Recombinant Lawsonia intracellularis ATP synthase subunit c (atpE)

What is the biological significance of ATP synthase subunit c (atpE) in Lawsonia intracellularis?

ATP synthase subunit c (atpE) is a membrane-bound component of the F0 sector of ATP synthase that plays an essential role in proton translocation and ATP production. In Lawsonia intracellularis, this protein consists of 84 amino acids and functions as part of the stator complex in the ATP synthase machinery . The protein is critical for bacterial energy metabolism, as it helps maintain the proton gradient necessary for ATP synthesis. Understanding its function provides insights into bacterial survival mechanisms within host cells and potential targets for therapeutic intervention against proliferative enteropathy.

How does the amino acid sequence of Lawsonia intracellularis atpE compare with that of other bacterial species?

The amino acid sequence of Lawsonia intracellularis atpE shows high conservation within the Lawsonia genus but demonstrates significant differences from other bacterial genera. According to genome sequence data, the full-length protein contains 84 amino acids with the sequence: MVKLDPSSLGLAIFGCAIGMALAALGCGIGQGLGLKGACEGIARNPEASGKIQVALILGLAFIESLAIYALVINLIILFANPFV . This sequence shows approximately 88-91% similarity with atpE from non-tuberculosis mycobacteria species and less similarity with other bacterial genera . These sequence variations provide the basis for species-specific detection methods and potentially selective therapeutic targeting.

What expression systems are most effective for producing recombinant Lawsonia intracellularis atpE?

E. coli is the most effective and widely used expression system for producing recombinant Lawsonia intracellularis atpE. Specifically, recombinant full-length Lawsonia intracellularis ATP synthase subunit c (atpE) has been successfully expressed with an N-terminal His-tag in E. coli . The expression construct typically includes the complete coding sequence (1-84 amino acids) with appropriate regulatory elements. For optimal expression, researchers should consider:

This approach yields recombinant protein that can be purified using immobilized metal affinity chromatography (IMAC) .

What purification challenges are specific to recombinant Lawsonia intracellularis atpE and how can they be addressed?

Purification of recombinant Lawsonia intracellularis atpE presents several challenges due to its hydrophobic nature and membrane-associated properties. Key challenges and their solutions include:

• Protein Solubility: The hydrophobic nature of atpE often leads to aggregation and inclusion body formation. This can be addressed by using mild detergents during cell lysis and throughout the purification process.

• Maintaining Native Conformation: Preserving the native structure is challenging but essential for functional studies. Adding stabilizing agents such as 6% trehalose to storage buffers has shown efficacy in maintaining proper protein conformation .

• Purification Yield: Typical yields are often low due to the protein's membrane-associated nature. Optimizing buffer conditions (pH 8.0 Tris/PBS-based buffers) and using stepwise elution protocols during affinity chromatography can improve yields .

• Protein Aggregation: Post-purification aggregation can be minimized by storing the protein in appropriate buffers with stabilizers. Lyophilization of the purified protein with proper excipients has been successful in maintaining stability .

• Storage Stability: Recombinant atpE should be stored at -20°C/-80°C, with aliquoting recommended to avoid repeated freeze-thaw cycles that could compromise protein integrity .

What methodologies are most effective for assessing the functional activity of recombinant Lawsonia intracellularis atpE?

Assessing the functional activity of recombinant Lawsonia intracellularis atpE requires specialized approaches that account for its role within the ATP synthase complex. Effective methodologies include:

• ATP Hydrolysis/Synthesis Assays: When incorporated into liposomes or nanodiscs with other ATP synthase components, the ATP synthesis/hydrolysis rates can be measured using methods similar to those employed for E. coli ATP synthase . This requires reconstitution of the protein into membrane-like environments.

• Proton Translocation Assays: Using pH-sensitive fluorescent dyes in reconstituted systems to monitor proton movement across membranes, similar to the approach used in other bacterial systems .

• Binding Studies with Known Inhibitors: As demonstrated with other bacterial atpE proteins, measuring the binding affinity of known ATP synthase inhibitors can indirectly assess functional integrity .

• Cross-linking Experiments: Chemical cross-linking can assess proper interactions with neighboring subunits, providing evidence for correct structural integration .

• Structural Integrity Assessment: Circular dichroism spectroscopy to evaluate secondary structure content, ensuring the recombinant protein maintains the expected α-helical properties characteristic of functional atpE.

These methodologies allow researchers to verify that recombinant atpE maintains functional properties similar to those of the native protein.

How can researchers distinguish between properly folded and misfolded recombinant Lawsonia intracellularis atpE?

