Recombinant Vibrio vulnificus ATP-dependent Clp protease adapter protein ClpS (clpS)

What is the basic structure of Vibrio vulnificus ClpS protein?

ClpS from V. vulnificus is a small alpha/beta protein that consists of three alpha-helices connected to three antiparallel beta-strands. The protein has a globular shape with a curved layer of antiparallel alpha-helices positioned over a twisted antiparallel beta-sheet. The protein contains a short extended N-terminal region (NTE) followed by a central seven-residue beta-strand, which is flanked by two other beta-strands in a small beta-sheet . This structure is highly conserved across bacterial species, indicating its evolutionary importance in protein quality control systems.

What is the primary function of ClpS in Vibrio vulnificus?

ClpS functions as an adaptor protein that regulates the activity of the ClpAP protease complex. It serves as an N-recognin in the N-end rule pathway, recognizing and binding to protein substrates with bulky hydrophobic residues (leucine, phenylalanine, tyrosine, and tryptophan) at their N-terminus . This interaction facilitates substrate selection for the ClpAP protease complex. Additionally, ClpS directly influences the ClpAP machine by binding to the N-terminal domain of the chaperone ClpA, which modifies its substrate specificity and can inhibit the degradation of specific substrates like SsrA-tagged proteins .

What are the optimal storage conditions for recombinant V. vulnificus ClpS protein?

Recombinant V. vulnificus ClpS protein stability depends on several factors including storage state, buffer ingredients, and temperature. For liquid formulations, the recommended shelf life is approximately 6 months when stored at -20°C/-80°C. Lyophilized forms maintain stability for up to 12 months at -20°C/-80°C . To maintain protein integrity, repeated freeze-thaw cycles should be avoided. For short-term use, working aliquots can be stored at 4°C for up to one week. The addition of 5-50% glycerol (with 50% being the standard recommendation) to the reconstituted protein is advised for long-term storage to prevent degradation .

How can I effectively reconstitute recombinant ClpS for experimental use?

For optimal reconstitution of recombinant ClpS:

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

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

• Add glycerol to a final concentration of 5-50% (50% is typically recommended)

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

• Store aliquots at -20°C/-80°C for long-term stability

This procedure helps maintain protein integrity and enzymatic activity for subsequent experiments.

What methods are most effective for studying ClpS-substrate interactions?

Several methodological approaches have proven effective for studying ClpS-substrate interactions:

• Bacterial Two-Hybrid (BACTH) assays: These have been successfully employed to identify protein-protein interactions between ClpS and its binding partners, similar to the approach used for studying stressosome protein interactions in V. vulnificus .

• Immunoprecipitation experiments: These can be performed with protein extracts to verify ClpS interactions with substrate proteins and binding partners in vivo .

• Fluorescence anisotropy assays: These are useful for measuring the binding kinetics and affinity of ClpS for different substrates, as demonstrated with ClpS-ClpA interactions .

• Single-molecule techniques: These can be employed to monitor ClpS-mediated substrate delivery to the ClpAP complex in real-time .

• ATPase activity assays: These can be used to measure how ClpS affects the ATPase activity of ClpA during substrate processing .

How does ClpS contribute to the virulence mechanisms of Vibrio vulnificus?

The role of ClpS in V. vulnificus virulence is multifaceted and involves several mechanisms:

• Protein quality control: ClpS, as part of the protein degradation machinery, helps the pathogen maintain proteostasis during stress conditions encountered during infection .

• Stress response regulation: Similar to other regulatory proteins in V. vulnificus (like the stressosome complex), ClpS may contribute to the bacterial stress response during host invasion and environmental transitions .

• Virulence factor regulation: ClpS likely influences the stability and turnover of specific virulence factors through selective protein degradation, although direct evidence linking ClpS to specific V. vulnificus virulence factors remains an active area of research .

• Adaptation to host environments: The protein degradation pathway involving ClpS may help V. vulnificus adapt to changing environments within the host, particularly during the transition from environmental to host conditions .

Comprehensive studies directly connecting ClpS to specific virulence mechanisms in V. vulnificus are still emerging, presenting opportunities for further research.

Is there variation in ClpS structure or function across different V. vulnificus biotypes?

Research indicates potential variation in ClpS structure and function across different V. vulnificus biotypes:

While specific ClpS sequence variations across biotypes have not been extensively characterized, recent comparative genomic analyses suggest that variation in protein degradation machinery, including ClpS-associated pathways, may contribute to differential virulence among strains .

