Recombinant Shigella dysenteriae serotype 1 ATP synthase subunit c (atpE)

What is Shigella dysenteriae serotype 1 ATP synthase subunit c (atpE)?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of the bacterial F-type ATP synthase complex. In Shigella dysenteriae serotype 1, atpE (UniProt ID: Q329S6) is a small, highly hydrophobic membrane protein consisting of 79 amino acids . The protein functions as part of the membrane-embedded proton channel that couples proton translocation to ATP synthesis. Also known as "Lipid-binding protein," atpE contains multiple transmembrane domains that form the c-ring structure within the F0 sector . This protein plays a fundamental role in the energy metabolism of the pathogen, making it potentially significant for both basic research and applied studies targeting bacterial survival mechanisms.

What expression systems are recommended for recombinant Shigella dysenteriae serotype 1 atpE?

For optimal expression of recombinant Shigella dysenteriae serotype 1 atpE, E. coli expression systems are most commonly employed due to their genetic compatibility with Shigella species . When expressing highly hydrophobic membrane proteins like atpE, consider these methodological approaches:

• Vector selection: pET series vectors with N-terminal His-tag facilitate downstream purification while minimizing interference with protein folding

• Host strain considerations: BL21(DE3) or C41(DE3)/C43(DE3) strains specifically optimized for membrane protein expression

• Induction conditions: Lower temperatures (16-25°C) and reduced IPTG concentrations (0.1-0.5 mM) to promote proper folding

• Membrane extraction: Proper detergent selection (DDM, LDAO) for efficient extraction from membrane fractions

Current protocols have successfully produced recombinant atpE with N-terminal His-tag in E. coli with high purity (>90% as determined by SDS-PAGE) .

What are the optimal storage and reconstitution conditions for recombinant atpE protein?

For maximum stability and activity of recombinant Shigella dysenteriae serotype 1 atpE protein, the following storage and reconstitution protocols are recommended based on experimental validation :

Storage conditions:

• Store lyophilized protein at -20°C to -80°C upon receipt

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

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

• Long-term storage requires 5-50% glycerol (final concentration) and storage at -20°C/-80°C

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

• Add glycerol to a final concentration of 50% for optimal stability

• For membrane protein functionality studies, consider reconstitution into liposomes using appropriate lipid compositions

A Tris/PBS-based buffer with 6% trehalose at pH 8.0 provides optimal stability for the protein in its reconstituted form .

How can recombinant atpE be used in studying Shigella pathogenesis mechanisms?

Recombinant atpE can serve as a valuable tool for investigating various aspects of Shigella pathogenesis through several methodological approaches:

• Energy metabolism studies:

  • Measure ATP synthesis rates in reconstituted systems

  • Investigate how energy metabolism contributes to virulence

  • Analyze the effects of metabolic inhibitors on bacterial survival

• Protein-protein interaction analysis:

  • Identify binding partners using pull-down assays with His-tagged atpE

  • Investigate interactions with other ATP synthase components

  • Explore potential interactions with host cellular factors during infection

• Differential expression analysis:

  • Compare atpE expression levels across different infection sites (e.g., intestinal vs. bloodstream)

  • Analyze expression changes under varying environmental conditions (pH, oxygen, nutrient limitation)

  • Correlate expression with virulence factor production

Research has demonstrated that Shigella species exhibit varying levels of gene expression depending on the infection site, with different expression profiles observed between bacteria isolated from stool versus blood samples . This suggests that atpE expression and function may be regulated in response to specific host environments during infection progression.

What techniques are optimal for structural and functional characterization of atpE?

For comprehensive structural and functional characterization of Shigella dysenteriae serotype 1 atpE, researchers should employ a multi-technique approach:

Structural characterization:

• Cryo-electron microscopy (cryo-EM) to resolve the c-ring structure

• NMR spectroscopy for dynamic structural analysis in membrane environments

• X-ray crystallography for high-resolution structural data (challenging for membrane proteins)

• Molecular dynamics simulations to predict conformational changes during proton translocation

Functional characterization:

• Proteoliposome reconstitution assays to measure proton translocation

• ATP synthesis assays in reconstituted systems

• Site-directed mutagenesis to identify essential residues

• Electrophysiological techniques to measure proton channel activity

Expression analysis:

• qRT-PCR for transcript quantification under various conditions

• Western blotting with anti-His antibodies for protein detection

• Transcriptomics to analyze differential expression patterns, similar to approaches used in analyzing S. dysenteriae gene expression at different infection sites

These techniques enable researchers to establish structure-function relationships and understand atpE's role in bacterial physiology and pathogenesis.

How can atpE be evaluated as a potential antimicrobial target?

