Recombinant Yersinia pseudotuberculosis serotype IB ATP synthase subunit c (atpE)

What is the basic function of ATP synthase subunit c (atpE) in Yersinia pseudotuberculosis?

ATP synthase subunit c (atpE) in Y. pseudotuberculosis is a critical component of the F0 sector of F-type ATP synthase. This protein forms the c-ring structure in the membrane domain that facilitates proton translocation across the bacterial membrane, which is essential for ATP synthesis. The protein functions as a lipid-binding component that participates in the coupling of proton movement to rotational catalysis, ultimately converting the proton motive force into chemical energy in the form of ATP . Within the bacterial energy metabolism network, atpE serves as a crucial node linking the tricarboxylic acid (TCA) cycle and electron transport chain to cellular energetics.

What expression systems are optimal for producing recombinant Yersinia pseudotuberculosis atpE?

Multiple heterologous expression systems have been successfully employed to produce recombinant Y. pseudotuberculosis atpE, each with distinct advantages depending on research objectives:

• E. coli expression system: Provides high yield and is suitable for structural studies, though proper folding of membrane proteins may be challenging. This system is widely used for basic biochemical characterization .

• Yeast expression: Offers eukaryotic post-translational modifications while maintaining reasonable yields. This system may be preferable when investigating protein-protein interactions within more complex cellular environments .

• Baculovirus expression: Delivers superior folding for complex proteins and is ideal for functional studies requiring native-like protein conformation .

• Mammalian cell expression: Provides the most sophisticated post-translational modification profile and is suitable for immunological studies, though with lower yields .

The choice of expression system should be guided by the specific research questions being addressed. For structural studies, the E. coli system often proves most practical, while functional studies may benefit from the more sophisticated folding machinery available in eukaryotic systems.

What are the optimal conditions for solubilizing and purifying membrane-associated atpE protein?

As a membrane-associated protein, atpE requires specialized techniques for effective solubilization and purification:

• Solubilization: Employ a stepwise detergent screening approach beginning with mild detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin. Initial solubilization should be performed at 4°C with gentle agitation for 1-2 hours. The protein-detergent ratio is critical, with 1:10-1:50 (protein:detergent) ratios typically yielding optimal results.

• Purification strategy: Implement a multi-step purification protocol:

  • Initial capture via affinity chromatography (utilizing the specific protein tag)

  • Intermediate purification via ion exchange chromatography

  • Final polishing via size exclusion chromatography to ensure proper oligomeric state

• Buffer optimization: Maintain stable pH (typically 7.0-8.0) with adequate ionic strength (150-300 mM NaCl) throughout purification to prevent aggregation. Consider incorporating amphipols or nanodiscs for downstream functional studies requiring a more native-like membrane environment.

Purity assessment should be conducted via SDS-PAGE, with expected purity exceeding 85% for most research applications .

What role does atpE play in Yersinia pseudotuberculosis virulence and pathogenicity?

The atpE protein contributes to Y. pseudotuberculosis pathogenicity through multiple mechanisms:

• Metabolic adaptation: AtpE functions at a critical juncture in the pyruvate-TCA cycle metabolic node, which has been identified as a focal point for controlling host colonization . This metabolic adaptation allows the bacterium to thrive in diverse host environments.

• Stress response integration: The protein participates in the bacterial response to environmental stressors encountered during infection, including pH fluctuations and nutrient limitation.

• Virulence regulation network: Evidence suggests that atpE-dependent ATP production influences the expression and secretion of virulence factors through energy-dependent regulatory cascades .

Recent research has demonstrated that mutations affecting ATP synthase function can significantly impact bacterial colonization efficiency and persistence in mouse infection models. The pyruvate metabolism and TCA cycle nexus, to which atpE contributes, has been specifically implicated in virulence regulation, revealing atpE as more than merely a housekeeping gene but rather an integrated component of the pathogenicity machinery .

How does atpE interact with the Cpx two-component regulatory system in Yersinia pathogenesis?

The Cpx two-component regulatory system, comprising CpxA (sensor kinase) and CpxR (response regulator), responds to extracytoplasmic stress and influences virulence gene expression in Y. pseudotuberculosis. Research has revealed several important interactions:

• Metabolic integration: The Cpx system detects perturbations in membrane integrity and energy status, potentially sensing atpE-related functions.

• Regulatory cross-talk: Phosphorylated CpxR (CpxR∼P) has been shown to modulate the expression of virulence factors, including the global regulator RovA . This regulatory network likely integrates metabolic signals from the ATP synthase complex.

• Stress adaptation mechanism: The Cpx system and atpE both contribute to adaptation to environmental stresses, with evidence suggesting coordinated regulation during host infection.