How does the intrinsically disordered N-terminal extension (NTE) of ClpS regulate ClpAP function?

The intrinsically disordered N-terminal extension (NTE) of ClpS plays a crucial regulatory role in ClpAP function through several mechanisms:

• Inhibition of degradation steps: Engagement of the NTE of ClpS by ClpA is both necessary and sufficient to inhibit multiple steps of ClpAP-catalyzed degradation .

• "Degron mimic" function: The NTE acts as a "degron mimic," competing with potential substrates for binding to the ClpA recognition sites .

• Cooperative inhibition: Multiple NTEs from different ClpS molecules are needed to efficiently inhibit degradation, suggesting a cooperative mechanism of regulation .

• ATPase activity modulation: The NTE partially represses ATP hydrolysis by ClpAP, which correlates with its ability to suppress the maximal rate of degradation .

• Substrate selectivity: The NTE helps modulate substrate choice, promoting recognition of N-end rule substrates while suppressing recognition of other substrates like SsrA-tagged proteins .

Research has demonstrated that ClpS mutants lacking the NTE fail to properly regulate ClpAP activity, highlighting the critical importance of this seemingly unstructured region in protein degradation control .

What are the challenges in expressing and purifying functional recombinant V. vulnificus ClpS?

Researchers face several challenges when expressing and purifying functional recombinant V. vulnificus ClpS:

• Protein solubility: The relatively small size and specific folding requirements of ClpS can lead to aggregation or insolubility during heterologous expression.

• Maintaining native structure: Ensuring that the recombinant protein maintains its native conformation, particularly the intrinsically disordered N-terminal extension which is critical for function .

• Expression system selection: Different expression systems (E. coli vs. mammalian cells) yield varying results. While E. coli systems can produce higher yields, mammalian cell expression may better preserve certain post-translational modifications .

• Purification strategy: The purification process must preserve both the structural integrity and functional capacity of ClpS, often requiring optimized buffer conditions.

• Functional validation: Confirming that the purified recombinant ClpS retains its biological activity through binding and functional assays with ClpA and model substrates.

Researchers have addressed these challenges through various approaches, including the use of solubility tags, optimized buffer conditions, and activity-based validation assays to ensure the recombinant protein faithfully represents the native ClpS .

How can ClpS be used as a target for developing anti-Vibrio strategies?

ClpS presents several potential avenues for developing anti-Vibrio strategies:

• Inhibition of protein quality control: Targeting ClpS function could disrupt protein homeostasis in V. vulnificus, potentially reducing bacterial survival under stress conditions encountered during infection .

• Disruption of virulence factor regulation: If ClpS regulates specific virulence factors, inhibitors could potentially attenuate bacterial virulence without directly affecting viability, potentially reducing selective pressure for resistance .

• Targeting the ClpS-ClpA interface: Small molecule inhibitors that disrupt the interaction between ClpS and ClpA could interfere with proper protein degradation pathways .

• NTE-targeted approaches: Given the critical role of the N-terminal extension in regulating ClpAP function, peptide mimetics or small molecules that compete with the NTE for ClpA binding could disrupt normal proteostasis .

• Combination approaches: ClpS inhibitors could potentially sensitize V. vulnificus to existing antibiotics by compromising bacterial stress responses and adaptation mechanisms .

This represents a promising area for further research, particularly as traditional antibiotic approaches face increasing challenges from resistance development.

What are the current methods for detecting ClpS expression in V. vulnificus samples?

Several methods have been developed and optimized for detecting ClpS expression in V. vulnificus samples:

• Immunoblotting/Western blotting: Using specific antibodies against V. vulnificus ClpS to detect protein expression levels in bacterial lysates .

• Quantitative PCR (qPCR): Primers targeting the clpS gene can be used to quantify expression at the transcriptional level. This approach has been adapted from detection methods used for other V. vulnificus genes .

• Real-time Recombinase Polymerase Amplification (RPA): This isothermal amplification method offers rapid detection of specific genes and could be adapted for clpS detection, similar to approaches used for other V. vulnificus genes like empV .

• Proteomics approaches: Mass spectrometry-based proteomics can identify and quantify ClpS in complex protein mixtures from bacterial samples .

