Recombinant Vibrio vulnificus 33 kDa chaperonin (hslO)

What is the predicted sequence and structure of Vibrio vulnificus 33 kDa chaperonin?

While the exact sequence of V. vulnificus hslO is not provided in the search results, we can draw insights from the related Clostridium botulinum 33 kDa chaperonin. The C. botulinum chaperonin (hslO) is a full-length protein consisting of 296 amino acids . The sequence alignment between V. vulnificus and C. botulinum hslO would likely reveal conserved functional domains essential for chaperone activity.

Researchers should:

• Perform comparative sequence analysis with other bacterial hslO proteins

• Analyze the protein for conserved domains typical of the HSP33 family

• Conduct structure prediction using tools like AlphaFold or homology modeling

• Examine the sequence for redox-sensitive motifs that might regulate activity

How does V. vulnificus hslO expression change under different stress conditions?

Based on research with other V. vulnificus virulence factors, expression of stress response proteins is likely regulated by environmental conditions. The stress sigma factor RpoS has been shown to regulate gene expression during host interactions in V. vulnificus . To study hslO expression:

• Design real-time PCR (qPCR) primers specific to the hslO gene

• Normalize expression to a housekeeping gene like recA (as used in V. vulnificus studies)

• Test expression under various conditions:

  • Standard laboratory media (control)

  • Heat shock (42°C for 30 minutes)

  • Oxidative stress (H₂O₂ exposure)

  • Host cell contact or host cell lysate media

  • Serum exposure

The protocol should follow established qPCR methodology as demonstrated in V. vulnificus research, where researchers measured expression of virulence genes using the ΔΔCt method .

What experimental controls are essential when working with recombinant V. vulnificus hslO?

When conducting experiments with recombinant V. vulnificus 33 kDa chaperonin, the following controls are critical:

• Expression controls:

  • Empty vector control to assess background

  • Known chaperonin (e.g., E. coli DnaK or GroEL) as positive control

  • Inactive mutant (e.g., cysteine-to-alanine substitution) for activity studies

• Purification controls:

  • SDS-PAGE analysis to confirm purity (target >85% as established for similar recombinant proteins)

  • Western blot verification with anti-His or specific antibodies

  • Mass spectrometry to confirm molecular identity

• Activity assays:

  • Heat-denatured hslO as negative control

  • Substrate-only and chaperone-only controls

  • ATP-depleted conditions to assess ATP-dependence

• Storage stability:

  • Fresh vs. stored protein activity comparison

  • Different storage conditions evaluation (similar to protocol for C. botulinum hslO)

What is the optimal expression system for producing high-quality recombinant V. vulnificus hslO?

Based on protocols used for similar bacterial chaperonins, researchers should consider:

• Expression systems comparison:

• Expression parameters:

  • Induction conditions (temperature, inducer concentration)

  • Cell lysis methods (sonication vs. chemical lysis)

  • Codon optimization for expression host

• Tag selection:

  • His-tag for IMAC purification

  • GST or MBP for improved solubility

  • TEV or other protease cleavage sites for tag removal

The expression system should be selected based on downstream applications and required protein quality .

What purification protocol yields the highest functional activity of V. vulnificus hslO?

A multi-step purification approach is recommended for obtaining high-purity, functional V. vulnificus 33 kDa chaperonin:

• Initial capture:

  • Affinity chromatography (Ni-NTA for His-tagged protein)

  • Buffer composition: 50 mM phosphate buffer, pH 7.4, 300 mM NaCl, 10-20 mM imidazole

• Intermediate purification:

  • Ion exchange chromatography based on theoretical pI

  • Size exclusion chromatography to remove aggregates

• Quality assessment:

  • SDS-PAGE to confirm >85% purity

  • Dynamic light scattering to assess homogeneity

  • Circular dichroism to confirm proper folding

• Activity preservation:

  • Add reducing agents if cysteine residues are present

  • Consider adding glycerol (5-50%) for stability

  • Aliquot and flash-freeze to prevent freeze-thaw cycles

• Storage recommendations:

  • For liquid form: -20°C to -80°C (6-month shelf life)

  • For lyophilized form: -20°C to -80°C (12-month shelf life)

  • Working aliquots at 4°C for up to one week

How can researchers troubleshoot poor expression or insolubility of V. vulnificus hslO?

When encountering expression or solubility issues:

• Expression troubleshooting:

  • Optimize codon usage for expression host

  • Test different promoter strengths

  • Vary induction parameters (temperature, inducer concentration, time)

  • Try autoinduction media

• Solubility enhancement strategies:

  • Reduce expression temperature (16-20°C)

  • Co-express with chaperones (GroEL/ES, DnaK/J/GrpE)

  • Try solubility-enhancing fusion tags (MBP, SUMO)

  • Optimize lysis buffer composition:

    • Add mild detergents (0.1% Triton X-100)

    • Include stabilizing agents (glycerol, arginine)

    • Test different salt concentrations

• Refolding approaches if inclusion bodies form:

  • Solubilize in 8M urea or 6M guanidine-HCl

  • Perform step-wise dialysis

  • Use pulse refolding with redox buffers

• Analytical techniques to identify issues:

  • Western blotting to confirm expression

  • Analytical SEC to assess aggregation state

  • Limited proteolysis to identify stable domains

What assays can reliably measure the chaperone activity of V. vulnificus hslO?

