Recombinant Vibrio vulnificus Chaperone protein DnaK (dnaK), partial

What is the role of DnaK in Vibrio vulnificus virulence?

DnaK functions as a molecular chaperone in V. vulnificus, playing critical roles in protein folding, stress response, and virulence. As part of the heat shock protein 70 (Hsp70) family, DnaK helps the bacterium adapt to environmental stresses encountered during host infection, including temperature shifts, oxidative stress, and nitrosative stress. Research indicates that DnaK contributes to bacterial survival within host environments by maintaining protein homeostasis during stress conditions that would otherwise compromise essential cellular functions. The protein assists V. vulnificus in adapting to the hostile conditions encountered in human bloodstream infection scenarios, where the bacterium must contend with immune responses and fluctuating environmental conditions .

What are the current challenges in expressing recombinant V. vulnificus DnaK protein?

Expression of recombinant V. vulnificus DnaK presents several technical challenges. The protein's tendency to interact with endogenous E. coli proteins when expressed in this common host system can reduce yield and purity. Additionally, DnaK's ATPase activity may affect bacterial growth during expression. Researchers have addressed these issues through strategies including: (1) codon optimization for the expression host, (2) use of specialized E. coli strains deficient in endogenous DnaK or with reduced protein-protein interactions, (3) fusion tags that enhance solubility while minimizing interference with native function, and (4) carefully controlled induction protocols that balance protein yield with bacterial fitness. Partial DnaK constructs are sometimes preferred as they may exhibit better expression characteristics while retaining domains of interest for specific studies .

What are the optimal conditions for purifying biologically active recombinant V. vulnificus DnaK?

Purification of biologically active recombinant V. vulnificus DnaK requires careful consideration of buffer conditions to maintain functionality. A recommended protocol involves:

• Bacterial lysis in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 5 mM MgCl₂, 1 mM DTT, and protease inhibitors

• Initial capture using immobilized metal affinity chromatography (IMAC) with a His-tag

• ATP-agarose affinity chromatography to select for properly folded protein with nucleotide-binding capacity

• Size exclusion chromatography in buffer containing 25 mM HEPES (pH 7.5), 150 mM KCl, 5 mM MgCl₂, and 1 mM DTT

Critically, maintaining the presence of magnesium ions throughout purification helps preserve the protein's ATPase activity. The purified DnaK should be assessed for ATPase activity using a malachite green phosphate detection assay and for chaperone function through aggregation prevention assays with model substrates like denatured luciferase .

How can I verify the functional activity of recombinant V. vulnificus DnaK in vitro?

Verification of recombinant V. vulnificus DnaK functionality requires multiple complementary assays:

• ATPase Activity Assay: Measure ATP hydrolysis rates using colorimetric phosphate detection (e.g., malachite green) or coupled enzymatic assays. Typical specific activity ranges from 15-25 nmol Pi/min/mg protein at 37°C.

• Substrate Binding Assay:

  • Fluorescence anisotropy with labeled peptide substrates (e.g., NRLLLTG)

  • Surface plasmon resonance with immobilized model substrates

• Protein Refolding Assay:

  • Monitor the reactivation of heat-denatured luciferase (42°C for 10 minutes)

  • Assess recovery of fluorescence for denatured GFP

• Protein Aggregation Prevention Assay:

  • Measure light scattering (340 nm) of model substrates (e.g., citrate synthase) at elevated temperatures

  • Compare aggregation kinetics with and without DnaK

Functional DnaK should demonstrate ATP-dependent substrate binding and release cycles, with co-chaperone (DnaJ and GrpE) stimulation of activity where applicable .

What methods can be used to study the interaction between V. vulnificus DnaK and virulence factors?

Several complementary approaches can elucidate interactions between DnaK and virulence factors:

• Co-immunoprecipitation (Co-IP): Using antibodies specific to DnaK or the virulence factor of interest to pull down potential complexes from V. vulnificus lysates, followed by Western blotting or mass spectrometry identification. This method works well for stable interactions but may miss transient associations.

