Recombinant Edwardsiella ictaluri ATP synthase subunit delta (atpH)

What is the ATP synthase subunit delta in Edwardsiella ictaluri and how does it differ from other organisms?

The ATP synthase subunit delta (atpH) in Edwardsiella ictaluri is a critical component of the bacterial ATP synthase complex, serving as part of the rotor mechanism between the F₁ and F₀ sectors. In bacterial systems like E. ictaluri, this component is designated as the delta subunit, whereas in mitochondrial ATP synthases it is known as the delta subunit and in chloroplasts as the epsilon subunit . The delta subunit plays a crucial role in the mechanical coupling between the membrane-embedded F₀ portion and the catalytic F₁ portion of ATP synthase. This coupling enables the conversion of proton gradient energy into the mechanical energy required for ATP synthesis.

What is the significance of studying E. ictaluri ATP synthase components in pathogen research?

Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish (ESC), which represents a major economic concern in commercial aquaculture, particularly in the United States where catfish farming constitutes a significant portion of fin fish aquaculture . Understanding the core metabolic components of this pathogen, including its ATP synthase machinery, provides valuable insights into its survival mechanisms within host cells and potential targets for therapeutic intervention. The ATP synthase complex is essential for energy production, and its components may play roles in bacterial virulence, adaptation to varying environmental conditions, and survival within host cells during infection.

What expression systems are commonly used for producing recombinant E. ictaluri ATP synthase subunit delta?

Recombinant E. ictaluri ATP synthase subunit delta (atpH) can be produced using various expression systems, with the most common being:

• E. coli expression systems - Offering high protein yields and straightforward cultivation

• Yeast expression systems - Providing eukaryotic post-translational modifications

• Baculovirus expression systems - Facilitating production in insect cells

• Mammalian cell expression systems - Offering the most authentic post-translational modifications

The choice of expression system depends on research requirements, including protein folding, post-translational modifications, and downstream applications. For structural studies or antibody production, bacterial expression systems are often preferred due to their high yield, while applications requiring functional activity might benefit from eukaryotic expression systems.

How should researchers optimize storage conditions to maintain the stability of recombinant E. ictaluri ATP synthase subunit delta?

Based on protocols for similar ATP synthase components, researchers should consider the following storage protocols for recombinant E. ictaluri ATP synthase subunit delta:

For optimal stability, the protein should be stored in a Tris-based buffer with 50% glycerol, as commonly used for similar ATP synthase components . Repeated freezing and thawing should be avoided as this can lead to protein degradation and loss of activity. Instead, prepare small working aliquots to minimize freeze-thaw cycles. For applications requiring extended storage, maintain the protein at -80°C in single-use aliquots.

What methods are effective for assessing the functional activity of recombinant ATP synthase subunit delta?

To evaluate the functional activity of recombinant ATP synthase subunit delta, researchers can employ several complementary approaches:

• ATPase inhibition assays - Since the C-terminal domain of ATP synthase delta/epsilon subunit has been shown to inhibit ATPase activity , researchers can measure the inhibitory effect of the recombinant protein on ATPase activity in reconstituted systems.

• Protein-protein interaction assays - Co-immunoprecipitation or pull-down assays to verify binding to other ATP synthase subunits, confirming proper folding and interaction capacity.

• Circular dichroism spectroscopy - To assess secondary structure formation and stability under various conditions, providing insights into proper folding.

• Reconstitution experiments - Incorporating the recombinant subunit into ATP synthase complexes lacking the delta component to measure restoration of coupling efficiency.

Each of these methods provides different insights into protein functionality and should be selected based on specific research questions and available equipment.

How can researchers investigate the role of ATP synthase subunit delta in pH-dependent energy metabolism of E. ictaluri?

E. ictaluri demonstrates acid tolerance mechanisms that are critical for survival in acidic environments, including the phagosome of host cells . To investigate the potential role of ATP synthase subunit delta in pH-dependent energy metabolism, researchers should consider the following methodological approach:

• pH-dependent activity assays - Compare ATP synthase activity in reconstituted systems containing wild-type versus modified delta subunits across pH gradients (pH 2.0-7.0), measuring ATP synthesis rates.