Experimental data indicates that CpxR∼P binds to the upstream regulatory region of the RovA gene, inhibiting its transcription . This molecular mechanism illustrates how metabolic status, potentially influenced by atpE activity, becomes integrated into virulence regulation networks.

What are the critical factors to consider when designing experiments involving recombinant atpE for studying bacterial metabolism?

When designing experiments to investigate bacterial metabolism using recombinant atpE, researchers should consider:

• Expression context: The native quaternary structure of ATP synthase involves multiple subunits. Consider whether isolated atpE or reconstituted ATP synthase complexes are more appropriate for your research question.

• Functional validation: Confirm that recombinant atpE retains native functionality through ATP synthesis/hydrolysis assays before proceeding with metabolic studies.

• Metabolic state manipulation:

  • Design experiments to capture the metabolic flux through the pyruvate-TCA cycle node

  • Consider using stable isotope labeling to track carbon flow

  • Employ both aerobic and microaerobic conditions to mimic infection environments

• Comparative approach: Include parallel analyses of wild-type and mutant strains (e.g., ΔyopK ΔyopJ Δasd mutants) to contextualize atpE function within broader metabolic networks .

• Temperature considerations: Y. pseudotuberculosis exhibits temperature-dependent regulation. Design experiments at both 25°C (environmental) and 37°C (host) temperatures to capture relevant metabolic adaptations .

A systems biology approach integrating gene expression profiles with metabolic pathway flux analysis has proven particularly informative for understanding the role of atpE in the broader context of Y. pseudotuberculosis pathogenesis .

What controls and validation steps are necessary when working with tagged versions of recombinant atpE?

When working with tagged versions of recombinant atpE, implement these validation steps:

• Functional equivalence testing:

  • Compare ATP synthesis rates between tagged and untagged versions

  • Assess membrane integration patterns

  • Verify oligomerization state using native PAGE or size exclusion chromatography

• Tag interference assessment:

  • For Avi-tagged biotinylated versions, confirm that biotinylation occurs specifically at the intended lysine residue using mass spectrometry

  • Verify that the tag does not disrupt interactions with other ATP synthase subunits

• Essential controls:

  • Include wild-type untagged protein as a positive control

  • Include non-relevant tagged protein as a specificity control

  • For each functional assay, include both positive and negative controls appropriate to the specific methodology

• Tag-specific considerations:

  • For Avi-tagged biotinylated versions, confirm that BirA ligase has catalyzed the specific amide linkage between biotin and the lysine of the AviTag

  • Verify subcellular localization remains consistent with native protein

Rigorous validation ensures that experimental observations reflect genuine biological phenomena rather than artifacts introduced by protein tagging.

How can recombinant atpE be utilized in vaccine development against Yersinia infections?

Recombinant atpE has potential applications in vaccine development through several approaches:

• Subunit vaccine strategy: While not directly demonstrated for atpE, the approach used with other Yersinia recombinant proteins suggests potential. The recombinant YopE Nt138-LcrV fusion protein delivered by attenuated Y. pseudotuberculosis strains provides a methodological template .

• Carrier protein application: AtpE could be engineered as a carrier for antigenic epitopes, leveraging its membrane-associated properties to enhance immunogenicity.

• Attenuated strain development: Strategic mutations in atpE could contribute to bacterial attenuation while preserving immunogenicity, similar to the approach with ΔyopK ΔyopJ Δasd triple mutations .

• Methodology for immunological assessment:

  • Evaluate both humoral (serum IgG, secretory IgA) and cell-mediated immune responses

  • Challenge models should include both subcutaneous and intranasal routes

  • Histopathological analysis should be conducted to confirm attenuation without tissue damage

Experimental evidence from related Yersinia vaccine approaches demonstrates that properly designed recombinant protein delivery systems can elicit robust immune responses providing protection against challenge with virulent strains .

How does atpE function integrate with the Type 3 Secretion System (T3SS) during Yersinia infection?

The integration between atpE function and the Type 3 Secretion System (T3SS) represents a sophisticated aspect of Yersinia pathogenesis:

• Energetic requirements: The T3SS is highly energy-dependent, requiring ATP for assembly and operation. AtpE-dependent ATP generation likely provides essential energy for T3SS function.

• Regulatory coordination: Evidence suggests coordinated regulation between metabolic status (influenced by ATP synthase activity) and T3SS expression/activation:

  • T3SS components are synthesized and secreted under specific conditions (37°C, calcium deprivation)

  • These conditions also influence metabolic flux through pathways connected to ATP synthase function

• Experimental approach to study this integration:

  • Utilize T3SS secretion assays under controlled metabolic conditions

  • Employ ATP synthase inhibitors to assess impact on T3SS function

  • Monitor both protein synthesis and secretion of T3SS effectors in atpE mutants

The research methodology demonstrated with YopE fusion proteins, which are secreted via the T3SS under calcium-deprived conditions at 37°C, provides a valuable experimental approach for investigating this integration . This secretion system carries important virulence factors directly into host cells, making the energy supply from ATP synthase a critical factor in pathogenesis.

