Recombinant Vibrio vulnificus ATP-dependent Clp protease proteolytic subunit (clpP)

What is the role of ClpP in Vibrio vulnificus virulence mechanisms?

ClpP protease plays an important role in the proteostasis of prokaryotic cells, including Vibrio vulnificus. As a serine protease, it participates in the degradation and regulation of proteins essential for bacterial survival and pathogenesis. Alteration of ClpP function has been demonstrated to affect the virulence and infectivity of numerous pathogens . In V. vulnificus specifically, ClpP likely contributes to pathogenesis by regulating the expression of various virulence factors through targeted protein degradation, similar to its function in other bacterial species.

The methodological approach to studying this connection typically involves:

• Creation of clpP deletion mutants in V. vulnificus

• Comparative virulence assessment between wild-type and mutant strains in animal models

• Transcriptomic and proteomic analyses to identify differentially expressed virulence factors

• Complementation studies with recombinant ClpP to confirm phenotype restoration

How is the ClpP protease structured and regulated in V. vulnificus compared to model organisms?

The V. vulnificus ClpP likely forms a tetradecameric complex composed of two heptameric rings, creating a barrel-shaped structure with the proteolytic active sites sequestered in an internal chamber, similar to ClpP in other organisms. The substrate selection for V. vulnificus ClpP relies on specific AAA+ unfoldases, which filter, unfold, and introduce proteins into the proteolytic chamber according to cellular needs .

In contrast to the well-studied E. coli ClpP system, the V. vulnificus ClpP complex may have unique structural and functional adaptations. For example, in cyanobacteria, researchers have identified a novel ClpP3/R complex with unique configurations and functional properties . The V. vulnificus ClpP might similarly display species-specific adaptations related to its role in marine environments and pathogenesis.

Regulatory control of V. vulnificus ClpP activity likely involves:

• Association with specific AAA+ unfoldases (e.g., ClpX, ClpA, or ClpC homologs)

• Interaction with adaptor proteins that influence substrate selection

• Potential anti-adaptor proteins that add an extra layer of regulation

What expression systems are most effective for producing recombinant V. vulnificus ClpP?

When expressing recombinant V. vulnificus ClpP, researchers should consider the following methodological approaches:

E. coli expression systems:

• BL21(DE3) strains are commonly used for recombinant protease expression

• pET vector systems with IPTG-inducible promoters provide controlled expression

• Co-expression vectors like pACYC Duet (as used with ClpP3/R complexes) facilitate simultaneous expression of ClpP with its partner unfoldases

Expression optimization strategies:

• Lower induction temperatures (16-25°C) to enhance proper folding

• Addition of 5-10% glycerol to stabilize the protein

• Use of fusion tags (His6, GST, MBP) to improve solubility and facilitate purification

• Codon optimization for the expression host

The effectiveness of the expression system should be evaluated through:

• SDS-PAGE analysis of soluble and insoluble fractions

• Western blotting for target protein identification

• Enzymatic activity assays using fluorogenic peptide substrates similar to those described for other ClpP proteins

How can researchers effectively purify and maintain the activity of recombinant V. vulnificus ClpP?

Purification of recombinant V. vulnificus ClpP requires careful consideration of protein stability and oligomeric state preservation. Based on established methods for other ClpP systems, the following approach is recommended:

Purification protocol:

• Affinity chromatography using His6-tag or other fusion partners

• Ion exchange chromatography to remove contaminants

• Size exclusion chromatography to ensure tetradecameric assembly integrity

• Confirmation of oligomeric state by native PAGE or analytical ultracentrifugation

Buffer optimization considerations:

• Inclusion of 10-20% glycerol to maintain protein stability

• Addition of reducing agents (1-5 mM DTT or β-mercaptoethanol)

• Physiological salt concentrations (75-150 mM NaCl)

• Neutral pH (7.0-7.5) with Tris or HEPES buffers

• Inclusion of 10 mM MgCl₂ to stabilize the complex

Long-term storage recommendations include flash freezing purified ClpP in liquid nitrogen with 20% glycerol and storing at -80°C in small aliquots to avoid freeze-thaw cycles that may disrupt the oligomeric structure.

What assays reliably measure the proteolytic activity of recombinant V. vulnificus ClpP?

