Recombinant Vibrio vulnificus Chaperone protein hscA homolog (hscA), partial

What is the function of the HscA chaperone protein in Vibrio vulnificus?

The HscA chaperone protein in Vibrio vulnificus functions as a molecular chaperone involved in protein folding, preventing aggregation of misfolded proteins, and facilitating proper protein assembly under stress conditions. HscA belongs to the heat shock protein 70 (Hsp70) family and plays a critical role in bacterial stress response, particularly during host infection when the pathogen faces various environmental stresses. In Vibrio vulnificus, HscA is involved in maintaining protein homeostasis under stressful conditions such as temperature shifts, oxidative stress, and host immune responses. This protein is particularly important for virulence as it helps the bacterium adapt to the hostile environment within the human host during infection .

How does the Vibrio vulnificus HscA homolog compare structurally to HscA proteins in other bacterial species?

The Vibrio vulnificus HscA homolog shares significant structural similarities with other bacterial HscA proteins, particularly those from related Vibrio species. The protein contains a highly conserved N-terminal nucleotide-binding domain (NBD) responsible for ATP binding and hydrolysis, and a C-terminal substrate-binding domain (SBD) that recognizes and binds to client proteins. Sequence alignment studies indicate approximately 50-66% identity with HscA proteins from other Vibrio species, similar to the homology observed between the HupA and HutA proteins in V. vulnificus and V. cholerae, respectively . The highest sequence conservation is typically found in the NBD region, while the SBD shows greater variability, potentially reflecting adaptation to species-specific substrate proteins. The linker region connecting these domains allows for the conformational changes necessary for chaperone function.

What expression systems are most effective for producing recombinant Vibrio vulnificus HscA protein?

For the recombinant expression of Vibrio vulnificus HscA protein, Escherichia coli-based expression systems have proven most effective, particularly BL21(DE3) strains containing pET expression vectors. These systems provide high yield and relatively straightforward purification protocols. The optimal expression conditions include induction with 0.5-1.0 mM IPTG at mid-log phase (OD600 of 0.6-0.8) and growth at 25-30°C for 4-6 hours post-induction, rather than the standard 37°C, to enhance soluble protein production and reduce inclusion body formation. The pET28a vector system incorporating a His-tag for simplified purification has shown particularly good results with yields of 15-20 mg/L of culture. For researchers requiring native protein, tobacco etch virus (TEV) protease cleavage sites can be engineered between the tag and protein to allow tag removal after purification .

What purification strategy yields the highest purity and activity for recombinant Vibrio vulnificus HscA?

A multi-step purification strategy provides the highest purity and activity for recombinant Vibrio vulnificus HscA. The recommended protocol begins with immobilized metal affinity chromatography (IMAC) using a Ni-NTA column, capitalizing on the engineered His-tag. This is followed by ion-exchange chromatography (typically DEAE or Q-Sepharose) to separate the target protein from contaminants with different charge properties. Size exclusion chromatography (Superdex 200) serves as the final polishing step to achieve >95% purity and remove aggregates. For optimal activity maintenance, all buffers should contain 5 mM ATP, 10 mM MgCl₂, and 1 mM DTT to stabilize the protein's native conformation. This protocol typically yields 8-10 mg of high-purity HscA per liter of bacterial culture with specific ATPase activity of 4.2-5.8 μmol Pi/min/mg protein when measured at 37°C using standard ATPase assays .

How can researchers effectively measure the chaperone activity of purified HscA protein?

Researchers can effectively measure the chaperone activity of purified HscA protein through multiple complementary assays. The primary method is the prevention of substrate protein aggregation, which can be monitored by light scattering at 320-360 nm. In this assay, a model substrate protein (such as luciferase or citrate synthase) is heat-stressed at 43°C, and the ability of HscA to prevent aggregation is quantified by comparing turbidity in the presence and absence of the chaperone. Additionally, ATPase activity assays provide indirect measurement of chaperone function, as ATP hydrolysis drives the chaperone cycle. This can be measured using malachite green assays to detect inorganic phosphate release. For more advanced analysis, researchers should employ substrate refolding assays that monitor the recovery of enzymatic activity of denatured model proteins. The ratio of functional to total substrate recovery provides a quantitative measure of chaperone efficiency. Optimal conditions for these assays include 25 mM HEPES (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, and 2 mM ATP at 37°C .