What are the key considerations when interpreting metabolic flux data involving atpE in different Yersinia strains?

When analyzing metabolic flux data involving atpE across different Yersinia strains, consider these methodological approaches:

• Context-dependent analysis:

  • Interpret flux data within the broader metabolic network, particularly focusing on the pyruvate-TCA cycle node identified as crucial for virulence

  • Compare wild-type and mutant strains under identical conditions

  • Account for temperature-dependent metabolic shifts (25°C vs. 37°C)

• Integration with multi-omics data:

  • Correlate flux data with transcriptomic profiles to identify regulatory patterns

  • Consider proteomic data to account for post-transcriptional regulation

  • Integrate with virulence phenotype data to establish causative relationships

• Statistical considerations:

  • Apply appropriate normalization for biomass differences

  • Account for growth phase-dependent metabolic shifts

  • Utilize statistical methods appropriate for compositional data

• Experimental design for robust interpretation:

  • Include technical and biological replicates

  • Validate key findings using orthogonal methodologies

  • Employ isotope labeling to track specific metabolic routes

The systems biology approach demonstrated in the analysis of Y. pseudotuberculosis metabolic adaptations provides a methodological framework that can be extended to specifically investigate atpE's role within these networks .

How should researchers analyze the impact of atpE mutations on bacterial fitness during infection?

Analysis of atpE mutations' impact on bacterial fitness during infection requires specialized methodologies:

This methodological approach has been demonstrated with virulence factor mutants of Y. pseudotuberculosis, revealing significant differences in tissue colonization and persistence compared to wild-type strains . Similar approaches would be valuable for specifically assessing atpE mutation impacts.

What emerging technologies could advance our understanding of atpE's role in bacterial pathogenesis?

Several cutting-edge approaches show promise for deeper insights into atpE function:

• Cryo-electron microscopy (Cryo-EM):

  • Enables visualization of the complete ATP synthase complex in near-native conditions

  • Provides structural insights into how atpE contributes to the c-ring formation

  • Allows comparison between different bacterial species to identify structural adaptations

• Real-time metabolic imaging:

  • FRET-based ATP sensors for live-cell imaging of ATP dynamics during infection

  • Correlative light and electron microscopy to link ATP synthase localization with function

  • Intravital microscopy to monitor metabolic adaptations during in vivo infection

• Single-cell approaches:

  • Single-cell RNA-seq to capture heterogeneity in metabolic states

  • Microfluidics combined with time-lapse microscopy to track individual bacterial responses

  • Mass cytometry for multi-parameter analysis of bacterial populations

• Genome editing techniques:

  • CRISPR interference for precise modulation of atpE expression

  • Site-specific mutagenesis to introduce subtle modifications to test structure-function hypotheses

  • Inducible expression systems for temporal control of ATP synthase activity

These technological approaches will help resolve outstanding questions regarding the integration of energy metabolism with virulence mechanisms in Y. pseudotuberculosis pathogenesis.

What are the most promising therapeutic strategies targeting atpE and ATP synthase in Yersinia infections?

Research into therapeutic strategies targeting atpE presents several promising directions:

• Small molecule inhibitors:

  • Develop compounds that specifically target the c-subunit of bacterial ATP synthase

  • Exploit structural differences between bacterial and human ATP synthase for selectivity

  • Consider combination approaches targeting both ATP synthesis and virulence factors

• Peptide-based inhibitors:

  • Design peptides that disrupt c-ring assembly or rotation

  • Utilize cell-penetrating peptides for improved delivery

  • Consider phage display libraries to identify high-affinity binders

• Immunotherapeutic approaches:

  • Develop antibodies targeting accessible portions of ATP synthase

  • Explore vaccine approaches using attenuated strains with modified atpE

  • Consider the methodology demonstrated with YopE fusion proteins for antigen delivery

• Metabolic reprogramming strategies:

  • Target the pyruvate-TCA cycle node identified as critical for virulence

  • Develop compounds that synergize with ATP synthase inhibition

  • Consider host-directed therapies that alter the metabolic environment for the pathogen

• Delivery strategies for therapeutic agents:

  • Nanoparticle-based delivery to improve targeting and reduce side effects

  • Exploit bacterial secretion systems for delivery of inhibitory molecules

  • Consider dual-targeting approaches combining atpE inhibition with disruption of virulence mechanisms

The integration of structural biology, metabolic analysis, and immunological approaches provides a comprehensive framework for developing these therapeutic strategies.