ATP synthase components, including atpE, represent promising targets for novel antimicrobial development. To evaluate atpE as a potential antimicrobial target, researchers should follow these methodological approaches:

• Target validation studies:

  • Generate conditional knockdowns to confirm essentiality

  • Perform growth inhibition studies with known ATP synthase inhibitors

  • Assess metabolic consequences of atpE inhibition

• Inhibitor screening approaches:

  • Develop high-throughput biochemical assays using purified recombinant atpE

  • Design in silico screening methods targeting the c-ring structure

  • Apply similar computational screening approaches as used for T3SS ATPase inhibitors

• Mechanism of action studies:

  • Determine inhibition kinetics (competitive vs. noncompetitive mechanisms)

  • Measure effects on membrane potential and ATP synthesis

  • Assess specificity against mammalian ATP synthase homologs

• Resistance development assessment:

  • Monitor for spontaneous resistance mutations

  • Analyze cross-resistance with other antimicrobials

  • Evaluate fitness costs associated with resistance mutations

Research on other Shigella ATPases has demonstrated the feasibility of identifying noncompetitive inhibitors with high specificity and low cytotoxicity, achieving inhibition rates of 87.9 ± 10.5% with IC50 values as low as 25 ± 20 μM . Similar approaches could be applied to atpE as a target.

What is the potential of atpE as a vaccine candidate compared to other Shigella antigens?

While atpE has not been extensively studied as a vaccine candidate for Shigella, several methodological approaches can be employed to evaluate its immunogenic potential:

Comparison of vaccine approaches:

Evaluation methodology:

• In silico analysis:

  • Predict B and T-cell epitopes using immunoinformatics tools

  • Assess conservation across Shigella serotypes and species

  • Evaluate potential cross-reactivity with human proteins

• Immunogenicity testing:

  • Measure antibody responses in animal models

  • Assess cellular immune responses

  • Determine protection in challenge studies

• Adjuvant optimization:

  • Test various adjuvant formulations to enhance immunogenicity

  • Optimize delivery systems for membrane proteins

The reverse vaccinology approach used to identify TolC as a vaccine candidate against S. flexneri could serve as a methodological template for evaluating atpE, with similar assessment of conservation, antigenicity, and protective efficacy .

How does atpE contribute to antimicrobial resistance mechanisms in Shigella?

ATP synthase components have emerging roles in antimicrobial resistance (AMR) mechanisms. For atpE specifically:

• Direct resistance mechanisms:

  • Mutations in atpE can confer resistance to specific ATP synthase inhibitors

  • Altered expression may compensate for energy deficits caused by other resistance mechanisms

• Indirect contributions to AMR:

  • ATP production supports energy-dependent efflux pump activity

  • Maintains membrane potential required for resistance to certain antimicrobials

  • Provides energy for repair mechanisms against antimicrobial damage

• Research approaches:

  • Transcriptomic analysis comparing atpE expression in resistant vs. susceptible strains

  • Correlation with expression of known resistance genes (dhfr1A, sulII, blaOXA, blaCTX-M-1, qnrS)

  • Functional studies of ATP synthase activity in resistant isolates

Recent studies have identified multiple antimicrobial resistance genes in clinical Shigella isolates, including resistance to quinolones, beta-lactams, and sulfonamides . The role of energy metabolism genes like atpE in supporting these resistance mechanisms requires further investigation, particularly in extensively drug-resistant (XDR) Shigella strains .

What are the implications of genomic diversity in Shigella for atpE-targeted therapeutics?

Understanding genomic diversity is crucial for developing effective atpE-targeted therapeutics:

• Conservation analysis:

  • Evaluate sequence conservation of atpE across clinical isolates

  • Compare with genomic diversity patterns observed in recent large-scale studies

  • Assess potential for resistance development through genetic variation

• Strain variation considerations:

  • Analyze atpE sequence variations across different Shigella species and serotypes

  • Identify conserved regions as optimal therapeutic targets

  • Design broad-spectrum inhibitors targeting highly conserved regions

• Translational implications:

  • Consider genomic diversity in drug design to minimize resistance emergence

  • Develop combination approaches targeting multiple essential proteins

  • Monitor evolving resistance patterns in clinical isolates

Recent genomic analyses of 1,246 Shigella isolates from seven countries revealed significant diversity and adaptive capacity, particularly in S. flexneri, which could generate vaccine escape variants in less than 6 months . Similar considerations would apply to atpE-targeted therapeutics, necessitating careful sequence conservation analysis.

How can systems biology approaches enhance understanding of atpE's role in Shigella pathophysiology?

Integrative systems biology approaches offer powerful tools for contextualizing atpE's role:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Correlate atpE expression with global metabolic profiles

  • Identify regulatory networks controlling atpE expression

• Host-pathogen interaction mapping:

  • Analyze differential expression patterns between intestinal and systemic infection sites

  • Identify host factors influencing bacterial energy metabolism

  • Determine how energy status affects virulence factor expression

• Metabolic flux analysis:

  • Measure ATP synthesis rates under varying conditions

  • Quantify energy allocation during different infection stages

  • Model energy requirements for virulence factor production and secretion

• Research methodology:

  • Implement isotope labeling studies for metabolic pathway analysis

  • Develop computational models integrating transcriptomic and metabolomic data

  • Apply experimental designs similar to those used for differential gene expression studies in Shigella

These approaches can reveal how atpE and ATP synthase function within the broader context of bacterial adaptation to host environments, potentially identifying novel intervention points.