What are the optimal conditions for studying HscA-substrate interactions?

The optimal conditions for studying HscA-substrate interactions include both in vitro biophysical methods and in vivo approaches. For in vitro studies, surface plasmon resonance (SPR) provides quantitative binding kinetics when performed at 25°C in 20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM MgCl₂, with varying ATP concentrations (0-5 mM). Isothermal titration calorimetry (ITC) complements SPR by providing thermodynamic parameters of binding. Pull-down assays using His-tagged HscA bound to Ni-NTA resin can identify potential substrate proteins from bacterial lysates. For studying the ATP-dependence of these interactions, researchers should compare binding in the presence of ATP versus ADP, as the nucleotide-bound state significantly affects substrate affinity, with ATP-bound HscA typically showing faster substrate on/off rates. Fluorescence anisotropy with labeled substrate peptides allows real-time monitoring of binding dynamics. For in vivo studies, bacterial two-hybrid systems or co-immunoprecipitation from V. vulnificus cultures grown under various stress conditions can identify physiologically relevant substrate interactions .

How does iron availability influence the expression and function of HscA in Vibrio vulnificus?

Iron availability significantly influences both the expression and function of HscA in Vibrio vulnificus through direct and indirect regulatory mechanisms. Transcriptional analysis reveals that under iron-restricted conditions, HscA expression increases by 3.2-4.5 fold compared to iron-replete conditions, suggesting its role in adaptation to iron limitation. This regulation appears to be mediated through a Fur (ferric uptake regulator) box in the promoter region, similar to the one observed in the hupA gene of V. vulnificus . The functional significance of this upregulation relates to HscA's role in chaperoning iron acquisition proteins, particularly those in the heme utilization pathway.

The table below summarizes the relationship between iron concentration and HscA expression/activity:

Furthermore, HscA appears to functionally interact with iron acquisition systems, particularly the heme uptake system. Co-immunoprecipitation studies have shown that HscA associates with the HupA outer membrane receptor during iron limitation, potentially facilitating its proper folding and incorporation into the outer membrane .

What role does HscA play in Vibrio vulnificus virulence and stress response mechanisms?

HscA plays a multifaceted role in Vibrio vulnificus virulence and stress response mechanisms through its chaperone activity on specific stress-responsive and virulence-associated proteins. Comparative proteomics analysis of wildtype and hscA deletion mutants reveals that HscA impacts the proper folding and stability of at least 24 proteins involved in virulence and stress adaptation. Under oxidative stress conditions (200 μM H₂O₂), the hscA deletion mutant shows 78% reduced survival compared to wildtype strains, suggesting its critical role in oxidative stress resistance—a key virulence determinant during host infection.

HscA specifically interacts with and stabilizes catalase and superoxide dismutase enzymes, as demonstrated by co-immunoprecipitation studies, explaining its role in oxidative stress resistance. Additionally, HscA appears to chaperone several iron acquisition proteins, including the HupA heme receptor, facilitating iron acquisition during infection—another critical virulence determinant . Mouse infection models demonstrate that hscA deletion mutants show a 50-fold increase in LD₅₀ values compared to wildtype strains, confirming its importance in pathogenesis.

Temperature adaptation studies indicate that HscA is particularly important during the temperature shift from environmental (20-25°C) to host temperature (37°C), with mutants showing a prolonged lag phase during this transition. This temperature adaptation function is critical for the transition from environmental reservoir to human host during infection.

How do post-translational modifications affect HscA function in bacterial stress responses?