Distinguishing between properly folded and misfolded recombinant Lawsonia intracellularis atpE is crucial for ensuring experimental validity. Several complementary techniques can be employed:

• Circular Dichroism (CD) Spectroscopy: This technique analyzes the secondary structure content, with properly folded atpE exhibiting characteristic α-helical signatures in the far-UV spectrum (208-222 nm).

• Intrinsic Fluorescence Spectroscopy: Changes in the local environment of aromatic residues can indicate conformational differences between folded and misfolded states.

• Limited Proteolysis: Properly folded proteins typically show distinct, limited digestion patterns when exposed to proteases, while misfolded variants often display more extensive digestion.

• Size Exclusion Chromatography: Properly folded atpE will typically elute at volumes corresponding to its monomeric or oligomeric state, while misfolded variants often form higher-order aggregates.

• Thermal Shift Assays: Monitoring protein unfolding as temperature increases can provide insights into structural stability, with properly folded proteins typically showing cooperative unfolding transitions.

• Antibody Recognition: Conformational antibodies raised against the native protein can often distinguish properly folded recombinant protein from misfolded variants.

Researchers should employ multiple methods for comprehensive assessment rather than relying on a single approach.

How can recombinant Lawsonia intracellularis atpE be utilized in vaccine development?

Recombinant Lawsonia intracellularis atpE offers significant potential as a component in vaccine development through several strategic approaches:

• Subunit Vaccine Development: Purified recombinant atpE can be formulated with appropriate adjuvants as part of a subunit vaccine. This approach has shown promise in studies of similar recombinant proteins against Lawsonia intracellularis, with significant humoral and cellular immune responses observed in vaccinated animals .

• Multi-antigen Formulations: Research indicates that combining multiple antigens provides enhanced protection against Lawsonia intracellularis. Recombinant atpE can be incorporated into chimeric constructs with other immunogenic proteins, potentially including outer membrane proteins and secreted antigens .

• Adjuvant Co-formulation: Studies have demonstrated that co-formulation of Lawsonia intracellularis antigens with porcine IFN-α significantly enhances antibody responses and provides better protection against challenge infection .

• Immune Response Characterization: Recombinant atpE can be used to monitor vaccine-induced immune responses through ELISA and other immunological assays, providing valuable data on vaccine efficacy .

• Protective Efficacy Assessment: Animal studies show that properly formulated recombinant antigen vaccines can provide protection against experimental Lawsonia intracellularis infection, as evidenced by post-mortem histopathological analysis of intestinal tissues .

This recombinant protein-based approach represents a faster, more scalable alternative to traditional vaccines that rely on whole-cell preparations requiring demanding bacterial culture conditions .

What role does atpE play in Lawsonia intracellularis pathogenesis?

The role of atpE in Lawsonia intracellularis pathogenesis is multifaceted and extends beyond its canonical function in ATP synthesis:

• Energy Production During Infection: As a component of ATP synthase, atpE contributes to bacterial energy metabolism, which is critical during intracellular replication within intestinal epithelial cells .

• Adaptation to Intracellular Environment: Comparative transcriptional analysis between pathogenic and non-pathogenic Lawsonia intracellularis variants has shown that genes involved in membrane transport and ATP-binding cassette (ABC) transporters are differentially expressed, suggesting that energy metabolism components like atpE may play roles in adaptation to the intracellular niche .

• Potential Virulence Factor: ATP synthase components may contribute to virulence beyond energy production. Studies with other bacterial pathogens suggest that ATP synthase can influence bacterial survival under stress conditions encountered during infection .

• Host-Pathogen Interaction: Research on Lawsonia intracellularis gene expression during infection indicates that genes encoding ATP synthase components may be regulated in response to host cell signals, suggesting a role in host-pathogen interactions .

• Nutrient Acquisition: Studies of host gene expression during Lawsonia intracellularis infection show downregulation of genes associated with nutrient acquisition, suggesting that bacterial energy metabolism components like atpE may operate in competition with host systems .

Understanding these roles provides insights into bacterial survival strategies and potential intervention points for controlling proliferative enteropathy.

How can recombinant Lawsonia intracellularis atpE be used for developing specific diagnostic tools?

Recombinant Lawsonia intracellularis atpE offers several advantages for developing specific diagnostic tools:

• Serological Diagnostic Development: Purified recombinant atpE can serve as an antigen in ELISA-based serological tests to detect antibodies in infected animals. This approach complements existing diagnostic methods for Lawsonia intracellularis infection .