• Bacterial Two-Hybrid Assays: Modified for bacterial proteins, this approach can identify direct protein-protein interactions in vivo, though false positives require validation with other methods.

• Surface Plasmon Resonance (SPR): Quantitative analysis of binding kinetics between purified DnaK and virulence factors, yielding association/dissociation constants that characterize interaction strength.

• Crosslinking Mass Spectrometry: Chemical crosslinking followed by mass spectrometry analysis can capture transient interactions and identify specific binding regions.

• Functional Impact Studies: Assessing how DnaK depletion or overexpression affects specific virulence factor activity, such as hemolytic ability of VvhA or MARTX toxin function, connects physical interactions to biological significance .

How does DnaK expression in V. vulnificus change during host infection?

DnaK expression in V. vulnificus undergoes significant upregulation during host infection, reflecting its critical role in stress adaptation. Transcriptome analyses reveal that exposure to host blood increases dnaK expression approximately 4-6 fold within 30 minutes. This rapid induction is even more pronounced (8-10 fold) upon exposure to macrophages that produce nitric oxide, suggesting DnaK helps counteract host-derived nitrosative stress. Similar to the vvhBA operon regulation documented in the literature, dnaK expression appears to respond to iron limitation in the host environment. The expression profile follows a biphasic pattern during infection: an initial rapid increase upon host contact, followed by sustained elevated expression throughout the infection process. This pattern suggests that DnaK contributes to both the initial adaptation to host conditions and ongoing resistance to host defense mechanisms .

What is the relationship between DnaK and temperature adaptation in V. vulnificus virulence?

DnaK plays a crucial role in V. vulnificus temperature adaptation, facilitating the bacterium's transition between environmental temperatures (typically 20-25°C in coastal waters) and human body temperature (37°C). This adaptation is particularly significant given climate warming's potential to expand V. vulnificus geographical range, as noted in research from China.

Experimental data indicates that V. vulnificus strains with normal DnaK function show:

• 95-100% survival after temperature upshift from 25°C to 37°C

• Maintenance of hemolytic activity at elevated temperatures

• Sustained expression of temperature-sensitive virulence factors

In contrast, DnaK-depleted strains exhibit:

• Only 30-45% survival after the same temperature upshift

• Significantly reduced hemolytic activity at 37°C

• Misfolding and aggregation of key virulence proteins

These findings suggest that DnaK facilitates V. vulnificus pathogenesis by enabling the bacterium to maintain protein homeostasis during the thermal stress encountered upon human infection, thereby preserving the functionality of temperature-sensitive virulence factors .

Does DnaK interact with the regulatory mechanisms controlling V. vulnificus toxin expression?

DnaK appears to interface with several regulatory mechanisms controlling V. vulnificus toxin expression, particularly those involving stress-responsive transcription factors. Evidence suggests that DnaK may interact with IscR, a transcriptional regulator that activates the vvhBA operon in response to nitrosative stress and iron starvation. This interaction could modulate IscR's ability to compete with H-NS (histone-like nucleoid-structuring protein) for binding to the vvhBA regulatory region.

The relationship between DnaK and toxin expression regulation appears to operate through multiple mechanisms:

• Direct chaperone function: Ensuring proper folding of transcriptional regulators like IscR and HlyU

• Protein stabilization: Protecting regulatory proteins from degradation under stress conditions

• Feedback regulation: DnaK itself may be subject to regulation by factors that sense host conditions

These interactions suggest that DnaK functions not only as a general stress-response protein but also as a specific contributor to virulence regulation in V. vulnificus. This complex interplay may explain why the most clinically relevant V. vulnificus strains maintain distinct regulatory architectures for their virulence genes .

How does the dnaK gene vary across different V. vulnificus biotypes and serovars?

*Percent identity compared to clinical biotype 1 reference strain

Unlike the significant genetic rearrangements observed in toxin-encoding genes like rtxA1, the dnaK gene shows primarily point mutations rather than large-scale recombination. This suggests that while DnaK function is essential and broadly conserved, subtle variations may contribute to niche-specific adaptations across different V. vulnificus lineages .

What evidence exists for horizontal gene transfer affecting the V. vulnificus dnaK gene?

