Recombinant Staphylococcus aureus Triosephosphate isomerase (tpiA)

What is Triosephosphate isomerase (TpiA) in Staphylococcus aureus?

Triosephosphate isomerase (TpiA) in S. aureus is a glycolytic enzyme that catalyzes the fifth step of the glycolytic pathway, reversibly isomerizing glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. The molecular weight of the TpiA monomer is approximately 27,000 Da, and it functions primarily as a dimer. Beyond its canonical role in energy production within bacterial cells, TpiA exhibits moonlighting functions outside the cell, particularly plasminogen binding, which may contribute to bacterial virulence .

How does S. aureus TpiA compare structurally to TpiA from other bacterial species?

S. aureus TpiA shares structural similarities with TpiA from other bacterial species, as it is a highly conserved enzyme across various organisms. Like other TpiA homologs, S. aureus TpiA functions as a dimer and contains the characteristic TIM barrel fold. Structurally, it is closely related to Streptococcus pneumoniae TpiA, which has also been identified as a plasminogen-binding protein . Both staphylococcal and streptococcal TpiA proteins represent examples of moonlighting proteins that perform additional functions beyond their primary metabolic roles when released extracellularly.

What are the primary and secondary functions of TpiA in S. aureus pathogenesis?

The primary function of TpiA in S. aureus is intracellular, where it catalyzes an essential step in glycolysis, contributing to energy production. Its secondary function occurs extracellularly, where it acts as a moonlighting protein that binds to host plasminogen. This plasminogen-binding activity is thought to be relevant for staphylococcal invasion of host tissues . When TpiA binds to plasminogen, it can promote the conversion of plasminogen to plasmin, a serine protease that degrades fibrin clots and extracellular matrix components, potentially facilitating bacterial spread through host tissues .

How effective is recombinant TpiA as a vaccine candidate against S. aureus infections?

Recombinant TpiA has shown significant promise as a vaccine candidate against S. aureus infections in experimental models. In murine studies, immunization with recombinant TpiA (rTPI) elicited high titers of IgG antibodies recognizing the staphylococcal TpiA protein. This immunization provided significant protection against S. aureus bacteremia when mice were challenged with the MSSA strain ATCC29213 . The efficacy appears to be related to TpiA's non-redundant and essential function in S. aureus, as well as its accessibility to antibodies when released extracellularly. The protective effect of anti-TpiA antibodies suggests that targeting this protein could be a valuable strategy for vaccine development against S. aureus infections.

What is the role of monoclonal antibodies against TpiA in protection against S. aureus?

Monoclonal antibodies (mAbs) raised against TpiA, specifically TPI-H8, have demonstrated significant protective efficacy against S. aureus infection in mouse models. The TPI-H8 mAb recognizes a large discontinuous epitope on TpiA . When administered to mice, these antibodies significantly improved survival in a murine sepsis model, indicating their potential therapeutic value. This protection mechanism likely involves neutralization of TpiA's extracellular functions, particularly its plasminogen-binding activity, which may interfere with S. aureus invasion and spread within host tissues. The success of these monoclonal antibodies supports the "reverse vaccinology 2.0" concept, where mAbs are used to distinguish protective from non-protective epitopes and to guide focused vaccine design .

How does TpiA compare to other moonlighting proteins as vaccine targets for S. aureus?

TpiA represents one of several moonlighting proteins identified as potential vaccine candidates against S. aureus. Another notable example is coproporphyrinogen III oxidase (CgoX), which has shown similar protective effects in immunization studies . When compared to other moonlighting proteins, TpiA offers several advantages: (1) it performs a non-redundant, essential function in S. aureus metabolism, (2) it has an accessible extracellular component that can be targeted by antibodies, and (3) targeting its extracellular function is not expected to drive the development of escape mutants since its intracellular essential function remains inaccessible to antibodies . Additionally, unlike some other vaccine candidates that may induce disease-enhancing antibodies, TpiA immunization has demonstrated a clear protective effect without evidence of immune enhancement.

What are the optimal protocols for expressing and purifying recombinant S. aureus TpiA?

For optimal expression and purification of recombinant S. aureus TpiA, the following methodology has proven successful:

• Cloning: The tpiA gene should be amplified from S. aureus genomic DNA and cloned into an expression vector such as pET-based vectors with a His6-tag for purification.

• Expression: Transform the construct into E. coli expression strains (typically BL21(DE3) or derivatives). Culture the transformed bacteria in LB medium with appropriate antibiotics at 37°C until reaching OD600 of 0.6-0.8, then induce protein expression with IPTG (typically 0.5-1 mM) for 3-4 hours at 30°C to minimize inclusion body formation .