• Molecular Detection Systems: The atpE gene sequence can be targeted for specific PCR-based detection systems. Similar approaches have been successful with other bacterial pathogens, where atpE gene targets offered high specificity for detection .

• Immunohistochemistry Applications: Antibodies raised against recombinant atpE can be used for immunohistochemical detection of Lawsonia intracellularis in tissue samples, providing a specific diagnostic tool for histopathological examination .

• Point-of-Care Test Development: Recombinant atpE can be incorporated into lateral flow assays or other rapid diagnostic platforms for field use, potentially improving disease surveillance in swine herds.

• Reference Standard Material: Purified recombinant atpE of known concentration can serve as a valuable standard for calibrating and validating various diagnostic assays, improving test standardization across laboratories.

Using recombinant atpE for diagnostics could enhance detection specificity, particularly when combined with other diagnostic markers, addressing the current challenges in detecting Lawsonia intracellularis infection during early stages .

How can structural analysis of recombinant Lawsonia intracellularis atpE inform drug design strategies?

Structural analysis of recombinant Lawsonia intracellularis atpE can significantly inform drug design strategies through several sophisticated approaches:

• Homology Modeling and Structure Determination: Similar to approaches used with Mycobacterium tuberculosis atpE, researchers can develop three-dimensional models of Lawsonia intracellularis atpE based on homology modeling, which can then be refined through molecular dynamics simulations . This model development typically follows a protocol that includes:

  • Alignment of the target sequence with template structures

  • Model building using software like Modeller9.16

  • Energy minimization and refinement with molecular dynamics

  • Validation using tools like Ramachandran plots and ERRAT

• Binding Site Identification: Computational analysis of the refined structure can identify potential binding pockets for small molecule inhibitors. These include sites involved in proton translocation and subunit interactions .

• Virtual Screening Approaches: Docking studies against large compound libraries can identify potential inhibitors with high binding affinities. This approach has identified compounds binding to mycobacterial atpE with energies ranging from -8.69 to -8.44 kcal/mol .

• Structure-Based Optimization: Lead compounds identified through screening can be optimized based on their interactions with key residues in the binding site, enhancing both potency and specificity.

• Resistance Mutation Analysis: Structural mapping of potential resistance mutations can guide the design of inhibitors less susceptible to resistance development, similar to approaches used with bedaquiline resistance in Mycobacterium tuberculosis .

This structure-based approach has proven successful in identifying ATP synthase inhibitors for other bacterial pathogens and could lead to novel therapeutic agents against Lawsonia intracellularis .

What are the current challenges in studying atpE's role in ATP synthesis within Lawsonia intracellularis?

Studying atpE's role in ATP synthesis within Lawsonia intracellularis presents several significant challenges that require innovative research approaches:

• Obligate Intracellular Nature: As an obligate intracellular pathogen, Lawsonia intracellularis cannot be cultured on conventional media, making it exceptionally difficult to study its metabolic functions in isolation . This necessitates complex cell culture systems for propagation.

• Genetic Manipulation Limitations: The lack of established genetic manipulation tools for Lawsonia intracellularis prevents direct gene knockout or modification studies of atpE in its native context. This contrasts with more genetically tractable bacterial systems.

• Functional Reconstitution: Recombinant atpE must be studied as part of the complete ATP synthase complex to fully understand its function, requiring co-expression with other subunits and complex reconstitution protocols.

• In vivo Relevance: Determining how in vitro findings with recombinant atpE translate to in vivo infection scenarios remains challenging, requiring sophisticated animal models such as the recently developed C57BL/6 mouse model .

• Technical Limitations in Protein Analysis: The hydrophobic nature of atpE creates technical challenges for structural studies, including crystallization and NMR analysis, limiting high-resolution structural information.

• Expression Level Variability: Studies of gene expression during infection indicate that ATP synthase components may be differentially regulated during various infection stages, complicating the interpretation of functional studies .

Addressing these challenges requires integrative approaches combining recombinant protein studies with cell culture infection models and appropriate animal systems.

How do mutations in atpE affect ATP synthase function and potentially confer resistance to inhibitors?

Mutations in atpE can significantly impact ATP synthase function and confer resistance to inhibitors through several molecular mechanisms:

• Binding Site Alterations: Point mutations in atpE can directly modify inhibitor binding sites, reducing affinity and efficacy. In Mycobacterium tuberculosis, mutations resulting in amino acid substitutions at positions 28, 61, 63, and 66 of atpE have been associated with resistance to bedaquiline, an ATP synthase inhibitor . Similar mechanisms could potentially occur in Lawsonia intracellularis.