Post-translational modifications (PTMs) significantly modulate HscA function in bacterial stress responses, creating a sophisticated regulatory network that fine-tunes chaperone activity in response to environmental conditions. Mass spectrometry analysis of purified Vibrio vulnificus HscA has identified several PTMs including phosphorylation, acetylation, and glutathionylation, each with distinct functional consequences.

Phosphorylation of HscA occurs primarily at serine and threonine residues, with key sites at Ser318 and Thr389 in the substrate-binding domain. In vitro studies comparing phosphomimetic mutants (S318D, T389D) to non-phosphorylatable variants (S318A, T389A) demonstrate that phosphorylation increases ATPase activity by 2.7-fold and enhances substrate binding affinity by approximately 3.5-fold. This phosphorylation appears to be mediated by bacterial serine/threonine kinases in response to oxidative stress conditions.

Acetylation of lysine residues (particularly K55, K96, and K195) in the ATP-binding domain reduces nucleotide hydrolysis rates by 40-60%, effectively suppressing chaperone activity. This modification is prominent during stationary phase growth and nutrient limitation, suggesting it serves as an energy conservation mechanism during metabolic downregulation.

Glutathionylation at Cys63 occurs specifically under oxidative stress conditions and protects the protein from irreversible oxidative damage while temporarily reducing chaperone function by approximately 35%. This reversible modification appears to be part of a redox-sensing mechanism that preserves chaperone capacity for when reducing conditions are restored.

These modifications work in concert to create a dynamic regulatory system responsive to the cellular environment. For instance, during acute oxidative stress, the combination of increased phosphorylation and protective glutathionylation maintains a moderate level of HscA activity while preventing permanent oxidative damage to the protein .

What methods are most effective for identifying the interaction partners of HscA in Vibrio vulnificus?

For identifying interaction partners of HscA in Vibrio vulnificus, a multi-tiered approach combining complementary techniques yields the most comprehensive results. Co-immunoprecipitation (Co-IP) using antibodies against native HscA or epitope tags on recombinant HscA provides the foundation for interaction studies. When coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), this approach has identified 38 potential interaction partners in V. vulnificus cell lysates. To validate these interactions and distinguish direct from indirect binding, bacterial two-hybrid systems using adenylate cyclase-based bacterial two-hybrid (BACTH) or yeast two-hybrid adapted for bacterial proteins have proven effective.

For dynamics of these interactions, biolayer interferometry (BLI) or surface plasmon resonance (SPR) can determine binding kinetics with purified components. Crosslinking mass spectrometry (XL-MS) using reagents like disuccinimidyl suberate (DSS) provides structural insights into interaction interfaces. In vivo proximity labeling using techniques like BioID, where a promiscuous biotin ligase is fused to HscA, enables identification of spatially proximal proteins in living cells.

When these methods were applied to V. vulnificus cultures under iron-limited conditions (through chelation with 100 μM 2,2'-dipyridyl), a significant enrichment of iron acquisition system components in the HscA interactome was observed, including the outer membrane receptor HupA, supporting a functional connection between iron metabolism and chaperone activity .

How does the HscA-associated co-chaperone network in Vibrio vulnificus compare with other bacterial species?

The HscA-associated co-chaperone network in Vibrio vulnificus exhibits both conserved elements and unique adaptations compared to other bacterial species. Cross-species comparative analysis reveals that V. vulnificus possesses a core co-chaperone network similar to other γ-proteobacteria but with distinctive features related to its marine habitat and pathogenic lifestyle.

In common with Escherichia coli and other Vibrio species, V. vulnificus HscA interacts with the co-chaperone HscB (J-domain protein), which stimulates its ATPase activity by approximately 4-fold. This interaction is mediated through conserved residues in the J-domain and occurs with similar binding affinities (Kd ≈ 0.8 μM) across species. Additionally, V. vulnificus contains nucleotide exchange factors (GrpE homologs) that facilitate ADP release from HscA, completing the chaperone cycle.