• Purification: Harvest cells by centrifugation, lyse by sonication in binding buffer containing imidazole (10-20 mM), and purify using nickel affinity chromatography. Elute the His6-tagged TpiA with an imidazole gradient (typically 250-500 mM) .

• Quality Control: Assess purity and integrity by SDS-PAGE and confirm identity by Western blot analysis using anti-His antibodies or specific anti-TpiA antibodies. Dialyze the purified protein against PBS to remove imidazole for downstream applications .

What immunization protocols are most effective for generating protective immunity against S. aureus using recombinant TpiA?

Based on successful murine studies, the following immunization protocol has demonstrated effectiveness for generating protective immunity against S. aureus using recombinant TpiA:

• Initial Immunization: Administer 80 μg of purified recombinant TpiA intraperitoneally (i.p.) with Freund's complete adjuvant.

• Boost Regimen: Provide two booster immunizations with 40 μg of antigen subcutaneously (s.c.) with incomplete Freund's adjuvant at days 33 and 56 after the initial immunization.

• Immunity Assessment: Monitor antibody response by ELISA to confirm the development of high titers of IgG antibodies recognizing recombinant S. aureus TpiA.

• Challenge: Challenge immunized animals with a virulent S. aureus strain (such as MSSA strain ATCC29213) intravenously 8 days after the second boost to assess protection .

This protocol has been shown to induce significant protection against S. aureus bacteremia in mouse models. For translational research toward human applications, alternative adjuvants approved for human use (like aluminum salts or MF59) would need to be evaluated.

How can the plasminogen-binding activity of TpiA be quantitatively assessed?

The plasminogen-binding activity of TpiA can be quantitatively assessed using several complementary techniques:

• Surface Plasmon Resonance (SPR/Biacore): This technique provides real-time binding kinetics between TpiA and plasminogen. Purified TpiA is immobilized on a sensor chip, and varying concentrations of plasminogen are flowed over the surface. This allows determination of association (ka) and dissociation (kd) rate constants, as well as equilibrium dissociation constants (KD) .

• Ligand Blot Analysis: Purified TpiA proteins are separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with purified plasminogen. After washing, bound plasminogen is detected using anti-plasminogen antibodies followed by appropriate secondary antibodies and visualization systems .

• Enzyme-Linked Immunosorbent Assay (ELISA): Microtiter plates are coated with TpiA, blocked, and incubated with increasing concentrations of plasminogen. Bound plasminogen is detected using specific antibodies and quantified spectrometrically .

• Plasminogen Activation Assay: This functional assay measures TpiA's ability to enhance plasminogen activation by tissue plasminogen activator (tPA) or urokinase. The generation of plasmin is monitored using chromogenic substrates specific for plasmin activity .

How does S. aureus release TpiA extracellularly, and what triggers this process?

S. aureus releases TpiA extracellularly through mechanisms that likely involve bacterial autolysis, similar to what has been observed with S. pneumoniae TpiA. In S. pneumoniae, the release of TpiA into the culture medium has been shown to be dependent on autolysin . This suggests that programmed cell lysis contributes significantly to the extracellular presence of this otherwise cytoplasmic enzyme.

The triggers for TpiA release might include:

• Growth Phase Dependence: TpiA release may increase during late exponential or stationary phase when autolysis naturally increases.

• Environmental Stress: Conditions such as antibiotic exposure, nutrient limitation, or host immune factors might enhance autolysis and subsequent TpiA release.

• Quorum Sensing: The agr system, which regulates many virulence factors in S. aureus, might indirectly influence TpiA release by modulating autolysin expression .

• Host Factors: Certain host immune components or matrix proteins might trigger increased autolysis and TpiA release during infection.

Further research is needed to fully elucidate the specific triggers and regulatory mechanisms controlling TpiA release in S. aureus under different environmental conditions and during infection.

What is the mechanism by which anti-TpiA antibodies provide protection against S. aureus infections?

Anti-TpiA antibodies likely provide protection against S. aureus infections through several complementary mechanisms:

• Inhibition of Plasminogen Binding: Antibodies may directly block the interaction between TpiA and host plasminogen, preventing the recruitment and activation of this host protease system. This would inhibit S. aureus' ability to degrade extracellular matrix components and spread through tissues .

• Enhanced Opsonization: Anti-TpiA antibodies may bind to surface-associated or released TpiA, tagging the bacteria for recognition and phagocytosis by immune cells such as neutrophils and macrophages.

• Complement Activation: Bound antibodies can activate the classical complement pathway, leading to the formation of membrane attack complexes and enhanced clearance of S. aureus.

• Neutralization of Moonlighting Functions: Beyond plasminogen binding, TpiA may have additional uncharacterized extracellular functions that contribute to virulence, which could be neutralized by specific antibodies.