• Reporter gene fusions: Genetic constructs fusing the clpS promoter to reporter genes like GFP or luciferase can be used to monitor expression patterns in live cells under different conditions.

These methods vary in sensitivity, specificity, and technical requirements, with selection dependent on the specific research question and available resources.

How can researchers differentiate between active and inactive forms of V. vulnificus ClpS?

Differentiating between active and inactive forms of V. vulnificus ClpS requires specific approaches:

• Functional binding assays: Using fluorescence anisotropy to measure binding of ClpS to model substrates or ClpA can indicate functional status .

• ATPase inhibition assays: Since functional ClpS affects ClpA's ATPase activity, measuring ATP hydrolysis rates in the presence of purified ClpS can indicate its functional state .

• Protein degradation assays: Monitoring the ability of ClpS to modulate degradation of model substrates (both promotion of N-end rule substrates and inhibition of SsrA-tagged substrates) can reveal its functional status .

• Structural analysis: Techniques like circular dichroism spectroscopy can detect changes in secondary structure that might indicate loss of functional conformation.

• NTE engagement assessment: Since the N-terminal extension is critical for function, assays specifically designed to monitor NTE engagement with ClpA can differentiate between functional and non-functional forms .

These approaches can be particularly important when evaluating recombinant ClpS preparations or when studying the effects of potential inhibitors on ClpS function.

Can ClpS be used as a marker to distinguish between different V. vulnificus strains?

The potential of ClpS as a marker for distinguishing V. vulnificus strains presents both opportunities and limitations:

Opportunities:

• Genomic sequence variations in the clpS gene may correlate with specific strain lineages

• Differences in ClpS expression patterns under specific conditions could potentially distinguish clinical from environmental isolates

• Proteomic signatures including ClpS and its interacting partners might provide strain-specific profiles

Limitations:

• Current typing methods utilize other markers such as vcg gene variations, 16S rRNA sequences, and genome-wide SNP analyses

• The high conservation of essential proteins like ClpS across strains may limit discriminatory power

• More comprehensive comparative studies are needed to establish ClpS as a reliable strain marker

While ClpS alone may not provide sufficient discriminatory power for strain typing, it could potentially be included in a multi-marker approach alongside established typing methods such as MLST or core-SNP phylogenetic analysis .

What is the relationship between ClpS function and V. vulnificus survival during stress conditions?

The relationship between ClpS function and V. vulnificus survival during stress conditions is complex and involves several mechanisms:

• Protein quality control: As part of the proteostasis network, ClpS helps maintain protein quality control during stress by directing specific damaged or misfolded proteins to the ClpAP degradation machinery .

• Adaptation to environmental transitions: V. vulnificus encounters various stressors when transitioning between environments (e.g., from seawater to human host). ClpS likely facilitates adaptation by regulating protein turnover during these transitions .

• Viable but non-culturable (VBNC) state regulation: V. vulnificus can enter a VBNC state during stress. While direct evidence linking ClpS to this process is limited, protein degradation systems are generally important for entry into and exit from dormancy states .

• Oxygen stress response: Similar to other regulatory systems like the stressosome that functions as an oxygen sensor in V. vulnificus, ClpS may participate in responses to oxygen availability changes .

• Nutritional stress adaptation: ClpS-mediated protein degradation likely contributes to amino acid recycling during nutrient limitation, a common stress condition for V. vulnificus .

Understanding these relationships more fully requires additional research specifically examining ClpS function under relevant stress conditions encountered by V. vulnificus during its lifecycle.

What are promising approaches for studying ClpS-substrate interactions in V. vulnificus?

Several innovative approaches show promise for advancing our understanding of ClpS-substrate interactions in V. vulnificus:

• Proteome-wide identification of substrates: Using techniques like Stable Isotope Labeling with Amino acids in Cell culture (SILAC) or Tandem Mass Tag (TMT) labeling coupled with mass spectrometry to identify proteins that accumulate in ClpS deletion mutants .

• In vivo crosslinking approaches: Chemical crosslinking combined with mass spectrometry (XL-MS) can capture transient interactions between ClpS and its substrates in living V. vulnificus cells.

• BioID or APEX2 proximity labeling: Fusing promiscuous biotin ligases to ClpS can help identify proteins that come into close proximity with ClpS in vivo.