To assess the functional activity of recombinant V. vulnificus 33 kDa chaperonin:

• Protein aggregation prevention assays:

  • Monitor thermal aggregation of model substrates (citrate synthase, luciferase)

  • Measure light scattering at 320-360 nm over time at elevated temperatures

  • Calculate percent protection compared to substrate-only controls

• Protein refolding assays:

  • Measure recovery of enzymatic activity for denatured substrates

  • Compare refolding kinetics with and without hslO

  • Assess concentration dependence of chaperone activity

• ATP hydrolysis measurement:

  • If ATP-dependent, use malachite green or other phosphate detection assays

  • Correlate ATPase activity with chaperone function

• Redox sensitivity assessment:

  • Compare activity under reducing and oxidizing conditions

  • Test activation by specific oxidants (H₂O₂, HOCl)

  • Measure conformational changes using intrinsic tryptophan fluorescence

• Data presentation:

How can researchers investigate the interaction between V. vulnificus hslO and substrate proteins?

To study hslO-substrate interactions:

• Physical interaction methods:

  • Co-immunoprecipitation with anti-hslO antibodies

  • Pull-down assays using tagged hslO

  • Surface plasmon resonance for binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

• Crosslinking approaches:

  • Chemical crosslinking with BS3 or formaldehyde

  • Photo-crosslinking with modified amino acids

  • Mass spectrometry analysis of crosslinked complexes

• Identification of natural substrates:

  • Immunoprecipitation followed by mass spectrometry

  • Comparative proteomics of wild-type vs. hslO mutant under stress

  • Affinity purification of stress-denatured proteins

• Structural analysis of complexes:

  • Cryo-electron microscopy

  • X-ray crystallography of co-crystals

  • NMR for mapping interaction interfaces

What is the relationship between V. vulnificus hslO and other stress-response mechanisms?

To investigate how hslO integrates with broader stress response networks:

• Transcriptional regulation studies:

  • Analyze if hslO is regulated by RpoS, similar to virulence factors in V. vulnificus

  • Perform qPCR to measure co-regulation with other stress genes

  • Use reporter fusions to monitor expression under different stresses

• Functional interaction analysis:

  • Create single and double mutants (hslO/rpoS) to assess genetic interactions

  • Compare phenotypes under various stress conditions

  • Test for compensatory mechanisms when hslO is deleted

• Stress response network:

  • Construct strain with rpoS mutation to test effect on hslO expression

  • Determine if hslO complements other chaperone mutants

  • Assess overlapping substrate specificity with other chaperones

• Experimental approach for RpoS regulation study:

  • Generate promoter-reporter fusion constructs

  • Compare expression in wild-type and rpoS mutant strains

  • Use methods similar to those demonstrated for rtxA1 gene regulation in V. vulnificus

How does V. vulnificus hslO contribute to bacterial virulence and host adaptation?

While specific data on hslO's role in V. vulnificus pathogenesis is not provided in the search results, a systematic approach to investigate this question would include:

• Genetic approaches:

  • Generate hslO deletion mutant in V. vulnificus

  • Create complemented strain expressing hslO from plasmid

  • Develop regulatable expression system to modulate hslO levels

• Virulence phenotype assessment:

  • Measure cytotoxicity using LDH release assay (as used for rpoS studies)

  • Observe morphological changes in host cells (cell rounding, shrinkage, blebbing)

  • Quantify survival in serum, phagocytes, and stress conditions

• In vivo virulence:

  • Compare wild-type and hslO mutant in infection models

  • Measure bacterial load in tissues

  • Assess survival rates and inflammatory responses

• Mechanisms of contribution:

  • Identify virulence factors that might be hslO substrates

  • Test if hslO is required for RtxA1 toxin function

  • Investigate role in stress adaptation during host transition

What methods can be used to study V. vulnificus hslO expression during infection?

To monitor hslO expression during host interaction:

• In vitro models:

  • Culture bacteria in host cell lysate media (as used for V. vulnificus gene expression studies)

  • Expose to serum-free DMEM to simulate host environment

  • Co-culture with host cells with physical separation (transwell system)

• Expression analysis approaches:

  • Real-time PCR with specific primers for hslO

  • Normalize to recA as established for V. vulnificus studies

  • Calculate relative expression using the ΔΔCt method

• Reporter systems:

  • Construct hslO promoter-GFP fusion

  • Use flow cytometry to measure expression at single-cell level

  • Image bacteria during cell infection to visualize expression

• Environmental triggers:

  • Test expression at different temperatures (37°C vs. environmental temperature)

  • Evaluate response to host iron limitation

  • Assess impact of innate immune factors (antimicrobial peptides, reactive oxygen species)

Based on V. vulnificus research, expression analysis during host contact should include appropriate time points (30, 60, 90, 120, 180 minutes) to capture dynamic changes .