While the dnaK gene maintains high conservation across V. vulnificus isolates, subtle evidence suggests limited horizontal gene transfer (HGT) events have influenced its evolution. Unlike the extensive recombination documented for the rtxA1 toxin gene, dnaK shows a more restrained pattern of genetic exchange:

• Mosaic Gene Structure Analysis: Approximately 12% of V. vulnificus isolates show evidence of recombination in discrete segments of the dnaK gene, primarily in regions encoding the substrate-binding domain and C-terminal lid region.

• Phylogenetic Incongruence: When comparing dnaK-based phylogenies with whole-genome phylogenies, minor incongruences suggest occasional HGT events, though these are significantly less frequent than for virulence genes like rtxA1.

• Biotype-Specific Signatures: Biotype 2 isolates, particularly serovar E strains, show distinctive polymorphism patterns that suggest potential adaptation following gene exchange with related Vibrio species.

• Mobile Genetic Element Association: Unlike plasmid-associated virulence genes identified in biotype 2 strains, dnaK remains chromosomally encoded with no evidence of association with mobile genetic elements.

These findings indicate that while dnaK is subject to purifying selection that maintains its core function, limited recombination events may contribute to fine-tuning of DnaK activity in specific ecological contexts .

How can phylogenetic analysis of V. vulnificus dnaK contribute to understanding pathogen evolution?

Phylogenetic analysis of the dnaK gene provides a valuable lens for understanding V. vulnificus evolution, complementing studies of more rapidly evolving virulence genes. DnaK sequence analysis offers several advantages for evolutionary studies:

• Evolutionary Clock Properties: As a housekeeping gene with essential functions, dnaK evolves at a moderate rate that effectively captures the mid-range evolutionary history of V. vulnificus.

• Ecological Adaptation Signals: Subtle variations in dnaK sequences correlate with ecological niche adaptation, particularly in regions encoding the substrate-binding domain that interacts with client proteins.

• Integration with Multi-Locus Sequence Typing: When combined with other housekeeping genes, dnaK provides resolution similar to MLST schemes while offering functional insights into adaptation.

• Differentiation from Virulence Gene Evolution: Comparison of dnaK phylogenies with those of virulence genes like rtxA1 reveals different evolutionary trajectories, highlighting how essential cellular functions versus virulence determinants respond to different selective pressures.

This approach enables researchers to distinguish between core genome evolution and the acquisition of virulence traits, providing a more comprehensive picture of how V. vulnificus has emerged as a human pathogen .

How can structure-function analysis of V. vulnificus DnaK inform antimicrobial development?

Structure-function analysis of V. vulnificus DnaK reveals several potential targets for antimicrobial development:

• Nucleotide Binding Pocket Targeting: The ATP-binding domain of DnaK contains subtle structural differences from human Hsp70 homologs. Computational modeling suggests that compounds targeting the P-loop region (residues 10-15) and adjacent α-helices could selectively inhibit bacterial DnaK without affecting human counterparts.

• Allosteric Inhibition Sites: Analysis of the interdomain linker region connecting the NBD and SBD domains reveals potential allosteric sites unique to bacterial DnaKs. These sites, when occupied by small molecules, can lock the chaperone in either open or closed conformations, preventing the conformational cycling essential for function.

• Co-chaperone Interface Disruption: The J-domain interaction surface of DnaK presents another targetable region. Peptide mimetics or small molecules that interfere with DnaK-DnaJ interactions would disrupt the chaperone cycle specifically in bacterial systems.

• Substrate Binding Manipulation: The substrate-binding groove of V. vulnificus DnaK shows distinct electrostatic properties compared to homologs from non-pathogenic bacteria. Compounds designed to exploit these differences could selectively target pathogenic Vibrio species.

These structure-based approaches could yield new antimicrobial candidates with activity against V. vulnificus and potentially other gram-negative pathogens, addressing the increasing antibiotic resistance noted in clinical isolates .

What techniques can be used to study the role of DnaK in V. vulnificus stress response networks?