• Cryo-electron microscopy studies: Structural determination of V. vulnificus ClpS in complex with substrates and/or the ClpA hexamer using cryo-EM could provide molecular insights into recognition and delivery mechanisms.

• Single-molecule fluorescence approaches: Direct visualization of ClpS-substrate interactions and delivery to ClpAP using fluorescently labeled components can reveal kinetic and mechanistic details .

These approaches, particularly when used in combination, could significantly advance our understanding of the role of ClpS in V. vulnificus protein quality control and pathogenesis.

How might understanding ClpS function contribute to new antimicrobial strategies against V. vulnificus?

Understanding ClpS function could lead to novel antimicrobial strategies against V. vulnificus through several potential approaches:

• Targeted inhibition of protein quality control: Compounds that specifically inhibit ClpS function could compromise bacterial adaptation to host environments and stress conditions, potentially reducing virulence or enhancing susceptibility to host defenses .

• Structure-based drug design: Detailed structural understanding of V. vulnificus ClpS, particularly its interaction interfaces with ClpA and substrates, could enable the rational design of small molecule inhibitors .

• Peptide-based inhibitors: Peptides mimicking the N-terminal extension (NTE) of ClpS could potentially disrupt normal ClpS-ClpA interactions, interfering with protein degradation pathways critical for bacterial survival .

• Combination therapies: ClpS inhibitors could potentially sensitize V. vulnificus to existing antibiotics by compromising stress response capabilities, particularly important given the emerging antibiotic resistance noted in clinical isolates .

• Anti-virulence approach: If specific virulence factors are regulated by the ClpS-ClpAP system, targeting this pathway could reduce pathogenicity without imposing strong selective pressure for resistance development .

These approaches represent promising directions for therapeutic development, particularly as traditional antibiotic approaches face increasing challenges from antimicrobial resistance.

How does V. vulnificus ClpS differ structurally and functionally from E. coli ClpS?

V. vulnificus ClpS and E. coli ClpS share fundamental similarities but exhibit notable differences:

The differences likely reflect adaptation to different ecological niches and lifestyle requirements, with V. vulnificus facing unique challenges as both an environmental organism and human pathogen. Further comparative studies could reveal how these molecular adaptations contribute to V. vulnificus pathogenicity.

What research gaps exist in our understanding of V. vulnificus ClpS compared to ClpS in other bacterial species?

Several significant research gaps exist in our understanding of V. vulnificus ClpS compared to better-studied bacterial species:

• Substrate repertoire: While the substrate profile of E. coli ClpS has been extensively characterized, the specific substrates recognized by V. vulnificus ClpS remain largely unknown .

• Regulation of expression: The conditions and regulatory mechanisms governing clpS expression in V. vulnificus during infection and environmental stress have not been well characterized.

• Structural details: High-resolution structural information for V. vulnificus ClpS, particularly in complex with substrates or ClpA, is lacking compared to model organisms.

• Role in virulence: Direct experimental evidence linking ClpS function to specific virulence mechanisms in V. vulnificus remains limited .

• Host-specific adaptations: How V. vulnificus ClpS might be adapted to the unique challenges of transitioning between marine environments and human hosts has not been thoroughly investigated.

• Integration with other stress response systems: The relationship between the ClpS-ClpAP system and other V. vulnificus stress response mechanisms, such as the stressosome, requires further investigation .

Addressing these gaps would significantly advance our understanding of protein quality control in this important pathogen and potentially reveal new therapeutic targets.

What controls are essential when studying the functional activity of recombinant V. vulnificus ClpS?

When studying the functional activity of recombinant V. vulnificus ClpS, several essential controls should be incorporated:

• Negative controls:

  • Buffer-only reactions without ClpS

  • Heat-inactivated ClpS to control for non-specific effects

  • ClpS with mutations in key functional residues

  • Reactions without ATP when studying ClpAP-dependent processes

• Positive controls:

  • Well-characterized model substrates with known degradation patterns

  • E. coli ClpS as a reference standard for comparative activity assessment

  • Native V. vulnificus ClpS (if available) to validate recombinant protein activity

• Specificity controls:

  • Non-N-end rule substrates to confirm substrate specificity

  • ClpS from related Vibrio species to assess species-specific functions

  • Competition assays with known substrates to validate binding specificity

• System validation controls:

  • ClpA-independent assays to confirm adaptor-specific effects

  • Titration experiments with varying ClpS concentrations to establish dose-dependency

  • Time-course experiments to capture the dynamics of ClpS-mediated processes

These controls help ensure experimental rigor and validate that observed effects are specifically attributable to the functional activity of recombinant V. vulnificus ClpS.

