Recombinant Yersinia pestis bv. Antiqua ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Yersinia pestis bv. Antiqua?

ATP synthase subunit c (atpE) in Y. pestis bv. Antiqua is a critical component of the F-type ATP synthase complex, specifically located in the F0 sector embedded in the bacterial membrane. This protein functions as a lipid-binding protein and plays an essential role in proton translocation during ATP synthesis. The full-length protein consists of 79 amino acids and has several alternative names, including F-type ATPase subunit c, F-ATPase subunit c, and lipid-binding protein . The protein is encoded by the atpE gene, which in Y. pestis bv. Antiqua (strain Angola) corresponds to the locus tag YpAngola_A4207 .

What expression systems are commonly used for recombinant Y. pestis atpE production?

For the production of recombinant Y. pestis bv. Antiqua ATP synthase subunit c, E. coli is the predominant expression system employed in research settings. The recombinant protein is typically expressed with affinity tags (such as His-tag) to facilitate purification . E. coli is preferred because:

• It allows for high-yield protein expression with relatively simple culture conditions

• The genetic manipulation techniques for E. coli are well-established

• It enables the addition of affinity tags, such as His-tags, for simplified purification

• The bacterial expression system is particularly suitable for membrane proteins that require post-translational modifications similar to those in the native prokaryotic host

When expressing recombinant atpE in E. coli, researchers should optimize codon usage, induction conditions, and growth parameters to maximize protein yield while minimizing the formation of inclusion bodies, which is a common challenge with membrane proteins.

How can recombinant Y. pestis atpE be structurally characterized?

Structural characterization of recombinant Y. pestis atpE requires a multi-technique approach due to its membrane protein nature. The methodological workflow typically includes:

• Protein Purification and Preparation:

  • Affinity chromatography using His-tagged recombinant protein

  • Size exclusion chromatography for increased purity

  • Detergent selection for membrane protein solubilization (common detergents include DDM, LDAO, and OG)

• Biophysical Characterization:

  • Circular dichroism (CD) spectroscopy to determine secondary structure content

  • Differential scanning calorimetry (DSC) for thermal stability assessment

  • Dynamic light scattering (DLS) to evaluate homogeneity and oligomeric state

• High-Resolution Structural Analysis:

  • X-ray crystallography (challenging for membrane proteins, requires lipidic cubic phase or detergent micelles)

  • Cryo-electron microscopy (cryo-EM) for structure determination without crystallization

  • Nuclear magnetic resonance (NMR) for dynamics studies of smaller fragments

• Computational Approaches:

  • Homology modeling based on related bacterial ATP synthase structures

  • Molecular dynamics simulations to predict conformational changes and lipid interactions

The choice of detergent is particularly crucial for maintaining the native structure of atpE during purification and subsequent analyses, as inappropriate detergents can denature or destabilize the protein.

What role does atpE play in Y. pestis pathogenicity?

While ATP synthase is primarily known for its role in energy metabolism, emerging research suggests potential connections between atpE and bacterial pathogenicity through several mechanisms:

• Energy Provision for Virulence:

  • ATP synthase generates the energy required for various virulence mechanisms, including protein secretion systems and flagellar movement

  • During host infection, Y. pestis must adapt to different nutritional environments, requiring efficient energy conversion systems

• Biofilm Formation:

  • Y. pestis forms biofilms that are crucial for transmission by fleas and survival in harsh environments

  • Research has shown that CRP (cAMP receptor protein) regulates biofilm formation in Y. pestis

  • While direct evidence linking atpE to biofilm formation is limited, the energy production through ATP synthase likely supports the metabolic requirements of biofilm development

• Potential Role in pH Homeostasis:

  • ATP synthase contributes to maintaining cytoplasmic pH, which is essential for bacterial survival in acidic host environments

  • The ability to maintain pH homeostasis may contribute to Y. pestis survival within macrophages

• Possible Drug Target:

  • The essential nature of ATP synthase makes it a potential target for antimicrobial development

  • Inhibitors specifically targeting bacterial ATP synthase could disrupt energy metabolism in Y. pestis

Further research using Y. pestis mutants with altered atpE expression or specific inhibitors of ATP synthase could help elucidate the precise contribution of this protein to pathogenicity and virulence.

What are the optimal methods for reconstituting lyophilized recombinant Y. pestis atpE?