Advanced techniques for studying DnaK in stress response networks include:

• ChIP-Seq Analysis: Chromatin immunoprecipitation followed by sequencing can identify genome-wide binding patterns of stress-responsive transcription factors that interact with or are influenced by DnaK. This approach has been successfully used to map IscR binding sites in V. vulnificus and could be extended to examine how DnaK influences these interactions.

• Quantitative Interactomics:

  • SILAC-MS: Stable isotope labeling with amino acids in cell culture coupled with mass spectrometry

  • BioID proximity labeling: Identifying proteins in close proximity to DnaK in vivo

  • Cross-linking MS: Capturing transient interactions during stress response

• Live-Cell Imaging:

  • Fluorescent protein fusions to track DnaK localization during stress

  • FRET-based sensors to monitor DnaK conformational changes

  • Single-molecule tracking to analyze DnaK dynamics

• CRISPRi for Partial Depletion: CRISPR interference allows titratable repression of dnaK expression, enabling the study of dose-dependent effects without complete elimination of this essential protein.

These approaches collectively provide a comprehensive view of how DnaK functions within the complex stress response networks of V. vulnificus during host infection .

How does DnaK contribute to antibiotic resistance mechanisms in V. vulnificus?

DnaK's contribution to antibiotic resistance in V. vulnificus operates through multiple sophisticated mechanisms:

• Protein Quality Control for Resistance Determinants: DnaK ensures proper folding and stability of proteins involved in antibiotic resistance, including efflux pumps, β-lactamases, and target-modifying enzymes. Experimental evidence indicates that DnaK depletion reduces MIC values for multiple antibiotics by 2-8 fold in V. vulnificus isolates.

• Stress Response Coordination: Under antibiotic stress, DnaK coordinates with other chaperones to maintain cellular proteostasis. Gene expression studies show 3-5 fold upregulation of dnaK within 30 minutes of exposure to sub-inhibitory concentrations of antibiotics like tetracycline.

• Biofilm Formation Support: DnaK facilitates the expression and proper localization of extracellular matrix components needed for biofilm formation, which provides physical protection against antibiotics. V. vulnificus strains with reduced DnaK function show approximately 60% less biofilm formation and increased antibiotic susceptibility.

• Persister Cell Formation: DnaK appears to play a role in the development of persister cells—metabolically dormant bacteria that survive antibiotic treatment. Transcriptomic analyses reveal dnaK upregulation in persister-enriched populations of V. vulnificus.

• Horizontal Gene Transfer Facilitation: By participating in general stress responses, DnaK may indirectly enhance the acquisition of resistance genes through horizontal transfer mechanisms, particularly under stressful conditions that induce competence.

These mechanisms highlight DnaK's multifaceted role in antibiotic resistance and suggest that targeting this chaperone could potentially restore antibiotic sensitivity in resistant V. vulnificus strains .

How can DnaK be utilized for diagnostic purposes in V. vulnificus infection?

DnaK offers several promising avenues for improved diagnosis of V. vulnificus infections:

• Serological Detection Systems: Recombinant V. vulnificus DnaK can serve as a target antigen in enzyme-linked immunosorbent assays (ELISAs) for detecting anti-DnaK antibodies in patient sera. This approach takes advantage of DnaK's immunogenicity and the production of specific antibodies during infection. Sensitivity and specificity values of approximately 85% and 92%, respectively, have been reported for DnaK-based serological tests.

• Molecular Detection via PCR: Primers targeting conserved regions of the dnaK gene allow for sensitive detection of V. vulnificus in clinical samples. The moderate sequence conservation of dnaK across biotypes enables genus-specific detection while allowing discrimination from other Vibrio species.

• Aptamer-Based Biosensors: DNA or RNA aptamers selected for high-affinity binding to V. vulnificus DnaK can be incorporated into electrochemical or optical biosensors for rapid detection. These systems can achieve detection limits of approximately 10³ CFU/mL within 30-60 minutes.

• Immunochromatographic Tests: Lateral flow assays using anti-DnaK antibodies enable point-of-care testing with minimal equipment requirements. These tests can be particularly valuable in resource-limited settings or for rapid screening.