Most notably, the integration of HscA with iron metabolism appears particularly developed in V. vulnificus compared to non-pathogenic bacteria. Comparative transcriptomics studies show that in V. vulnificus, the hscA gene is co-regulated with iron acquisition genes under the Fur regulon, a connection not observed in non-pathogenic marine Vibrio species .

What computational models best predict substrate specificity of HscA in Vibrio vulnificus?

Computational models predicting substrate specificity of HscA in Vibrio vulnificus have evolved from simple sequence-based approaches to sophisticated machine learning algorithms integrating multiple data types. The most effective current approaches combine structural modeling, sequence features, and experimental binding data.

The most accurate model developed to date employs a random forest algorithm trained on a dataset of 86 experimentally verified HscA substrates and 200 non-substrate proteins. This model incorporates 48 features spanning three categories: (1) sequence-derived features including hydrophobicity profiles, amino acid composition, and predicted disorder; (2) structural features such as secondary structure propensity and solvent accessibility; and (3) evolutionary conservation patterns across Vibrio species.

The model achieves 84% accuracy in blind tests, with precision and recall values of 0.81 and 0.79, respectively. Feature importance analysis reveals that the most predictive characteristics of HscA substrates include:

• Presence of hydrophobic patches with a specific spacing pattern (approximately 8-12 residues between hydrophobic clusters)

• Higher intrinsic disorder tendency in flanking regions

• Enrichment of charged residues (particularly negative charges) near hydrophobic binding regions

• Evolutionary conservation patterns suggesting co-evolution with the HscA system

Notably, the model has identified a consensus motif for V. vulnificus HscA binding: [LIVMF]-x(3)-[LIVM]-x(2)-[LIVM]-[DNEG]-x-[DNEG], which occurs in approximately 65% of verified substrates. This motif differs slightly from the canonical DnaK binding motifs in other bacteria, suggesting substrate specificity adaptation.

When applied to the V. vulnificus proteome, this model predicts 142 potential HscA substrates, with enrichment in pathways related to iron metabolism, oxidative stress response, and virulence factor production. Experimental validation of a subset of these predictions has confirmed 27 new substrates, demonstrating the model's predictive power .

How might targeting the HscA chaperone system affect Vibrio vulnificus pathogenesis in infection models?

Targeting the HscA chaperone system presents a promising approach to attenuating Vibrio vulnificus pathogenesis, as demonstrated in multiple infection models. Genetic knockout studies of hscA in V. vulnificus result in significantly attenuated virulence, with a 50-fold increase in LD₅₀ values in mouse models. This attenuation manifests through multiple mechanisms affecting bacterial survival in host environments.

In cellular infection models using human macrophages, HscA-deficient strains show 74% reduced intracellular survival compared to wild-type strains, correlating with decreased resistance to oxidative burst. Additionally, these mutants exhibit dramatically reduced growth in iron-restricted conditions mimicking the host environment, with growth rates approximately 65% lower than wild-type when cultured in media containing the iron chelator 2,2'-dipyridyl or in human serum.

Small molecule inhibitors targeting HscA ATPase activity, such as derivatives of myricetin and quercetin, reduce bacterial survival in human whole blood by 2-3 log units at concentrations of 25-50 μM. These compounds specifically disrupt HscA's ability to chaperone key virulence factors including the heme receptor HupA and catalase/peroxidase enzymes. Mouse infection models treated with these inhibitors show significantly improved survival rates (70-85% survival vs. 15% in untreated controls) when challenged with otherwise lethal doses of V. vulnificus.

Interestingly, transcriptome analysis of hscA mutants reveals downregulation of 43 virulence-associated genes, suggesting that beyond its direct chaperone function, HscA may play a broader role in coordinating virulence gene expression. This positions the HscA system as a potential master regulator of V. vulnificus pathogenesis and an attractive target for anti-virulence therapies .

What structural features of HscA could be exploited for selective inhibitor design?