The reconstitution of lyophilized recombinant Y. pestis atpE requires careful attention to buffer composition and protein handling to maintain structural integrity and functional activity:

Recommended Reconstitution Protocol:

• Initial Preparation:

  • Centrifuge the vial containing lyophilized protein briefly (30 seconds at 10,000 × g) to collect all material at the bottom before opening

  • Allow the vial to reach room temperature before opening to prevent condensation

• Buffer Selection and Reconstitution:

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • For long-term storage, add glycerol to a final concentration of 5-50% (typical recommendation is 50%)

  • For functional studies, consider reconstitution in Tris-based buffers (pH 7.5-8.0) containing:

    • 150 mM NaCl

    • 5-10% glycerol

    • 0.1-0.5% appropriate detergent (DDM or LDAO)

• Reconstitution Process:

  • Add buffer slowly to the vial while gently rotating to ensure even wetting of the protein

  • Allow the protein to hydrate at 4°C for 30 minutes

  • Mix by gentle pipetting or inversion (avoid vortexing which can denature membrane proteins)

  • For complete solubilization, allow the solution to stand at 4°C for 1-2 hours with occasional gentle mixing

• Post-Reconstitution Handling:

  • Centrifuge at 15,000 × g for 10 minutes at 4°C to remove any insoluble material

  • Prepare working aliquots to avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

  • Store remaining aliquots at -20°C or -80°C for extended storage

This protocol can be optimized based on the specific downstream applications and experimental requirements.

How can researchers validate the functional activity of purified recombinant Y. pestis atpE?

Validating the functional activity of recombinant Y. pestis atpE requires assessing both its structural integrity and its ability to participate in proton translocation and ATP synthesis. The following methodological approaches are recommended:

Structural Validation:

• SDS-PAGE and Western Blotting:

  • Confirms protein purity (>90% is typical for most applications)

  • Western blotting with anti-His antibodies verifies the presence of the tagged protein

• Circular Dichroism (CD) Spectroscopy:

  • Confirms proper secondary structure formation

  • Typical membrane proteins like atpE show characteristic alpha-helical patterns

  • Comparison with known ATP synthase subunit c structures provides reference data

Functional Validation:

• Reconstitution into Liposomes:

  • Incorporation of purified atpE into liposomes using methods such as:

    • Detergent dialysis

    • Rapid dilution

    • Direct incorporation during liposome formation

  • Liposome composition typically includes phosphatidylcholine and phosphatidylglycerol (7:3 ratio)

• Proton Translocation Assays:

  • Using pH-sensitive fluorescent dyes (e.g., ACMA or pyranine)

  • Monitoring pH changes upon addition of ionophores or ATP

• ATP Synthase Activity in Reconstituted Systems:

  • When reconstituted with other ATP synthase subunits:

    • ATP synthesis activity measurement using luciferase-based assays

    • ATP hydrolysis activity using colorimetric phosphate detection

• Binding Studies:

  • Isothermal titration calorimetry (ITC) to assess interaction with known binding partners

  • Surface plasmon resonance (SPR) for kinetic analyses of protein-protein interactions

The combination of these assays provides comprehensive validation of both structural integrity and functional capacity of the recombinant protein.

How can recombinant Y. pestis atpE be used in vaccine development research?

Recombinant Y. pestis atpE has potential applications in vaccine development research, although it has not been the primary antigen of focus compared to other Yersinia antigens like YopE and LcrV. The methodological approach for exploring atpE in vaccine development includes:

• Antigenicity Assessment:

  • Bioinformatic prediction of potential B-cell and T-cell epitopes within the atpE sequence

  • Screening for conservation across Y. pestis strains and variation compared to human ATP synthase

  • ELISA-based detection of antibodies against recombinant atpE in convalescent sera

• Immunization Strategies:

  • Recombinant protein delivery systems:

    • Direct protein immunization with appropriate adjuvants

    • Incorporation into nanoparticles or liposomes for enhanced delivery

  • Live attenuated vector systems:

    • Similar to the approach used with Y. pseudotuberculosis PB1+ strain (χ10069) that was engineered to deliver Y. pestis YopE-LcrV fusion protein

    • Integration of atpE or atpE epitopes into fusion constructs with known immunogenic proteins

• Protection Studies:

  • Animal models for Y. pestis infection:

    • Mouse models for pneumonic and bubonic plague

    • Assessment of survival rates, bacterial burden, and immune responses

  • Measurement of protection parameters:

    • Survival curves following challenge with virulent Y. pestis

    • Bacterial clearance rates

    • Correlates of protective immunity (antibody titers, T-cell responses)

• Combination Approaches:

  • Incorporation of atpE with other Y. pestis antigens:

    • Multi-epitope vaccines containing fragments from various proteins

    • Co-delivery with established protective antigens like F1 and V antigens

While direct evidence for atpE as a protective antigen is limited, the approach used with YopE-LcrV fusion proteins demonstrates the potential of such strategies, where mice vaccinated with recombinant attenuated Y. pseudotuberculosis expressing YopE-LcrV showed significant protection (80% survival) against intranasal challenge with 240 median lethal doses of Y. pestis .

