Recombinant Staphylococcus aureus 10 kDa chaperonin (groS)

What is the molecular function of the 10 kDa chaperonin (groS) in S. aureus?

The 10 kDa chaperonin, also known as groES, is a critical molecular chaperone that works in conjunction with groEL (a larger chaperonin) to facilitate proper protein folding in S. aureus. GroES forms a homoheptameric ring structure that binds to one or both ends of the groEL double barrel in the presence of adenine nucleotides, effectively capping it . This protein complex is essential for bacterial survival, particularly under stress conditions, as it prevents protein aggregation and facilitates proper folding of nascent or stress-denatured proteins. The folding of unfolded substrates initiates when they are bound to groEL and capped by groES, with the subsequent release of properly folded substrates depending on ATP binding and hydrolysis in the trans ring .

How does the structure of S. aureus groS relate to its function?

S. aureus groS forms a dome-shaped homoheptameric ring structure approximately 10 kDa in size. Each monomer contributes to the formation of a central cavity that is critical for its chaperoning function. The protein contains mobile loop regions that interact with groEL during the protein folding cycle. When ATP binds to groEL, conformational changes occur that allow groES to cap the groEL chamber, creating an enclosed environment where substrate proteins can fold protected from the cellular environment. This structural arrangement is highly conserved across bacterial species, reflecting the essential nature of this protein folding machinery.

What is the relationship between groS and S. aureus pathogenicity?

While the search results don't directly address the specific role of groS in S. aureus virulence, we can infer its importance based on general bacterial pathogenesis principles. S. aureus is a significant human pathogen responsible for various infections ranging from minor skin infections to life-threatening conditions like pneumonia, meningitis, and sepsis . As a chaperonin, groS likely contributes to pathogenicity by:

The ability of S. aureus to adapt to various environments within the human host depends partly on properly functioning chaperone systems, making groS an important indirect contributor to pathogenicity.

What are the optimal expression systems for recombinant S. aureus groS?

Several expression systems can be employed for recombinant S. aureus groS production, with E. coli being the most common host organism according to available research data . The following methodological considerations are important:

Expression Hosts:

• E. coli strains (BL21(DE3), Rosetta) are commonly used for high-yield production

• Yeast expression systems may be considered when bacterial expression proves challenging

Vector Selection:

• pET series vectors containing T7 promoters offer strong, inducible expression

• Vectors with fusion tags (His, GST) facilitate downstream purification

Expression Conditions Table:

The search results indicate that recombinant S. aureus groS proteins with His-tags are commercially available, suggesting this is a successful expression approach .

What purification strategies yield the highest purity and functional activity?

Purification of recombinant S. aureus groS typically follows a multi-step chromatographic approach:

• Affinity Chromatography:

  • For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins provides good initial purification

  • Different bacterial sources of recombinant groS, including S. aureus, are purified using this approach as shown in the product listings

• Ion Exchange Chromatography:

  • Due to its defined charge properties, groS can be further purified using anion or cation exchange chromatography

  • This step is particularly valuable for removing contaminating nucleic acids and bacterial proteins

• Size Exclusion Chromatography:

  • Critical for isolating properly assembled heptameric groS and removing any aggregates

  • Provides information about the oligomeric state of the purified protein

Buffer Optimization:

• Phosphate or HEPES buffers (pH 7.0-8.0)

• Moderate salt concentrations (100-300 mM NaCl)

• Addition of ATP or ADP (1-5 mM) stabilizes the protein

• Reducing agents (DTT or β-mercaptoethanol) prevent unwanted disulfide formation

How can researchers validate the correct folding and activity of purified groS?