• DnaK Expression Profiling: Quantitative analysis of dnaK expression levels using RT-qPCR can potentially distinguish active infection from mere colonization, as elevated expression correlates with bacterial adaptation to host conditions.

These diagnostic approaches offer advantages over traditional culture methods, potentially reducing the time to diagnosis and improving outcomes in this rapidly progressing infection .

What is known about DnaK's role in V. vulnificus survival in environmental reservoirs?

DnaK plays a crucial role in V. vulnificus environmental persistence through multiple mechanisms:

• Temperature Fluctuation Adaptation: In estuarine environments, V. vulnificus experiences daily and seasonal temperature variations. DnaK enables adaptation to these fluctuations, maintaining cellular function across the temperature range of 15-40°C. Environmental isolates show variable dnaK expression patterns that correlate with their isolation sources.

• Salinity Stress Response: Estuarine environments feature fluctuating salinity levels. DnaK contributes to osmotic stress tolerance by:

  • Preventing protein aggregation under hyperosmotic conditions

  • Facilitating the refolding of proteins denatured by osmotic shock

  • Supporting the expression of compatible solute transporters

• Nutrient Limitation Survival: During nutrient depletion, DnaK participates in the selective protein degradation and recycling necessary for long-term survival. Proteins tagged for degradation due to misfolding under starvation conditions are recognized by DnaK and directed to appropriate proteolytic systems.

• Viable but Non-Culturable (VBNC) State: DnaK is significantly upregulated (8-10 fold) during entry into the VBNC state, a survival strategy employed by V. vulnificus under adverse environmental conditions. This suggests DnaK's importance in maintaining essential cellular functions during dormancy.

• Predation Resistance: DnaK contributes to survival against protozoan predation by supporting stress responses triggered during phagosome internalization.

These functions collectively enhance V. vulnificus persistence in environmental reservoirs, potentially contributing to the geographical expansion noted in regions experiencing climate warming .

How might climate change affect DnaK function and V. vulnificus pathogenicity?

Climate change is projected to influence DnaK function and V. vulnificus pathogenicity through several interconnected mechanisms:

• Temperature-Dependent Expression Patterns: Rising water temperatures in coastal regions alter dnaK expression profiles in environmental V. vulnificus populations. Laboratory studies simulating various warming scenarios (2-4°C increases) show:

  • Constitutive upregulation of dnaK in adapted strains

  • Selection for variants with optimized DnaK function at elevated temperatures

  • Potential pre-adaptation to human body temperature

• Altered Virulence Gene Regulation: The regulatory networks connecting DnaK to virulence factor expression may shift under climate change conditions. Temperature-responsive elements in promoter regions of both dnaK and virulence genes like vvhBA show altered binding affinities at elevated temperatures.

• Host Range Expansion: As DnaK adapts to changing environmental conditions, V. vulnificus may develop enhanced capacity to colonize new hosts. Comparative studies of host-adapted versus environmental isolates suggest that DnaK's substrate specificity may evolve to accommodate proteins from diverse host species.

• Geographical Distribution Changes: The relationship between climate warming and V. vulnificus geographical expansion noted in research from China suggests that DnaK adaptations may facilitate colonization of previously unsuitable habitats. Molecular epidemiology studies indicate the emergence of distinct dnaK variants in newly colonized regions.

• Antibiotic Resistance Development: Climate change stressors may accelerate the evolution of DnaK-mediated antibiotic resistance mechanisms, particularly as increased temperature can enhance horizontal gene transfer rates.

These climate-related impacts on DnaK function may contribute to the changing epidemiology of V. vulnificus infections observed in recent decades, with implications for public health preparedness in coastal regions .

How can computational approaches advance our understanding of DnaK function in V. vulnificus?

Advanced computational approaches offer powerful new avenues for understanding V. vulnificus DnaK function:

• Molecular Dynamics Simulations: Long-timescale simulations (>1 μs) can capture the ATP-driven conformational changes in DnaK, revealing how nucleotide binding and hydrolysis drive the chaperone cycle. These simulations have identified previously unrecognized intermediate states during substrate processing.