How can researchers determine if V. vulnificus hslO interacts with host cellular components?

To investigate potential hslO-host interactions:

• Binding studies:

  • Express and purify recombinant hslO

  • Perform binding assays with host cell lysates

  • Identify binding partners by pull-down and mass spectrometry

• Immunological approaches:

  • Test if hslO is recognized by host pattern recognition receptors

  • Measure host cytokine responses to purified hslO

  • Assess antibody development against hslO during infection

• Cellular localization:

  • Use fluorescently tagged hslO to track during infection

  • Perform fractionation of infected cells to locate bacterial proteins

  • Use immunostaining to visualize hslO during cell infection

• Host cell effects:

  • Evaluate impact of purified hslO on host cell morphology

  • Measure changes in host cell signaling pathways

  • Assess effects on host cell stress response systems

• Comparative analysis:

  • Compare effects to those of known virulence factors like RtxA1

  • Determine if effects are dependent on chaperone activity

How can structural biology approaches advance understanding of V. vulnificus hslO function?

To elucidate the structure-function relationship of V. vulnificus 33 kDa chaperonin:

• Structure determination strategies:

  • X-ray crystallography of purified hslO

  • Cryo-electron microscopy for larger complexes

  • NMR for dynamic regions and ligand interactions

• Structural features to analyze:

  • Redox-sensing domain structure

  • Substrate binding sites

  • Conformational changes upon activation

  • Oligomerization interfaces

• Mutational analysis guided by structure:

  • Site-directed mutagenesis of key residues

  • Creation of chimeric proteins with other HSP33 homologs

  • Structure-based design of activity-modulating mutations

• Computational approaches:

  • Molecular dynamics simulations

  • Docking studies with potential substrates

  • Comparison with C. botulinum hslO structure

• Structure-guided functional studies:

  • Design substrate-trapping mutants

  • Create conformation-specific antibodies

  • Develop small molecule modulators of activity

What genomic approaches can reveal the evolution and conservation of hslO in Vibrio species?

To explore hslO evolution and conservation:

• Comparative genomic analysis:

  • Analyze V. vulnificus genome structure (two chromosomes)

  • Determine if hslO is located in core genome or variable regions

  • Compare with hslO in other Vibrio species

• Evolutionary studies:

  • Construct phylogenetic trees of hslO across bacterial species

  • Calculate selection pressure (dN/dS ratios) on different protein domains

  • Identify horizontally transferred regions

• Population genomics:

  • Examine hslO sequence variation among V. vulnificus clinical isolates

  • Compare environmental vs. clinical strain hslO sequences

  • Identify potential adaptive mutations

• Genomic context analysis:

  • Determine if hslO is part of the super-integron (SI) identified in V. vulnificus

  • Analyze surrounding genes for co-regulation patterns

  • Identify potential mobile genetic elements

• Correlation with pathogenicity:

  • Compare hslO sequences between pathogenic and non-pathogenic Vibrio strains

  • Identify sequence variants associated with virulence

  • Analyze regulatory elements for host-responsive elements

How can high-throughput approaches identify the complete substrate repertoire of V. vulnificus hslO?

To comprehensively identify hslO substrates:

• Proteome-wide interaction screening:

  • Protein microarray with purified hslO

  • Bacterial two-hybrid or split-reporter systems

  • Ribosome display with randomized peptide libraries

• Mass spectrometry-based approaches:

  • Stable Isotope Labeling with Amino acids in Cell culture (SILAC)

  • Thermal proteome profiling to identify stabilized proteins

  • Crosslinking mass spectrometry to capture transient interactions

• In vivo proximity labeling:

  • hslO-BioID fusion expression in V. vulnificus

  • APEX2-hslO for temporal mapping of interactions

  • Compare substrate profiles under different stress conditions

• Bioinformatic prediction of substrates:

  • Develop sequence/structure motifs from known substrates

  • Machine learning approaches trained on verified interactions

  • Network analysis to predict functional interactions

• Validation strategies:

  • Direct binding assays with candidate substrates

  • In vitro aggregation protection assays

  • In vivo co-expression studies

What is the potential of V. vulnificus hslO as a target for antimicrobial development?

To evaluate hslO as a therapeutic target:

• Target validation:

  • Determine essentiality under infection-relevant conditions

  • Compare hslO sequence between bacterial and human homologs

  • Assess impact of hslO inhibition on virulence (similar to rpoS studies)

• Screening approaches:

  • Develop high-throughput activity assays

  • Screen for inhibitors of chaperone function

  • Identify compounds that lock hslO in inactive conformation

• Rational drug design:

  • Structure-based design targeting unique features

  • Fragment-based screening for binding site identification

  • Peptide inhibitors based on substrate binding motifs

• Therapeutic potential assessment:

  • Test candidate inhibitors in V. vulnificus infection models

  • Evaluate combination therapy with conventional antibiotics

  • Assess potential for resistance development

• Advantages as antimicrobial target:

  • Potential to reduce virulence without selection pressure of bactericidal agents

  • Possible broad-spectrum activity against multiple Vibrio pathogens

  • Novel mechanism compared to conventional antibiotics