Validation of correctly folded and functionally active recombinant S. aureus groS can be accomplished through multiple complementary approaches:

• Structural Validation:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure

  • Size exclusion chromatography to verify heptameric assembly

  • Dynamic light scattering to assess homogeneity and oligomeric state

  • Negative-stain electron microscopy to visualize the characteristic ring structure

• Functional Validation:

  • In vitro protein folding assays using model substrates (malate dehydrogenase, citrate synthase)

  • ATPase activity assays in conjunction with groEL

  • Thermal shift assays to assess protein stability

  • Surface plasmon resonance to measure binding kinetics with groEL

• Biological Validation:

  • Complementation assays in groS-deficient bacterial strains

  • Protection assays against heat or chemical denaturation of client proteins

A combination of these approaches provides comprehensive validation of the structural integrity and functional activity of recombinant groS.

How can recombinant S. aureus groS contribute to vaccine development efforts?

S. aureus is a significant public health concern with increasing antibiotic resistance, and despite considerable research, no vaccine has been approved . Recombinant groS could potentially contribute to vaccine development in several ways:

• As a Carrier Protein:

  • The search results mention glycoengineering technology for creating multicomponent staphylococcal vaccines, where genes encoding S. aureus capsular polysaccharide biosynthesis were coexpressed with a protein carrier

  • GroS could potentially serve as a carrier protein in such conjugate vaccine approaches

• As an Immunomodulator:

  • Bacterial heat shock proteins including chaperonins can have immunomodulatory properties

  • These properties might enhance immune responses to other S. aureus antigens in a multicomponent vaccine

• As a Conserved Antigen:

  • The relatively conserved nature of groS across S. aureus strains makes it a potentially valuable component in a multi-antigen vaccine

  • Single-component vaccines targeting S. aureus have failed to show efficacy in clinical trials , suggesting multicomponent approaches incorporating proteins like groS may be more promising

• In Combination Approaches:

  • The search results mention a novel glycoengineering technology for creating a multicomponent staphylococcal vaccine

  • Such approaches could potentially incorporate groS as part of a comprehensive multi-antigen strategy

What methodological approaches can elucidate groS interactions with other S. aureus proteins?

Several methodological approaches can be employed to study groS interactions with other S. aureus proteins:

• Co-Immunoprecipitation Studies:

  • Using antibodies against groS to pull down protein complexes from S. aureus lysates

  • Mass spectrometry analysis of co-precipitated proteins to identify interaction partners

• Bacterial Two-Hybrid Screening:

  • Systematic screening of S. aureus genomic libraries to identify proteins that interact with groS

  • Validation of interactions through targeted follow-up experiments

• Crosslinking Mass Spectrometry:

  • Chemical crosslinking of protein complexes followed by mass spectrometry analysis

  • This approach can capture transient interactions and provide structural information

• Surface Plasmon Resonance:

  • Quantitative measurement of binding kinetics between purified groS and candidate interacting proteins

  • Real-time analysis of association and dissociation rates

• Cryo-Electron Microscopy:

  • Structural analysis of groES-groEL-substrate complexes

  • Visualization of conformational changes during the chaperonin cycle

These approaches can reveal both the client proteins that depend on groS for folding and other potential regulatory interactions within the S. aureus proteome.

How does groS conservation across S. aureus lineages inform evolutionary studies?

The search results indicate that S. aureus can be sorted into ten dominant human lineages with numerous minor lineages . Approximately 22% of the S. aureus genome is non-coding and can differ between bacteria . Analysis of groS conservation across these lineages can provide valuable evolutionary insights:

• Sequence Conservation Analysis:

  • Highly conserved proteins like chaperonins can serve as molecular clocks for evolutionary studies

  • Comparing groS sequences across the dominant S. aureus lineages can reveal evolutionary relationships

  • The rate of synonymous versus non-synonymous mutations in groS can indicate selective pressures

• Functional Constraints:

  • The essential nature of groS likely imposes functional constraints that limit variation

  • Any observed variations might indicate adaptations to specific ecological niches or host environments

• Co-evolution Patterns:

  • Examining how groS and groEL co-evolve can reveal insights into the maintenance of chaperonin system functionality

  • Correlating groS evolution with changes in client proteins could identify co-evolutionary networks

• Horizontal Gene Transfer Assessment:

  • Determining whether groS shows evidence of horizontal gene transfer between S. aureus lineages or from other species

  • This could provide insights into the evolutionary mechanisms shaping the S. aureus genome

The evolutionary analysis of groS can contribute to our understanding of how S. aureus has adapted to diverse environments while maintaining essential cellular functions.