• Prevention of Tissue Invasion: By inhibiting the plasminogen activation cascade, anti-TpiA antibodies may restrict the ability of S. aureus to penetrate tissue barriers and establish systemic infection .

Research has demonstrated that immunization with rTPI elicits protective immunity against S. aureus bacteremia, supporting these proposed protective mechanisms .

How does TpiA's dual role as a glycolytic enzyme and virulence factor affect strategies for therapeutic targeting?

TpiA's dual role as an essential glycolytic enzyme and a virulence factor presents both opportunities and challenges for therapeutic targeting:

Advantages:

• Essential Function: As TpiA is essential for S. aureus glycolysis and energy production, resistance mutations that completely abolish TpiA function would be lethal, potentially reducing the emergence of resistance to TpiA-targeting therapies .

• Moonlighting Separation: The intracellular metabolic function of TpiA is physically separated from its extracellular virulence function, allowing antibodies to target the virulence function without affecting bacterial viability directly. This creates a potential "resistance-proof" targeting strategy that doesn't directly drive selection for resistant mutants .

• Conserved Structure: The high conservation of TpiA across S. aureus strains suggests that therapies targeting this protein could be broadly effective against diverse clinical isolates.

Challenges:

• Functional Redundancy: There may be functional redundancy in plasminogen binding among different S. aureus proteins, potentially limiting the efficacy of TpiA-only targeting approaches.

• Selective Pressure: While complete loss of TpiA function would be lethal, mutations that specifically affect antibody binding but preserve enzymatic function could emerge under selective pressure.

• Accessibility: Effective targeting requires TpiA to be accessible to antibodies or other therapeutic agents, which depends on the timing and extent of its release from bacteria.

Optimal therapeutic strategies might include combining TpiA targeting with other approaches, such as antibiotics or targeting of other virulence factors, to enhance efficacy and reduce the potential for resistance development.

How does S. aureus TpiA compare functionally to TpiA from Streptococcus pneumoniae?

S. aureus TpiA and S. pneumoniae TpiA share significant functional similarities but also exhibit some important differences:

Similarities:

Differences:

Understanding these similarities and differences is important for developing targeted therapies and determining whether findings from one bacterial species can be extrapolated to another.

What is the relationship between TpiA and other moonlighting proteins in S. aureus vaccine development?

In S. aureus vaccine development, TpiA is one of several moonlighting proteins that have been identified as potential vaccine candidates. The relationship between TpiA and other moonlighting proteins can be characterized as follows:

• Complementary Targets: TpiA and coproporphyrinogen III oxidase (CgoX) have both been identified as promising vaccine candidates in the same screening approaches. Both proteins induce protective immunity when used for immunization in mouse models of S. aureus infection .

• Structural and Functional Diversity: While TpiA is involved in glycolysis and plasminogen binding, other S. aureus moonlighting proteins serve different metabolic functions internally while contributing to virulence externally. This functional diversity could be advantageous in multi-target vaccine approaches.

• Epitope-Specific Immunity: The protective efficacy of these moonlighting proteins appears to be epitope-specific. For TpiA, a large discontinuous epitope recognized by the monoclonal antibody TPI-H8 was associated with protection. Similarly, for CgoX, a linear epitope of just 12 amino acids recognized by the CgoX-D3 mAb provided protective immunity .

• Vaccine Formulation Strategy: The recognition that multiple moonlighting proteins can provide protection suggests potential benefit in combining several such antigens in a multi-component vaccine to broaden protection. Alternatively, focusing on specific protective epitopes from each protein could result in a more defined epitope-focused vaccine with improved safety and efficacy .

This epitope-focused approach aligns with the "reverse vaccinology 2.0" concept, where monoclonal antibodies help distinguish protective from non-protective epitopes to guide more precise vaccine design .

What are the most promising directions for future research on S. aureus TpiA?

The most promising directions for future research on S. aureus TpiA include:

• Structural Elucidation of TpiA-Plasminogen Interaction: Determining the precise molecular interaction between TpiA and plasminogen through crystallography or cryo-EM would enable structure-based design of inhibitors or improved vaccine components.

• Identification of Additional Moonlighting Functions: Beyond plasminogen binding, TpiA may have other undiscovered extracellular functions that contribute to S. aureus pathogenesis. Comprehensive functional screens could reveal additional roles.

• Development of Epitope-Based Vaccines: Further characterization of protective TpiA epitopes could lead to the design of epitope-focused vaccines that elicit more precise immune responses with greater efficacy and safety profiles .

• Investigation of Synergistic Effects: Testing combinations of TpiA with other vaccine candidates like CgoX could reveal synergistic protection, potentially leading to more effective multi-component vaccines .

• Translation to Clinical Studies: Moving promising TpiA-based vaccine candidates from animal models to human clinical trials, with careful monitoring of safety and efficacy.

