Recombinant Shigella flexneri serotype 5b ATP synthase subunit a (atpB)

What is ATP synthase subunit a (atpB) in Shigella flexneri serotype 5b and what is its function?

ATP synthase subunit a (atpB) in S. flexneri serotype 5b is a membrane-embedded component of the F0 sector of ATP synthase. This 271-amino acid protein plays a crucial role in proton translocation across the bacterial membrane, which is essential for ATP synthesis. The protein functions as part of a complex that couples proton movement to energy production, making it essential for bacterial survival . The atpB protein contributes to intracellular pH regulation, which is particularly important for the bacterium's acid resistance mechanisms - a critical virulence determinant that allows S. flexneri to survive passage through the acidic environment of the human stomach .

How is recombinant S. flexneri atpB protein typically expressed and purified?

According to available data, recombinant S. flexneri serotype 5b atpB protein is commonly expressed in E. coli expression systems with an N-terminal His tag to facilitate purification . Standard protocols include:

• Expression using E. coli as the host organism

• Purification to >90% purity as determined by SDS-PAGE

• Production as a lyophilized powder for storage stability

• Reconstitution in deionized sterile water to concentrations of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (final concentration) for long-term storage

For optimal results, the reconstituted protein should be stored at -20°C/-80°C, with aliquoting recommended to avoid repeated freeze-thaw cycles. Working aliquots can be maintained at 4°C for up to one week .

How does the virulence plasmid (VP) affect ATP synthase expression and function in S. flexneri?

Research has revealed a complex relationship between the virulence plasmid (VP) of S. flexneri and ATP synthase expression. Blue Native/SDS-PAGE (BN/SDS-PAGE) and Isoelectric Focusing/SDS-PAGE (IEF/SDS-PAGE) 2-D electrophoresis analyses have demonstrated that deletion of the VP significantly increases the abundance of the ATP synthase complex, while the abundance of individual protein monomers remains relatively unchanged .

The presence of the VP reduces intracellular ATP synthesis by decreasing the expression or stability of the assembled ATP synthase complex. This was confirmed through ATP quantification assays, which showed that ATP concentrations were greatly increased in VP-deletion mutant strains . This regulatory mechanism may represent an adaptive strategy that balances energy production with virulence factor expression, optimizing bacterial fitness during infection.

Interestingly, the VP-mediated suppression of ATP synthase activity correlates with decreased bacterial survival in extremely acidic environments, such as the host stomach . This suggests a complex interplay between energy metabolism, acid resistance, and virulence that researchers must consider when studying S. flexneri pathogenesis.

What methodologies are most effective for studying ATP synthase complex formation in S. flexneri?

For investigating ATP synthase complex formation and function in S. flexneri, researchers have successfully employed several complementary approaches:

• Blue Native/SDS-PAGE (BN/SDS-PAGE): This technique preserves protein-protein interactions during electrophoresis, allowing visualization of intact ATP synthase complexes .

• Isoelectric Focusing/SDS-PAGE (IEF/SDS-PAGE): This 2-D electrophoresis approach separates proteins first by their isoelectric point and then by molecular weight, providing detailed expression profiles of protein complexes and monomers .

• Western blot analysis: This technique allows detection of specific ATP synthase subunits, as demonstrated with AtpD in previous studies .

• ATP activity assays: Luminescence-based ATP detection systems can quantify ATP levels as a functional readout of ATP synthase activity .

• Mass spectrometry: MALDI-TOF mass spectrometry has been used to identify differentially expressed proteins in ATP synthase complexes .

For more detailed structural studies, techniques such as cryo-electron microscopy, X-ray crystallography, or hydrogen-deuterium exchange mass spectrometry could provide valuable insights into complex assembly and conformational dynamics.

How can researchers effectively measure the impact of atpB mutations on S. flexneri virulence?

To effectively assess how atpB mutations affect S. flexneri virulence, researchers should employ a multi-faceted approach:

• Generation of atpB variants:

  • Site-directed mutagenesis targeting functional domains

  • Construction of conditional expression systems for essential residues

  • Generation of chimeric proteins to identify domain-specific functions

• In vitro phenotypic assays:

  • ATP synthesis/hydrolysis assays using luminescence detection systems

  • Acid resistance testing to measure survival at low pH (pH 2-3)

  • Intracellular pH measurements using fluorescent probes

  • Membrane potential assays to assess proton motive force

• Cellular infection models:

  • Epithelial cell invasion assays (similar to methods used for studying RACK1 in S. flexneri)

  • Intracellular replication quantification

  • Cell-to-cell spread assessment

  • Host cell cytokine response measurement

• Complex formation analysis:

  • BN/SDS-PAGE to evaluate ATP synthase assembly in mutants

  • Protein-protein interaction studies to identify altered subunit interactions

• In vivo virulence assessment:

  • Animal models of shigellosis to evaluate colonization, tissue damage, and bacterial burden

  • Competitive index assays comparing mutant and wild-type strains in vivo

This comprehensive approach would provide insights into how specific atpB mutations affect various aspects of S. flexneri pathophysiology, from basic energy metabolism to virulence expression.