• Site-directed mutagenesis - Introduce point mutations in conserved residues of the delta subunit to identify amino acids critical for pH-dependent conformational changes and activity regulation.

• Fluorescence resonance energy transfer (FRET) analysis - Label the delta subunit and interacting subunits with FRET pairs to measure conformational changes under varying pH conditions.

• Gene expression studies - Compare expression levels of atpH in E. ictaluri cultured under neutral versus acidic conditions to determine if expression is regulated in response to environmental pH.

• Delta subunit knockout studies - Create delta subunit-deficient strains and assess their ability to survive in acidic environments compared to wild-type strains.

These approaches would help elucidate whether the ATP synthase delta subunit contributes to the acid tolerance mechanisms that allow E. ictaluri to survive in the acidified phagosome environment of host cells.

What is the relationship between ATP synthase subunit delta and the Type III Secretion System (T3SS) in E. ictaluri pathogenesis?

While direct evidence linking ATP synthase subunit delta to T3SS function is limited, investigating potential functional relationships between these systems is valuable for understanding E. ictaluri pathogenesis. T3SS plays a crucial role in translocating effector proteins from E. ictaluri in the Edwardsiella-containing vacuole (ECV) to the host cell cytoplasm .

Researchers interested in exploring potential links between ATP metabolism and T3SS function could design experiments to:

• Compare ATP levels in wild-type versus delta subunit mutant strains during infection, particularly during the acidification and subsequent neutralization of the ECV that is required for effector translocation.

• Investigate whether ATP synthase activity influences the expression or assembly of T3SS components through global transcriptome analysis.

• Assess whether proton motive force, which is regulated by ATP synthase, affects T3SS function using protonophores or ATP synthase inhibitors.

• Examine co-localization of ATP synthase complexes and T3SS machinery during different stages of infection using fluorescence microscopy.

Understanding these relationships could provide insights into how E. ictaluri coordinates energy metabolism with virulence factor deployment during the infection process.

How can researchers overcome solubility issues when expressing recombinant E. ictaluri ATP synthase subunit delta?

Membrane-associated proteins like ATP synthase components can present solubility challenges during recombinant expression. To overcome these issues, researchers should consider:

When troubleshooting solubility issues, it's recommended to first optimize expression conditions before moving to more complex approaches like detergent screening or co-expression strategies. Each approach should be systematically evaluated for its impact on both protein yield and functional activity.

What strategies can address contradictory results in ATP synthase subunit delta structure-function studies?

Contradictory results in structure-function studies of ATP synthase components can arise from various sources. Researchers can address these discrepancies through the following strategies:

• Rigorous validation of experimental conditions - Ensure that all experimental parameters (pH, temperature, ionic strength) are carefully controlled and reported, as ATP synthase function is highly sensitive to these factors.

• Multiple structural characterization methods - Combine X-ray crystallography, NMR, and cryo-EM approaches to provide complementary structural data. For instance, NMR order parameters (S²) for backbone N-H bond vectors might seem contradictory to helical structure assessments, but can be reconciled through molecular dynamics simulations .

• Functional assays at multiple scales - Test protein function in isolated subunits, reconstituted complexes, membrane preparations, and intact cells to understand context-dependent effects.

• Molecular dynamics simulations - Use computational approaches to reconcile seemingly contradictory experimental data, particularly for understanding dynamic structural elements .

• Cross-laboratory validation - Establish collaborations to reproduce key findings using identical protocols but different equipment and personnel to ensure reproducibility.

When faced with contradictory results, researchers should consider whether the discrepancies reveal important regulatory mechanisms rather than experimental errors. ATP synthase components often exhibit conformational flexibility related to their function, which can lead to context-dependent structural and functional characteristics.

How should researchers analyze order parameters and structural dynamics of ATP synthase subunit delta?