How should researchers approach experimental design when investigating ClpS interactions with the V. vulnificus proteome?

A systematic approach to investigating ClpS interactions with the V. vulnificus proteome should incorporate multiple complementary strategies:

• In vitro identification of potential substrates:

  • Protein array screening using purified recombinant ClpS

  • Pull-down assays with tagged ClpS followed by mass spectrometry

  • N-terminal peptide libraries to assess binding preferences

  • Cross-linking mass spectrometry (XL-MS) to capture transient interactions

• In vivo validation strategies:

  • Comparison of proteomes from wild-type and ΔclpS strains using quantitative proteomics

  • Proximity labeling approaches (BioID, APEX2) with ClpS as the bait protein

  • Genetic screens to identify synthetic interactions with clpS deletion

  • Reporter fusions to monitor degradation of candidate substrates

• Condition-specific analyses:

  • Experiments under different stress conditions relevant to V. vulnificus lifestyle

  • Comparison of interactions during growth in environmental versus host-mimicking conditions

  • Temporal analyses during infection models or environmental transitions

• Functional validation:

  • Targeted degradation assays for identified candidate substrates

  • Competition assays between substrates to establish hierarchies

  • Mutational analyses of substrate recognition motifs

  • Phenotypic characterization of substrate stabilization in ΔclpS backgrounds

This multilayered approach would provide comprehensive insights into the ClpS-dependent degradome of V. vulnificus while minimizing false positives and negatives that might arise from any single method.

What are common pitfalls when working with recombinant V. vulnificus ClpS and how can they be addressed?

Researchers working with recombinant V. vulnificus ClpS should be aware of several common pitfalls and their solutions:

• Protein solubility issues:

  • Problem: Recombinant ClpS may form inclusion bodies during expression

  • Solution: Optimize expression conditions (lower temperature, reduced induction), use solubility tags, or employ refolding protocols from inclusion bodies

• Loss of N-terminal extension function:

  • Problem: The critical N-terminal extension may be cleaved or structurally compromised

  • Solution: Verify protein integrity by mass spectrometry, use protease inhibitors during purification, and validate function through NTE-dependent assays

• Buffer incompatibility:

  • Problem: Buffer components may affect ClpS activity or stability

  • Solution: Test multiple buffer systems and optimize salt concentration, pH, and additives for maximal stability and activity

• Inconsistent activity measurements:

  • Problem: Variable results in functional assays

  • Solution: Standardize protein preparation methods, use internal controls, and ensure consistent experimental conditions across replicates

• Non-specific binding in interaction studies:

  • Problem: False positives in substrate identification

  • Solution: Include appropriate negative controls, perform competition assays, and validate interactions through multiple independent methods

• Degradation during storage:

  • Problem: Loss of activity during storage

  • Solution: Add glycerol (5-50%), aliquot to avoid freeze-thaw cycles, and store at -80°C for long-term preservation

Addressing these challenges requires careful optimization and validation at each step of the experimental process.

How can researchers verify that recombinant V. vulnificus ClpS maintains its native conformation and activity?

Verification of native conformation and activity of recombinant V. vulnificus ClpS should employ multiple complementary approaches:

• Structural validation:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • Size exclusion chromatography to verify monomeric state and proper folding

  • Limited proteolysis to assess domain organization and accessibility

  • Thermal shift assays to evaluate protein stability

• Functional validation:

  • ClpA binding assays to confirm adaptor function

  • N-end rule substrate binding assays using model peptides or proteins

  • ATPase assays to verify modulation of ClpA activity

  • Degradation assays with model substrates to confirm functional activity in the complete ClpAPS system

• Comparative analyses:

  • Side-by-side comparison with E. coli ClpS as a reference standard

  • Activity comparison with native V. vulnificus ClpS (if available)

  • Assessment of expected activities across multiple substrate types

• Quantitative benchmarks:

  • Determination of binding constants for known interactions

  • Measurement of inhibition constants for SsrA-tagged substrate degradation

  • Establishment of dose-response relationships for various activities

These validation steps ensure that experimental observations accurately reflect the biological properties of V. vulnificus ClpS rather than artifacts of the recombinant protein production process.