What are common issues in recombinant groS expression and how can they be resolved?

Researchers frequently encounter several challenges when expressing recombinant S. aureus groS:

• Inclusion Body Formation:

  • Problem: Overexpressed groS aggregates in insoluble inclusion bodies

  • Solutions:

    • Lower induction temperature (16-20°C)

    • Reduce IPTG concentration (0.1-0.2 mM)

    • Co-express with groEL or other chaperones

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

• Low Expression Levels:

  • Problem: Poor yield of target protein

  • Solutions:

    • Optimize codon usage for the expression host

    • Try different promoter systems or expression vectors

    • Screen multiple E. coli strains for optimal expression

    • Adjust media composition and growth conditions

• Protein Instability:

  • Problem: Rapid degradation of expressed protein

  • Solutions:

    • Add stabilizing agents in buffers (glycerol, ATP)

    • Include protease inhibitors during purification

    • Maintain cold temperatures throughout processing

    • Consider storing the protein with its co-chaperonin groEL

• Improper Oligomerization:

  • Problem: Failure to form correct heptameric structure

  • Solutions:

    • Include molecular crowding agents during purification

    • Use native purification conditions

    • Develop refolding procedures that facilitate proper assembly

Each of these challenges requires systematic troubleshooting approaches and may necessitate customizing protocols for the specific properties of S. aureus groS.

How can researchers address data inconsistencies in groS functional assays?

When researchers encounter inconsistent results in groS functional assays, several methodological considerations can help resolve these discrepancies:

• Standardize Protein Quality:

  • Ensure consistent protein purity across experiments (>95% by SDS-PAGE)

  • Verify oligomeric state by size exclusion chromatography before each assay

  • Quantify protein concentration using multiple methods (Bradford, BCA, A280)

• Control Assay Conditions:

  • Maintain precise temperature control during assays (±0.5°C)

  • Prepare fresh ATP solutions for each experiment

  • Use the same buffer system across experiments with carefully controlled pH

• Assess Protein Stability:

  • Monitor protein stability over time under assay conditions

  • Determine if activity loss correlates with structural changes

  • Implement stability-enhancing additives if necessary

• Validate with Multiple Substrates:

  • Use different model substrate proteins to confirm activity patterns

  • Compare results with known literature values for similar chaperonin systems

  • Develop internal controls for day-to-day variability

• Consider Partner Proteins:

  • Ensure consistent quality of partner proteins (groEL) if used in assays

  • Standardize the ratio of groES to groEL in complex formation

  • Verify complex formation before functional testing

By systematically addressing these factors, researchers can identify the source of inconsistencies and develop more reliable assay protocols.

What strategies can improve yield and functional activity of recombinant groS?

To optimize both yield and functional activity of recombinant S. aureus groS, researchers can implement these methodology-focused strategies:

• Expression System Optimization:

  • Use strong, tightly regulated promoters (T7 or tac systems)

  • Select expression hosts with reduced protease activity (BL21(DE3) pLysS)

  • Consider co-expression with molecular chaperones

• Culture Condition Refinement:

  • Implement fed-batch fermentation to achieve higher cell densities

  • Optimize oxygen transfer rates in cultivation vessels

  • Use enriched media formulations supplemented with amino acids and vitamins

• Co-factor Supplementation:

  • Add metal ions required for structural stability (Mg²⁺)

  • Include ATP or ATP analogs during purification

  • Supply potential co-factors during expression and purification

• Post-translational Handling:

  • Minimize freeze-thaw cycles by aliquoting purified protein

  • Store with stabilizing additives (glycerol, ATP, reducing agents)

  • Consider lyophilization protocols developed for oligomeric proteins

• Activity Preservation Techniques:

  • Perform activity assays immediately after purification

  • Test various buffer compositions for long-term storage

  • Investigate protein immobilization techniques for enhanced stability

These methodological approaches, when systematically implemented and optimized for S. aureus groS, can significantly enhance both yield and functional activity of the recombinant protein.

