analysis approaches:

• Construction of comprehensive phylogenetic trees of UPF0102 family proteins

• Identification of conserved domains across bacterial species

• Analysis of co-evolution with other bacterial proteins

• Evaluation of selection pressure on specific residues

• Mapping of sequence conservation onto predicted structural models

Functional conservation assessment:

• Complementation studies using homologs from different species

• Comparison of phenotypes in respective knockout mutants

• Domain-swapping experiments to identify functional regions

• Cross-species binding studies with potential interaction partners

• Parallel characterization of biochemical activities

Genomic context analysis:

• Examination of gene neighborhood conservation across species

• Analysis of co-occurrence patterns with other genes

• Investigation of operon structures containing UPF0102 family genes

• Correlation with species-specific pathogenicity mechanisms

• Identification of regulatory elements controlling expression

This comparative approach can be particularly informative for UPF (Uncharacterized Protein Family) members, where direct functional information may be limited. The approach aligns with successful strategies used to characterize other bacterial virulence factors, establishing evolutionary relationships that inform functional predictions .