Analyzing order parameters and structural dynamics of ATP synthase components requires sophisticated approaches that can capture both static and dynamic aspects of protein structure. Based on methodologies used for similar studies , researchers should:

• Combine multiple experimental techniques:

  • NMR relaxation measurements to derive S² order parameters

  • Chemical shift data to identify secondary structure elements

  • Hydrogen-deuterium exchange to assess solvent accessibility and structural stability

• Implement molecular dynamics (MD) simulations:

  • Use S²-restraining MD simulations to reconcile seemingly contradictory data

  • Compare unrestrained and restrained simulations to identify regions of conformational flexibility

  • Analyze both backbone and side-chain dynamics over various time scales

• Apply statistical validation:

  • Calculate confidence intervals for order parameters

  • Implement model-free formalism to separate fast and slow motions

  • Use Bayesian statistical approaches to quantify uncertainty in structural models

• Correlate dynamics with function:

  • Identify regions with low order parameters that may function as hinges or switches

  • Map conservation of dynamic properties across homologous proteins

  • Correlate changes in dynamics with functional states (e.g., ATP-bound vs. free states)

Low S² order parameter values for backbone N-H bond vectors may seem contradictory to helical structure assessments but can indicate the presence of both α- and 310-helical hydrogen bonds involving the N-H group . This apparent paradox can be resolved through careful analysis of multiple experimental datasets and MD simulations.

What statistical approaches are appropriate for analyzing ATP synthase activity data across different pH conditions?

When analyzing ATP synthase activity across different pH conditions, particularly relevant for E. ictaluri which survives in acidified phagosomes, researchers should employ appropriate statistical methods:

For experimental designs involving acid tolerance assays similar to those described for E. ictaluri , a completely randomized design with factorial arrangement of treatments is recommended. This approach allows for analysis of both main effects (e.g., pH, protein variant) and interaction effects (e.g., specific variants showing altered pH profiles).

How can structural investigations of E. ictaluri ATP synthase subunit delta contribute to antimicrobial development?

ATP synthase has emerged as a potential target for antimicrobial development due to its essential role in bacterial energy metabolism. Future research on E. ictaluri ATP synthase subunit delta could contribute to this field through:

• Structural comparison with host homologs - Detailed structural analysis of E. ictaluri ATP synthase subunit delta compared to fish host homologs could reveal unique features that could be exploited for selective inhibition.

• Identification of allosteric sites - Beyond the catalytic sites, identifying regulatory sites specific to bacterial ATP synthase delta subunits could provide targets for allosteric inhibitors that don't affect host ATP synthases.

• Structure-based virtual screening - Using high-resolution structures to conduct in silico screening of compound libraries against potential binding pockets on the delta subunit.

• Fragment-based drug discovery - Identifying small molecular fragments that bind to the delta subunit and can be developed into larger, more potent inhibitors.

• Peptide inhibitors - Designing peptides that mimic natural binding interfaces of the delta subunit with other ATP synthase components, potentially disrupting complex assembly.

These approaches could ultimately lead to new therapeutic strategies for controlling E. ictaluri infections in aquaculture, addressing a significant economic challenge in the industry .

What emerging technologies could enhance our understanding of ATP synthase subunit delta dynamics in living bacterial systems?

Several cutting-edge technologies hold promise for advancing our understanding of ATP synthase dynamics in living systems:

• Cryo-electron tomography - Enables visualization of ATP synthase complexes within intact bacterial cells, providing insights into native arrangement and interactions.

• Live-cell single-molecule FRET - Allows real-time monitoring of conformational changes in the delta subunit during ATP synthesis/hydrolysis cycles in living bacteria.

• Time-resolved mass spectrometry - Provides detailed information about dynamic protein-protein interactions and conformational states with millisecond temporal resolution.

• In-cell NMR spectroscopy - Permits structural and dynamic characterization of isotopically labeled proteins within living cells, revealing physiologically relevant states.

• Optogenetic control systems - Enables light-triggered activation or inhibition of ATP synthase components to study their dynamic roles in bacterial physiology.

• Nanobody-based sensors - Development of conformation-specific nanobodies as probes for specific functional states of the ATP synthase complex.

Implementation of these technologies could address fundamental questions about how ATP synthase subunit delta contributes to the adaptation of E. ictaluri to varying environmental conditions, particularly the acidic environments encountered during host infection .