How can multi-omics approaches enhance our understanding of Y. pestis atpE function?

Multi-omics approaches provide powerful tools for understanding the complex role of atpE in Y. pestis biology and pathogenicity. The Yersiniomics database and other resources enable integrated analyses using the following methodological framework:

• Genomic Analysis:

  • Comparative genomics across Yersinia species and strains to identify:

    • Sequence conservation and variation in atpE

    • Genetic context and operon structure

    • Regulatory elements controlling atpE expression

  • The Yersiniomics database contains 200 genomic datasets that can be leveraged for such analyses

• Transcriptomic Approaches:

  • RNA-Seq and microarray data analysis to determine:

    • Expression patterns of atpE under different conditions

    • Co-expression networks involving atpE

    • Regulatory factors influencing atpE transcription

  • Integration with the 317 transcriptomic datasets available in Yersiniomics

• Proteomic Analysis:

  • Mass spectrometry-based proteomics to identify:

    • Post-translational modifications of atpE

    • Protein-protein interactions involving atpE

    • Abundance changes under different growth conditions or during infection

  • Correlation with the 62 proteomic datasets in Yersiniomics

• Metabolomic Integration:

  • Assessment of metabolic changes associated with atpE function:

    • Energy metabolism intermediates

    • Membrane lipid composition

    • Proton motive force measurements

• Systems Biology Integration:

  • Network analysis combining multi-omics data to identify:

    • Functional modules involving atpE

    • Regulatory circuits controlling ATP synthase expression and assembly

    • Metabolic dependencies on ATP synthase function

The integration of these multi-omics approaches provides a comprehensive understanding of atpE function beyond what can be achieved through single-technique approaches. For example, correlating transcriptomic data with proteomic data can reveal post-transcriptional regulation mechanisms affecting ATP synthase assembly and function.

What are common challenges in working with recombinant Y. pestis atpE and how can they be addressed?

Working with recombinant Y. pestis atpE presents several technical challenges due to its nature as a small, hydrophobic membrane protein. The following table outlines common issues and their solutions:

Addressing these challenges requires careful optimization of each step from expression to storage, with particular attention to maintaining the native-like environment for this membrane protein.

How should researchers design experiments to study the interaction between Y. pestis atpE and potential inhibitors?

Designing robust experiments to study interactions between Y. pestis atpE and potential inhibitors requires a multi-faceted approach combining biophysical, biochemical, and computational methods:

• Initial Screening Approaches:

  • In silico screening:

    • Molecular docking using homology models of Y. pestis atpE

    • Virtual screening of compound libraries against predicted binding pockets

    • Selection of compounds with predicted favorable binding energies

  • Thermal shift assays:

    • Differential scanning fluorimetry to detect stabilizing compounds

    • Comparison of melting temperatures (Tm) in presence vs. absence of compounds

    • High-throughput format suitable for initial compound screening

• Direct Binding Assays:

  • Isothermal Titration Calorimetry (ITC):

    • Quantitative measurement of binding thermodynamics

    • Determination of binding constants, stoichiometry, enthalpy, and entropy

    • Protocol modification for membrane proteins in detergent micelles

  • Surface Plasmon Resonance (SPR):

    • Real-time binding kinetics measurements

    • Association and dissociation rate constants determination

    • Immobilization strategies optimized for membrane proteins

• Functional Inhibition Assays:

  • Reconstituted liposome systems:

    • Measurement of proton translocation in presence of inhibitors

    • Detection of inhibition using pH-sensitive fluorescent dyes

    • Dose-response curves to determine IC50 values

  • Whole-cell ATP synthesis inhibition:

    • Assessment of cellular ATP levels using luciferase-based assays

    • Correlation between inhibitor concentration and ATP production

    • Control experiments with other energy-producing pathways

• Structural Studies of Inhibitor Binding:

  • X-ray crystallography or cryo-EM:

    • Co-crystallization or soaking with inhibitors

    • Structure determination of atpE-inhibitor complexes

    • Identification of binding sites and interaction mechanisms

  • NMR spectroscopy:

    • Chemical shift perturbation assays for mapping binding sites

    • STD-NMR for epitope mapping of bound inhibitors

    • HSQC experiments to monitor conformational changes

• Validation in Biological Systems:

  • Growth inhibition assays:

    • Correlation between atpE inhibition and bacterial growth inhibition

    • Specificity testing against other bacterial species

    • Cytotoxicity assessment against mammalian cells

  • Resistance development studies:

    • Serial passage in sub-inhibitory concentrations

    • Sequencing of atpE in resistant strains

    • Characterization of resistance mechanisms

This comprehensive experimental design framework enables thorough characterization of inhibitor interactions with Y. pestis atpE and assessment of their potential as antimicrobial agents.