Several methodological approaches can be employed to assess the proteolytic activity of recombinant V. vulnificus ClpP:

Peptide-based fluorogenic assays:

• Fluorogenic peptides such as N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin (AMC)

• Suc-Val-Lys-Met-AMC or Suc-Ile-Ile-Trp-AMC for substrate specificity analysis

• Standardized reaction conditions: 30 μM peptide and 1-5 μg ClpP in buffer containing 25 mM Tris/Cl (pH 7.5), 75 mM NaCl, 10 mM MgCl₂, 1 mM DTT

• Incubation at 37°C for 5-20 minutes with fluorescence monitoring (excitation 310-380 nm, emission 460 nm)

Protein substrate degradation assays:

• α-casein degradation monitored by SDS-PAGE and Coomassie Blue staining

• FITC-casein degradation measured by fluorescence (excitation 490 nm, emission 525 nm)

• GFP-tagged substrate degradation assessed by immunoblotting or fluorescence loss

• Reactions performed with 1 μM ClpP, appropriate AAA+ unfoldase, ATP regeneration system, and 0.1-1 μM substrate

Coupled ATPase activity measurements:

• Monitoring ATPase activity of the associated unfoldase (e.g., ClpC)

• Measuring release of inorganic phosphate during protein degradation

• Reaction conditions: 0.5 μM unfoldase with 1 μM ClpP in buffer containing 4 mM ATP

How do mutations in the catalytic triad affect V. vulnificus ClpP activity and structure?

The catalytic triad in ClpP consists of Ser, His, and Asp residues that are essential for its proteolytic function. Based on research on other ClpP systems, the following methodological approach can assess the effects of mutations:

Site-directed mutagenesis strategy:

• Replace the active site Ser residue with Ala to create a proteolytically inactive variant

• Create single mutations in His and Asp residues of the catalytic triad

• Design mutations in the binding interfaces between ClpP subunits to assess oligomerization

Functional analysis methods:

• Compare proteolytic activity of wild-type and mutant ClpP using peptide and protein substrates

• Assess oligomeric assembly by size exclusion chromatography and native PAGE

• Examine conformational changes using circular dichroism spectroscopy

• Analyze thermal stability through differential scanning fluorimetry

As demonstrated with other ClpP proteins, mutations in the catalytic triad are expected to abolish proteolytic activity while potentially maintaining the structural integrity of the complex. For example, replacing the active site Ser with Ala in ClpP3 eliminated proteolytic activity while preserving the ability to form complexes .

How does V. vulnificus ClpP compare across different phylogenetic lineages of the species?

V. vulnificus is classified into five distinct phylogenetic lineages (L1-L5), each associated with different ecological niches and virulence characteristics . Researchers interested in comparing ClpP across these lineages should consider:

Comparative genomic approach:

• Extract and align clpP gene sequences from V. vulnificus strains representing all five lineages

• Identify conserved regions and lineage-specific variations

• Assess selection pressure on clpP using dN/dS ratio analysis

• Examine regulatory regions to identify potential differences in expression control

Functional comparison methods:

• Express and purify recombinant ClpP from representative strains of each lineage

• Compare substrate specificity using standardized proteolytic assays

• Assess interaction with lineage-specific unfoldases and adaptor proteins

• Evaluate potential correlation with virulence phenotypes

The phylogenetic lineages of V. vulnificus display different virulence characteristics. For example, L1 comprises clinical and environmental Bt1 strains involved in human cases related to raw seafood ingestion, while L2 includes both Bt1 and Bt2 strains associated with diseased fish in aquaculture. L3 is linked to wound infections after farmed-fish handling. These ecological differences may reflect or influence ClpP function across lineages .

What is the relationship between ClpP activity and virulence plasmid pVvbt2 in V. vulnificus?

The virulence plasmid pVvbt2 confers fish pathogenicity to V. vulnificus Bt2 strains . Researchers investigating the relationship between ClpP and this plasmid should consider:

Research methodology:

• Compare clpP expression in isogenic strains with and without pVvbt2

• Identify potential plasmid-encoded substrates or regulators of ClpP

• Assess whether ClpP regulates the expression of plasmid-encoded virulence factors

• Determine if ClpP activity differs in response to environmental conditions relevant to fish pathogenicity

Experimental approaches:

• Transcriptomic analysis to identify differentially expressed genes in the presence/absence of pVvbt2

• Proteomics to detect changes in the substrate profile of ClpP

• In vitro and in vivo infection models to assess the role of ClpP in different host contexts

• Genetic complementation studies with clpP variants in different genetic backgrounds

The phylogenetic analysis indicates that pVvbt2 has been acquired independently by different V. vulnificus clones, particularly in aquaculture environments, with the zoonotic Bt2-Serovar E clone spreading worldwide . This suggests that ClpP may interact differently with plasmid-encoded factors depending on the genetic background.

How can recombinant V. vulnificus ClpP be utilized as a target for novel antimicrobial development?

ClpP has emerged as an attractive antimicrobial target due to its role in bacterial virulence and its structural differences from eukaryotic proteases . Researchers exploring V. vulnificus ClpP as a therapeutic target should consider:

Drug discovery methodology:

• High-throughput screening of compound libraries against recombinant V. vulnificus ClpP

• Structure-based drug design utilizing crystallographic data

• Development of compounds that either activate or inhibit ClpP function

• Evaluation of specificity against human mitochondrial ClpP to minimize off-target effects

Compound evaluation approach:

• In vitro activity assays using fluorogenic substrates

• Cell-based assays to assess compound penetration and toxicity

• Assessment of effects on V. vulnificus virulence in infection models

• Combination studies with established antibiotics

ClpP-targeting compounds would represent a new class of antibiotics with novel mechanisms of action, potentially avoiding cross-resistance to established antibiotic classes. When not targeting an essential function, these compounds might exert lower evolutionary selection pressure, providing a greater window of opportunity for the host immune system to clear the infection .