What is the relationship between atpB function and acid resistance in S. flexneri?

The relationship between atpB function and acid resistance in S. flexneri is intricate and physiologically significant. As part of the ATP synthase complex, atpB contributes to establishing and maintaining the proton motive force across the bacterial membrane, which is critical for intracellular pH homeostasis .

Experimental evidence indicates that deletion of the virulence plasmid (VP) increases ATP synthase complex formation and raises intracellular ATP levels . Counterintuitively, this VP deletion was associated with reduced bacterial survival in extremely acidic environments, such as the host stomach . This suggests that precise regulation of ATP synthase activity, rather than maximal expression, may be optimal for acid resistance.

The mechanism likely involves balancing proton influx through ATP synthase with proton efflux via other systems. Excessive ATP synthase activity might disrupt this balance, compromising pH homeostasis under acidic stress. Additionally, research has shown connections between ATP synthase and other acid resistance systems, such as the glutamate-dependent acid resistance system involving GadA/B proteins, which were differentially expressed in strains with altered ATP synthase levels .

This relationship has important implications for bacterial pathogenesis, as acid resistance is essential for S. flexneri to survive passage through the stomach and reach its site of infection in the intestine.

How does atpB expression and function compare between different S. flexneri serotypes?

While the search results don't provide direct comparative data on atpB expression between S. flexneri serotypes, genomic analyses have revealed significant differences between serotypes that may influence ATP synthase regulation and function. Comparative genomic studies between S. flexneri 5b strain 8401 and S. flexneri 2a strain 301 have identified "differences in the pathogenicity islands and chromosomal rearrangements between different serotypes" .

These genomic variations could potentially impact the regulation of energy metabolism genes, including atpB. Supporting this possibility, research has shown differential expression of acid resistance proteins (GadA/B) among different S. flexneri strains, with strain 2457T exhibiting notably lower levels compared to other strains . Since ATP synthase and acid resistance mechanisms are functionally interconnected, this suggests possible serotype-specific differences in ATP synthase regulation as well.

Research methodologies to investigate these differences would include:

• Comparative genomic analysis of atpB and ATP synthase regulatory regions

• Transcriptomic and proteomic profiling of ATP synthase components across serotypes

• Functional assays measuring ATP production and proton pumping efficiency

• Acid resistance comparisons between serotypes with emphasis on ATP synthase contribution

Understanding these serotype-specific differences could provide insights into the varied pathogenic potential of different S. flexneri strains.

What are the optimal conditions for reconstitution and storage of recombinant S. flexneri atpB protein?

For optimal reconstitution and storage of recombinant S. flexneri atpB protein, researchers should follow these evidence-based protocols:

• Initial handling:

  • Briefly centrifuge the vial containing lyophilized protein before opening to ensure all material is at the bottom

  • Maintain sterile conditions throughout the reconstitution process

• Reconstitution:

  • Use deionized sterile water as the primary reconstitution buffer

  • Reconstitute to a concentration of 0.1-1.0 mg/mL

  • Avoid introducing air bubbles that might cause protein denaturation

• Stabilization:

  • Add glycerol to a final concentration of 5-50%, with 50% being recommended for long-term storage

  • Consider adding protease inhibitors if protein degradation is observed

• Storage conditions:

  • For long-term storage, maintain at -20°C/-80°C

  • Create small working aliquots to avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

• Quality control:

  • Periodically verify protein stability and activity

  • Consider size exclusion chromatography to assess aggregation state

  • Validate functional activity before use in critical experiments

Adherence to these guidelines will help ensure the stability and activity of the recombinant protein for experimental applications.

What analytical techniques are most suitable for studying atpB structure-function relationships?