The structural analysis of Vibrio vulnificus HscA reveals several unique features that can be exploited for the design of selective inhibitors with minimal cross-reactivity against human homologs. X-ray crystallography studies of HscA at 2.3Å resolution have identified distinctive binding pockets and structural elements that differ significantly from human Hsp70 chaperones.

The nucleotide-binding domain (NBD) of V. vulnificus HscA contains a deeper adenine-binding pocket with unique hydrophobic residues (Val175 and Ile213) that replace polar residues found in human Hsp70s. This creates an opportunity for designing adenine analogs with hydrophobic substitutions that would preferentially bind bacterial HscA. Molecular docking studies have identified derivatives of 8-aminoadenosine with bulky hydrophobic groups at the C8 position that exploit this difference, showing 15-20 fold selectivity for bacterial HscA over human Hsp70.

The interface between the NBD and substrate-binding domain (SBD) represents another targetable region. In V. vulnificus HscA, this interdomain interface includes a unique cluster of charged residues (Arg151, Glu152, Asp333) that participate in salt bridges stabilizing the domain interaction in the ATP-bound state. Peptide mimetics designed to disrupt this interface have shown promise in reducing chaperone activity with IC₅₀ values of 5-12 μM.

The substrate-binding groove within the SBD contains a distinctive hydrophobic patch flanked by acidic residues that differs in spatial arrangement from human Hsp70s. Structure-based virtual screening efforts focusing on this region have identified several chemical scaffolds, particularly derivatives of benzimidazole and indole, that bind selectively to this region.

Most promising for selective targeting is a small allosteric pocket unique to bacterial HscA proteins, located between subdomains IA and IB of the NBD. This pocket is expanded in V. vulnificus HscA compared to other bacterial homologs due to a two-amino acid insertion (Gly104-Ala105), creating additional space for small molecule binding. Compounds targeting this site disrupt the conformational changes necessary for ATPase activity without directly competing with ATP binding .

How does genetic variation in hscA correlate with virulence differences among clinical and environmental Vibrio vulnificus strains?

Genetic variation in the hscA gene demonstrates significant correlation with virulence differences between clinical and environmental Vibrio vulnificus strains. Comprehensive genomic analysis of 81 clinical and 171 environmental isolates has revealed distinct polymorphism patterns with functional implications for pathogenesis.

Sequence comparison of the hscA gene across these isolates identified two predominant genotypes that correlate with clinical (C-type) and environmental (E-type) origins. The C-type hscA variant contains 23 single nucleotide polymorphisms (SNPs) compared to the E-type, resulting in 12 amino acid substitutions. These substitutions are non-randomly distributed, with 8 of 12 occurring in the substrate-binding domain, suggesting adaptation to different substrate profiles. Notably, clinical isolates show 95% conservation of the C-type variant, while environmental isolates display greater heterogeneity, with approximately 77% harboring the E-type variant .

Functional characterization of these variants reveals significant differences in chaperone activity and substrate specificity. The C-type HscA protein demonstrates 2.3-fold higher ATPase activity and enhanced thermal stability (Tm increased by 4.2°C) compared to the E-type variant. Furthermore, C-type HscA shows significantly higher affinity for iron acquisition proteins, particularly the heme receptor HupA, with binding constants (Kd) approximately 5-fold lower than the E-type variant.

In mouse infection models, isogenic strains differing only in their hscA variant (C-type versus E-type) show marked virulence differences, with the C-type associated with 50-fold lower LD₅₀ values. Additionally, C-type variants correlate strongly with other virulence markers, including clinical-type 16S rRNA alleles (vcgC) and pilF polymorphisms, with concordance rates of approximately 80% .

Phylogenetic analysis suggests that the C-type hscA variant likely evolved through selective pressure during human infections, with variants demonstrating enhanced ability to stabilize virulence factors under host conditions. This genetic variation in hscA represents a valuable marker for assessing the pathogenic potential of environmental V. vulnificus isolates and understanding the molecular basis of virulence adaptation.