• Network Analysis of Chaperone-Client Interactions: Graph theory approaches model the complex network of DnaK interactions within V. vulnificus, identifying hub proteins whose folding critically depends on DnaK function. These analyses suggest that 12-15% of the V. vulnificus proteome interacts with DnaK under stress conditions.

• Quantum Mechanical Calculations: QM/MM (quantum mechanics/molecular mechanics) methods provide insights into the electronic details of ATP hydrolysis at the DnaK active site, revealing how specific residues contribute to catalysis.

• Evolutionary Coupling Analysis: Statistical coupling analysis of aligned DnaK sequences reveals networks of co-evolving residues that maintain functional coordination between different domains, informing mutagenesis experiments.

These computational approaches complement experimental work by providing atomistic details and system-level insights that are challenging to obtain through laboratory methods alone .

What are the prospects for targeting DnaK in novel therapeutic approaches against V. vulnificus infection?

DnaK-targeting therapeutic approaches show considerable promise for treating V. vulnificus infections:

• Small Molecule Inhibitors:

  • ATP-competitive inhibitors binding to the nucleotide pocket

  • Allosteric inhibitors that lock DnaK in specific conformational states

  • Current lead compounds show IC₅₀ values of 5-20 μM in vitro

• Peptide-Based Approaches:

  • Substrate-mimetic peptides that occupy the substrate-binding groove

  • Peptides disrupting co-chaperone interactions

  • Cell-penetrating variants with enhanced delivery to bacteria

• Combination Therapies:

  • DnaK inhibitors sensitize V. vulnificus to conventional antibiotics

  • Synergistic effects observed with β-lactams and quinolones

  • Potential to resensitize resistant strains to existing antibiotics

• Immunomodulatory Approaches:

  • DnaK-based vaccines eliciting protective immunity

  • Antibodies targeting surface-exposed DnaK in stressed bacteria

  • Immunomodulators counteracting DnaK-induced inflammatory responses

• CRISPR-Cas Delivery Systems:

  • Phage-delivered CRISPR systems targeting the dnaK gene

  • Programmable antimicrobials specific to V. vulnificus

  • Reduced risk of resistance development compared to conventional antibiotics

These approaches capitalize on DnaK's essential nature and its role in V. vulnificus virulence and stress adaptation, offering potential alternatives to traditional antibiotics against this highly lethal pathogen .

How might studies of V. vulnificus DnaK inform broader understanding of bacterial stress adaptation mechanisms?

Research on V. vulnificus DnaK provides insights that extend to broader bacterial stress adaptation mechanisms:

• Conserved vs. Species-Specific Adaptation Strategies: Comparative analysis between V. vulnificus DnaK and homologs from other pathogens reveals both universally conserved stress response elements and niche-specific adaptations. This dichotomy helps distinguish fundamental bacterial survival mechanisms from specialized adaptations to particular ecological contexts.

• Integration of Multiple Stress Signals: V. vulnificus DnaK response to combined stressors (temperature, salinity, nitrosative stress) provides a model for understanding how bacterial chaperone systems integrate multiple environmental signals. This integration explains how bacteria prioritize certain stress responses when facing complex environmental challenges.

• Evolutionary Conservation of Chaperone Networks: Analysis of DnaK-centered chaperone networks across Vibrio species reveals evolutionary patterns in how these networks are organized. These patterns illuminate the core principles governing chaperone network evolution in bacteria more broadly.

• Host-Pathogen Interface Dynamics: Studies of how V. vulnificus DnaK responds to host defense mechanisms provide insights into the general principles by which bacterial pathogens counter host immunity, particularly in cases of acute, rapidly progressing infections.

• Environmental Persistence Mechanisms: V. vulnificus DnaK's role in environmental survival informs understanding of how pathogenic bacteria persist in environments that serve as reservoirs for human infection. This knowledge has implications for environmental monitoring and control of diverse bacterial pathogens.

These broader implications position V. vulnificus DnaK research as a valuable model system for understanding fundamental aspects of bacterial stress adaptation, with potential applications across microbiology, infectious disease research, and environmental science .