For comprehensive investigation of structure-function relationships in S. flexneri atpB, researchers should consider these analytical approaches:

• Structural analysis techniques:

  • X-ray crystallography (challenging for membrane proteins but provides atomic-level detail)

  • Cryo-electron microscopy (particularly valuable for membrane protein complexes)

  • Circular dichroism spectroscopy (for secondary structure characterization)

  • Nuclear magnetic resonance for specific domains or fragments

  • Molecular modeling based on homologous structures

• Functional analysis techniques:

  • ATP synthesis/hydrolysis assays using luminescence detection systems

  • Proton translocation measurements using pH-sensitive fluorophores

  • Membrane potential assays using voltage-sensitive dyes

  • Thermal stability assays to correlate stability with function

• Structure-function correlation approaches:

  • Site-directed mutagenesis targeting residues implicated in function

  • Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

  • Cross-linking mass spectrometry to map protein-protein interaction interfaces

  • Molecular dynamics simulations to study conformational changes

• Protein-protein interaction analysis:

  • Blue Native PAGE to analyze intact membrane protein complexes

  • Co-immunoprecipitation using His-tagged recombinant protein

  • Surface plasmon resonance for quantitative binding measurements

  • Fluorescence resonance energy transfer to study interactions in real-time

This multi-faceted approach would provide comprehensive insights into how the structure of atpB relates to its function in ATP synthesis, proton translocation, and its role in bacterial physiology.

How can researchers optimize expression systems for producing functional recombinant atpB?

Optimizing expression systems for functional recombinant atpB requires addressing several challenges specific to membrane proteins:

• Expression host selection:

  • E. coli has been successfully used for S. flexneri atpB expression

  • Consider specialized strains designed for membrane protein expression (C41/C43)

  • Evaluate different growth temperatures (often lower temperatures improve folding)

  • Test induction conditions (IPTG concentration, induction timing and duration)

• Expression construct design:

  • Include an N-terminal His-tag for purification as demonstrated in previous work

  • Consider fusion tags that enhance solubility (MBP, SUMO)

  • Optimize codon usage for the expression host

  • Include TEV or other protease cleavage sites for tag removal

• Membrane extraction and solubilization:

  • Screen multiple detergents for optimal extraction (DDM, LDAO, etc.)

  • Consider amphipols or nanodiscs for stabilizing the protein in solution

  • Optimize detergent-to-protein ratios to prevent aggregation

• Purification strategy:

  • Implement two-step purification for higher purity (affinity followed by size exclusion)

  • Maintain detergent above critical micelle concentration throughout

  • Include stabilizing agents (glycerol, specific lipids) in all buffers

• Functional validation:

  • Verify ATP synthase activity of the purified protein

  • Assess proton pumping ability in reconstituted systems

  • Compare activity to native ATP synthase as a benchmark

• Troubleshooting approaches:

  • Use GFP fusion constructs to monitor expression and membrane integration

  • Analyze expression using Western blotting with anti-His antibodies

  • Implement small-scale expression testing before scaling up

By systematically optimizing these parameters, researchers can improve the yield and functionality of recombinant S. flexneri atpB protein for structural and functional studies.

What is the potential of S. flexneri atpB as a therapeutic target?

The potential of S. flexneri atpB as a therapeutic target stems from several key characteristics:

• Essential function: ATP synthase is critical for bacterial energy metabolism, making it potentially indispensable for survival . Inhibiting its function could therefore have bactericidal effects.

• Role in pathogenesis: Research has established connections between ATP synthase activity and acid resistance, which is essential for S. flexneri to survive host stomach acid and cause infection . Targeting atpB could potentially reduce bacterial virulence.

• Structural features: As an integral membrane protein with multiple transmembrane domains, atpB contains potential binding pockets for small molecule inhibitors . The detailed sequence information available could guide structure-based drug design.

• Precedent in antimicrobial development: ATP synthase inhibitors have been explored as antimicrobials against other bacterial pathogens, suggesting feasibility for S. flexneri applications.

• Selectivity: ATP synthase is conserved across species, necessitating selective targeting of bacterial versus human homologs.

• Membrane accessibility: As a membrane protein, drug delivery to atpB may present challenges.

• Resistance development: The essential nature of ATP synthase could create strong selective pressure for resistance mutations.

Research suggests that comprehensive screening of therapeutic targets in S. flexneri has identified multiple promising candidates . While the search results don't specifically identify atpB among these, the essential nature of ATP synthase makes it a potential addition to the therapeutic target portfolio.

Could atpB serve as a vaccine candidate against S. flexneri infection?