How is groS being explored as a potential therapeutic target?

While the search results don't specifically address groS as a therapeutic target in S. aureus, we can infer potential approaches based on chaperonin research:

• Small Molecule Inhibitor Development:

  • Compounds that prevent groES binding to groEL could disrupt essential protein folding machinery

  • Molecules targeting the ATP binding site of the groEL-groS system

  • Allosteric modulators that alter groES conformation

• Combination Therapy Approaches:

  • Using groS inhibitors to sensitize S. aureus to existing antibiotics

  • This approach may be particularly valuable against methicillin-resistant S. aureus (MRSA), which is noted as a significant clinical problem

• Structure-Based Drug Design:

  • Using structural information about groES and its interactions to design targeted inhibitors

  • Virtual screening of compound libraries against the groES structure

• Novel Delivery Systems:

  • Nanoparticle-based delivery of inhibitors specifically to sites of S. aureus infection

  • Targeted delivery systems that recognize S. aureus biofilms

The essential nature of the groEL-groS system for bacterial survival makes it a potentially valuable target for novel antimicrobial development, particularly given the global challenge of antimicrobial resistance in S. aureus .

What is the potential role of groS in S. aureus adaptation to different host environments?

S. aureus is highly adaptable and can colonize diverse host environments. The potential role of groS in this adaptation includes:

• Stress Response Modulation:

  • GroS helps S. aureus survive temperature fluctuations, oxidative stress, and other host-imposed stresses

  • This may facilitate adaptation to different anatomical locations within the host

• Protein Quality Control:

  • Different host environments may require distinct sets of proteins for colonization

  • GroS ensures proper folding of proteins needed for adaptation to specific niches

• Biofilm Formation Support:

  • S. aureus forms biofilms that contribute to persistent infections

  • Chaperonins may play a role in maintaining protein functionality within the biofilm environment

• Host-Pathogen Interface:

  • GroS may help maintain the function of proteins involved in host-pathogen interactions

  • This could influence the bacterium's ability to evade host immune responses

Understanding groS's role in S. aureus adaptation could provide insights into the bacterium's remarkable ability to persist in various host environments and cause a wide spectrum of diseases .

How does the analysis of groS contribute to understanding antibiotic resistance mechanisms?

Antibiotic resistance in S. aureus, particularly methicillin-resistant strains (MRSA), is a major global health concern . Analysis of groS may contribute to understanding resistance mechanisms in several ways:

• Protective Functions:

  • GroS may help maintain the folding and function of proteins involved in antibiotic resistance

  • This could include enzymes that modify antibiotics or cell wall synthesis proteins that are targets for β-lactam antibiotics

• Stress Response Coordination:

  • Antibiotic exposure creates cellular stress that may upregulate chaperonin systems

  • GroS could be part of the adaptive response that helps bacteria survive antibiotic challenge

• Evolutionary Adaptations:

  • Comparing groS sequences and expression patterns between susceptible and resistant strains may reveal adaptations

  • Such differences could contribute to the enhanced fitness of resistant strains under antibiotic pressure

• Novel Intervention Strategies:

  • Understanding how groS contributes to resistance could inform new approaches to combat MRSA

  • Targeting chaperonin systems in combination with existing antibiotics might overcome resistance mechanisms

Given that S. aureus is "one of the leading pathogens for deaths associated with antimicrobial resistance" , understanding all aspects of its cellular machinery, including the role of groS, is critical for developing effective interventions.