• TpiA as a Biomarker: Exploring the potential of TpiA as a diagnostic biomarker for S. aureus infections, possibly through detection of circulating TpiA or anti-TpiA antibodies in patient samples.

• Development of TpiA-Targeting Therapeutics: Designing inhibitors that specifically target TpiA's moonlighting functions without affecting its essential metabolic role could provide novel therapeutic approaches with reduced selection for resistance.

These research directions collectively represent a comprehensive approach to understanding and exploiting TpiA for both preventive and therapeutic strategies against S. aureus infections.

What analytical techniques are most suitable for assessing TpiA structure-function relationships?

For comprehensive analysis of TpiA structure-function relationships, several complementary analytical techniques are recommended:

By combining these techniques, researchers can establish comprehensive structure-function relationships for both the enzymatic and moonlighting activities of TpiA.

How can epitope mapping be effectively performed for TpiA to identify protective versus non-protective epitopes?

Effective epitope mapping for TpiA to distinguish protective from non-protective epitopes can be achieved through a multi-method approach:

• Peptide Array Analysis: Synthesize overlapping peptides (typically 12-15 amino acids with 1-2 amino acid offsets) spanning the entire TpiA sequence. Screen these peptides for binding to protective monoclonal antibodies like TPI-H8. This approach is particularly useful for identifying linear epitopes.

• Alanine Scanning Mutagenesis: Systematically replace individual amino acids with alanine in regions of interest to identify critical residues required for antibody binding. This can be especially valuable for mapping discontinuous epitopes like those recognized by TPI-H8 .

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This technique can identify regions of TpiA that are protected from solvent exchange when bound to protective antibodies, helping to map conformational epitopes.

• X-ray Crystallography of Antibody-Antigen Complexes: Determining the crystal structure of TpiA in complex with the Fab fragment of protective antibodies provides the most detailed information about epitope-paratope interactions.

• Phage Display: Random peptide libraries displayed on phage can be screened against protective antibodies to identify mimotopes that may represent conformational epitopes.

• Competitive Binding Assays: These can determine if different antibodies recognize overlapping or distinct epitopes on TpiA, helping to create an epitope map.

• In Vivo Protection Studies: Test the protective efficacy of antibodies against mapped epitopes in animal models. This is crucial for distinguishing protective from non-protective epitopes .

• Epitope-Specific Immunization: Conjugate identified epitope peptides to carrier proteins and test their ability to elicit protective immunity in animal models, as demonstrated for the CgoX-D3 epitope .

This comprehensive approach can guide the development of epitope-focused vaccines with improved safety and efficacy profiles, aligning with the "reverse vaccinology 2.0" concept .

What are the key considerations for designing TpiA-based vaccines for clinical trials?

Designing TpiA-based vaccines for clinical trials requires careful consideration of multiple factors:

• Antigen Design and Selection:

  • Focus on protective epitopes identified through epitope mapping studies

  • Consider using full-length rTpiA versus selected epitope regions

  • Ensure proper protein folding and epitope presentation

  • Evaluate stability and shelf-life of the antigen formulation

• Adjuvant Selection:

  • Replace research-grade adjuvants like Freund's with clinical-grade alternatives

  • Test multiple adjuvants (e.g., aluminum salts, MF59, AS01, AS04) for optimal immunogenicity

  • Balance immunogenicity with safety profile

• Dosing and Formulation:

  • Optimize antigen dose based on preclinical dose-response studies

  • Determine optimal immunization schedule (primary and booster doses)

  • Develop stable formulation with appropriate buffer systems

• Safety Considerations:

  • Assess potential cross-reactivity with human proteins

  • Evaluate risk of disease enhancement (although not observed in preclinical studies)

  • Monitor for autoimmune responses

• Efficacy Parameters:

  • Define clear primary and secondary endpoints

  • Establish correlates of protection based on antibody titers or functional assays

  • Design appropriate challenge models for early-phase trials

• Target Population:

  • Define appropriate subject population (e.g., healthy adults, high-risk patients)

  • Consider potential differences in immune response across age groups

• Combination Approaches:

  • Evaluate potential synergy with other S. aureus antigens like CgoX

  • Consider inclusion in multi-component vaccine formulations

• Manufacturing Considerations:

  • Develop scalable GMP production processes

  • Ensure batch-to-batch consistency and purity

  • Implement appropriate quality control measures

• Regulatory Strategy:

  • Design trials in accordance with regulatory guidelines

  • Plan for appropriate safety monitoring and reporting

  • Prepare comprehensive Investigational New Drug (IND) application

By addressing these considerations systematically, researchers can enhance the likelihood of developing a safe and effective TpiA-based vaccine for S. aureus infections, addressing an important unmet medical need .