The potential of atpB as a vaccine candidate against S. flexneri requires evaluation through several key considerations:

• Antigenicity and immunogenicity:

  • While the search results don't directly address atpB's immunogenicity, methodologies used to evaluate TolC as a vaccine candidate against S. flexneri could be applied

  • In silico analysis should assess atpB for B-cell and T-cell epitopes

• Conservation and cross-protection:

  • The atpB protein is relatively conserved across S. flexneri serotypes, suggesting potential for broad protection

  • Comparative sequence analysis would be needed to confirm conservation across clinically relevant strains

• Accessibility to the immune system:

  • As a membrane-embedded protein, atpB may have limited exposed regions accessible to antibodies

  • Structural analysis would be needed to identify surface-exposed epitopes

• Recombinant protein production feasibility:

  • Current protocols for producing His-tagged recombinant atpB provide a foundation for vaccine development

  • Modifications may be needed to enhance solubility and stability for vaccine formulation

• Safety considerations:

  • Homology with human ATP synthase components could raise concerns about auto-immunity

  • Careful epitope selection would be essential to avoid cross-reactivity

• Evaluation methodology:

  • Following approaches similar to those used for TolC, researchers could:

    • Express and purify recombinant atpB protein

    • Administer to animal models via appropriate routes

    • Evaluate IgG production by indirect-ELISA

    • Perform challenge studies with virulent S. flexneri strains

While the search results don't specifically address atpB as a vaccine candidate, the methodological framework established for other S. flexneri antigens provides a template for evaluation. The reverse vaccinology approach described for TolC could be adapted to systematically assess atpB's vaccine potential .

What research gaps exist in our understanding of S. flexneri atpB function and regulation?

Several significant research gaps remain in our understanding of S. flexneri atpB function and regulation:

• Regulatory mechanisms:

  • While the virulence plasmid has been shown to influence ATP synthase complex formation , the specific molecular mechanisms of this regulation are not fully elucidated

  • The transcriptional and post-translational regulatory networks controlling atpB expression during infection remain poorly characterized

• Host-pathogen interactions:

  • How host factors directly interact with bacterial ATP synthase during infection is largely unknown

  • Whether host cells specifically target ATP synthase as part of antimicrobial defense mechanisms requires investigation

• Serotype-specific variations:

  • While genomic differences between S. flexneri serotypes have been identified , their specific effects on ATP synthase structure, function, and regulation are not well understood

  • The functional consequences of any sequence variations in atpB between serotypes need characterization

• Role in virulence beyond acid resistance:

  • ATP synthase's contributions to virulence mechanisms beyond acid resistance, such as intracellular survival or stress response, require further investigation

  • The metabolic adaptations linked to ATP synthase regulation during different infection stages are poorly defined

• Structural insights:

  • High-resolution structural data specifically for S. flexneri ATP synthase is lacking

  • Structure-function relationships unique to S. flexneri atpB compared to other bacterial homologs need characterization

• Therapeutic targeting:

  • The druggability of specific binding sites within atpB remains to be systematically evaluated

  • The potential for resistance development against ATP synthase inhibitors requires assessment

Addressing these knowledge gaps will require integrated approaches combining genetic, biochemical, structural, and in vivo infection studies to fully understand the complex role of atpB in S. flexneri pathobiology.

What emerging technologies could advance research on S. flexneri atpB?

Several emerging technologies hold promise for advancing our understanding of S. flexneri atpB:

• Cryo-electron microscopy advances:

  • Single-particle cryo-EM can now achieve near-atomic resolution for membrane protein complexes

  • This could enable detailed structural analysis of S. flexneri ATP synthase in different functional states

  • Tomographic approaches could visualize ATP synthase in its native membrane environment

• CRISPR-Cas9 genome editing:

  • Precise genome editing technologies enable creation of point mutations in the chromosomal atpB gene

  • CRISPRi approaches allow for fine-tuned repression to study essentiality

  • CRISPR-based screens could identify genetic interactions with atpB

• Single-molecule techniques:

  • Fluorescence resonance energy transfer (FRET) could track conformational changes during ATP synthesis

  • Single-molecule force spectroscopy might reveal mechanical aspects of ATP synthase function

  • Super-resolution microscopy could visualize ATP synthase distribution in bacterial cells

• Advanced protein engineering approaches:

  • Directed evolution could generate atpB variants with enhanced stability for structural studies

  • Computational protein design might identify stable, soluble fragments for crystallization

  • Split-protein complementation systems could investigate assembly of the ATP synthase complex

• Metabolomic and proteomic integrations:

  • Multi-omics approaches can correlate ATP synthase activity with global metabolic changes

  • Quantitative proteomics could track ATP synthase complex assembly under different conditions

  • Protein-protein interaction networks might reveal novel regulators of ATP synthase

• Microfluidic and organoid systems:

  • Microfluidic devices can create defined pH gradients to study ATP synthase function

  • Human intestinal organoids provide physiologically relevant infection models

  • Organ-on-chip approaches could investigate tissue-specific aspects of infection

Implementation of these emerging technologies would provide unprecedented insights into the structural, functional, and regulatory aspects of S. flexneri atpB, potentially leading to novel therapeutic strategies targeting this essential bacterial protein.