• Conformational Changes: Mutations can induce structural changes that propagate beyond the immediate binding site, altering the protein's dynamic properties and function. For example, the L49P mutation in atpE changes leucine to proline, potentially affecting helix formation and stability .

• Proton Translocation Efficiency: Some mutations may preserve ATP synthesis while altering the efficiency of proton translocation, thereby modifying the protein's susceptibility to inhibitors that target this process.

• Subunit Interactions: Mutations can disrupt interactions with neighboring subunits, as demonstrated in studies where N-terminal deletions in E. coli subunit a affected cross-linking with subunits b and c . Such alterations could impact both function and inhibitor binding.

• Compensatory Mechanisms: Some mutations may trigger compensatory changes in other ATP synthase components, preserving function while reducing inhibitor sensitivity through indirect mechanisms.

Understanding these mechanisms is critical for developing inhibitors with higher barriers to resistance and for predicting potential resistance pathways that might emerge during therapeutic use.

What novel experimental approaches could advance our understanding of atpE function in Lawsonia intracellularis?

Several innovative experimental approaches could significantly advance our understanding of atpE function in Lawsonia intracellularis:

• Single-Cell Analysis of Bacterial Metabolism: Applying cutting-edge techniques like single-cell RNA-seq to infected host cells could reveal how atpE expression varies among individual bacteria within the same host cell environment, providing insights into metabolic heterogeneity during infection.

• Cryo-Electron Tomography: This technique could provide structural information about the ATP synthase complex within intact bacteria, revealing native arrangements not observable in recombinant systems.

• Inducible Expression Systems: Developing conditional expression systems for use in cell culture models could allow temporal control of atpE expression during infection, revealing its stage-specific roles.

• Synthetic Biology Approaches: Engineering minimally modified bacteria that express Lawsonia intracellularis atpE could provide tractable models for studying function without the limitations of an obligate intracellular lifestyle.

• Proteome-Wide Interaction Mapping: Techniques like proximity labeling could identify novel interaction partners of atpE beyond the ATP synthase complex, potentially revealing moonlighting functions.

• In Situ Metabolic Imaging: Developing fluorescent sensors for ATP or proton gradient dynamics could allow real-time visualization of ATP synthase function during the infection process in cell culture models.

• Comparative Genomics Across Isolates: Analysis of atpE sequence variations across multiple clinical isolates could reveal adaptive changes related to host specificity or virulence, similar to the comparative approaches used in transcriptional analysis of pathogenic variants .

These approaches could provide unprecedented insights into the multifaceted roles of atpE in Lawsonia intracellularis biology and pathogenesis.

How might comparative studies between atpE from Lawsonia intracellularis and other bacterial pathogens inform evolutionary understanding and therapeutic approaches?

Comparative studies between atpE from Lawsonia intracellularis and other bacterial pathogens can yield valuable insights for both evolutionary understanding and therapeutic development:

• Evolutionary Conservation Analysis: Sequence comparison of atpE across bacterial species reveals conservation patterns that indicate functionally critical regions. The atpE gene from Lawsonia intracellularis shows 99-100% identity with other members of the Mycobacterium tuberculosis complex but only 88-91% similarity with non-tuberculosis mycobacteria , suggesting evolutionary constraints on certain regions while allowing divergence in others.

• Structure-Function Relationship: Comparing the structural features of atpE across species can identify domains uniquely important to Lawsonia intracellularis. This approach has been successful in identifying species-specific features in ATP synthase components that can be targeted for selective inhibition.

• Resistance Mechanism Prediction: By studying known resistance mutations to ATP synthase inhibitors in other bacteria (such as the mutations in Mycobacterium tuberculosis atpE that confer bedaquiline resistance ), researchers can predict potential resistance mechanisms in Lawsonia intracellularis and design inhibitors with higher barriers to resistance.

• Cross-Species Inhibitor Development: Compounds showing activity against atpE from multiple bacterial species could serve as broad-spectrum ATP synthase inhibitors. The methodologies used to identify inhibitors of mycobacterial atpE (including molecular docking, MD simulation, and MM-GBSA analyses ) could be applied to develop inhibitors effective against Lawsonia intracellularis.

• Host-Pathogen Co-evolution: Comparing bacterial atpE with host ATP synthase components can reveal evolutionary adaptations that allow bacterial ATP synthases to function in diverse host environments while maintaining essential activities.

These comparative approaches provide a foundation for understanding the broader evolutionary context of ATP synthase function and for developing targeted therapeutic strategies against multiple bacterial pathogens.