What techniques effectively measure V. vulnificus ClpP substrate specificity in the context of drug development?

Understanding substrate specificity is crucial for developing targeted ClpP modulators. Researchers should consider:

Substrate identification methods:

• Proteomics approaches comparing wild-type and clpP-deficient strains

• Pull-down assays using catalytically inactive ClpP as bait

• Peptide library screening to identify preferred cleavage motifs

• In silico prediction of potential substrates based on known degradation signals

Specificity assessment techniques:

• Fluorescence resonance energy transfer (FRET) peptide libraries

• Positional scanning peptide libraries

• Competition assays with known substrates

• Mass spectrometry-based identification of cleavage products

Substrate identification in model bacteria reveals the importance of ClpP function in regulating the bacterial proteome. The substrates identified are involved in various processes relevant to bacterial infectivity and virulence, supporting ClpP as a prime target for antivirulence drug development .

What factors affect the structural integrity of recombinant V. vulnificus ClpP during experimental procedures?

Maintaining the tetradecameric structure of ClpP is crucial for its proper function. Researchers should be aware of:

Critical factors affecting structural integrity:

• Buffer composition (ionic strength, pH, presence of divalent cations)

• Temperature during purification and storage

• Protein concentration effects on oligomerization

• Presence of detergents or other additives in buffers

Analytical methods to assess structural integrity:

• Size exclusion chromatography to confirm tetradecameric assembly

• Native PAGE to evaluate oligomeric state

• Dynamic light scattering to assess size distribution

• Electron microscopy to visualize complex formation

Based on studies with ClpP3/R complexes, successful purification can be achieved through co-expression of tagged and untagged subunits followed by metal affinity chromatography and size exclusion chromatography. The purified complex should be stored in buffer containing appropriate salt concentration (e.g., 75 mM NaCl) and stabilizing additives .

How can researchers effectively study the interaction between V. vulnificus ClpP and its AAA+ unfoldases?

The interaction between ClpP and its associated unfoldases is essential for proper substrate selection and degradation. Researchers should consider:

Methodological approaches:

• Co-expression and co-purification of ClpP with partner unfoldases

• Surface plasmon resonance or isothermal titration calorimetry to quantify binding affinities

• Cryo-electron microscopy to visualize the ClpP-unfoldase complex

• Cross-linking coupled with mass spectrometry to identify interaction surfaces

Functional assessment techniques:

• ATPase activity measurements of unfoldases in the presence/absence of ClpP

• Protein degradation assays with model substrates requiring unfoldase activity

• Competition assays with known adaptor proteins

• Mutagenesis of potential interaction surfaces to identify critical residues

The presence of diverse AAA+ unfoldases offers tight regulation of ClpP activity in cells. Furthermore, adaptors binding to the Clp ATPases upon specific signals or stresses influence substrate choice, providing additional control of this regulation. Examples include SspB, RssB, and UmuD for E. coli ClpX, ClpS for E. coli ClpA, or MecA for B. subtilis ClpC .

How might the function of V. vulnificus ClpP differ between clinical and environmental isolates?

V. vulnificus strains from different sources (clinical vs. environmental) may exhibit variations in ClpP function that could contribute to their pathogenic potential. Researchers exploring this question should consider:

Research approach:

• Comparative genomics of clpP sequences from clinical and environmental isolates

• Expression analysis of clpP under conditions mimicking human infection versus marine environments

• Substrate profiling to identify differences in protein targets between isolates

• Assessment of ClpP contribution to stress responses relevant to host adaptation

Genomic analyses indicate that despite differences in distribution between phylogenetic groups, high recombination rates and frequent exchange of mobile genetic elements and virulence factors occur between V. vulnificus populations. Microdiversity analyses have revealed unique genomic markers among C1 strains (clinical-associated alleles) with a potential direct role in virulence .

What role might ClpP play in V. vulnificus adaptation to different host environments?

V. vulnificus is a multi-host pathogen capable of infecting both humans and fish. The role of ClpP in this host adaptation process deserves investigation:

Experimental design considerations:

• Transcriptomic and proteomic profiling of V. vulnificus during growth in human serum versus fish blood

• Comparison of clpP mutant fitness in different host models

• Assessment of temperature-dependent regulation of ClpP activity (37°C for humans vs. lower temperatures for fish)

• Investigation of ClpP's role in degrading host-specific antimicrobial factors

Understanding how ClpP contributes to V. vulnificus adaptation across different hosts could provide insights into the evolution of this pathogen, particularly in the context of the five identified phylogenetic lineages that show different host associations and virulence characteristics .