Recombinant Shigella dysenteriae serotype 1 ATP synthase subunit b (atpF)

What is ATP synthase subunit b (atpF) in Shigella dysenteriae and how does it differ from other ATP synthase subunits?

ATP synthase subunit b (atpF) is part of the F₀ sector of the ATP synthase complex in Shigella dysenteriae serotype 1. While related to the ATP synthase subunit a (atpB), which consists of 271 amino acids and functions in the membrane-embedded F₀ sector, subunit b (atpF) is a distinct component of the same complex . The ATP synthase complex plays a crucial role in energy metabolism by generating ATP through oxidative phosphorylation. In Shigella, this energy production is particularly important for supporting the pathogen's rapid intracellular growth and virulence mechanisms .

How is recombinant Shigella dysenteriae serotype 1 ATP synthase subunit b typically expressed and purified for research purposes?

Recombinant expression of Shigella dysenteriae serotype 1 ATP synthase subunit b typically employs E. coli expression systems, similar to the methodology used for ATP synthase subunit a (atpB) . The expression protocol generally involves:

• Cloning the atpF gene into an expression vector with a suitable tag (commonly His-tag)

• Transformation into E. coli expression hosts

• Induction of protein expression under optimized conditions

• Cell lysis and protein extraction

• Purification using affinity chromatography (typically Ni-NTA for His-tagged proteins)

• Further purification steps such as ion exchange or size exclusion chromatography if needed

• Quality assessment using SDS-PAGE (>90% purity standard)

• Storage as lyophilized powder or in appropriate buffer with cryoprotectants

For optimal stability, the purified protein should be stored at -20°C/-80°C with aliquoting recommended to avoid repeated freeze-thaw cycles .

What are the structural characteristics of ATP synthase subunit b in Shigella dysenteriae serotype 1?

While the specific crystal structure of Shigella dysenteriae serotype 1 ATP synthase subunit b has not been directly reported in the provided search results, we can infer its structural properties based on related ATP synthase components. As part of the F₀ sector, subunit b likely contains transmembrane domains that anchor it to the bacterial membrane, with additional domains that interact with other subunits of the ATP synthase complex.

The related ATP synthase subunit a (atpB) in Shigella dysenteriae serotype 1 is characterized by:

• 271 amino acids in length

• Hydrophobic transmembrane regions

• Functional domains that participate in proton translocation

ATP synthase subunit b is expected to have structural features optimized for its role in the stator that connects the F₁ and F₀ sectors of the ATP synthase complex.

What are the recommended protocols for reconstituting and handling recombinant Shigella dysenteriae ATP synthase subunit b protein?

Based on established protocols for similar ATP synthase subunits:

• Reconstitution procedure:

  • Briefly centrifuge the vial containing lyophilized protein before opening

  • 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% (optimally 50%)

  • Aliquot and store at -20°C/-80°C

• Handling precautions:

  • Avoid repeated freeze-thaw cycles as they compromise protein integrity

  • For working stocks, store aliquots at 4°C for up to one week

  • Maintain sterile conditions throughout handling

  • Use appropriate buffers (typically Tris/PBS-based, pH 8.0) for dilution and experiments

• Quality control:

  • Verify protein integrity via SDS-PAGE before experiments

  • Assess functional activity using ATP hydrolysis assays when appropriate

How can researchers effectively incorporate ATP synthase subunit b in functional studies of Shigella metabolism and virulence?

Researchers can employ multiple approaches to study the functional significance of ATP synthase subunit b:

• Genetic manipulation strategies:

  • Generate knockout mutants (ΔatpF) using methods similar to those used for other Shigella genes like pta and ackA

  • Create complemented strains by cloning the atpF coding region with its native promoter into vectors like pACYC184

  • Design point mutations in critical functional residues identified through structural analysis

• Functional assays:

  • Assess growth rates of wild-type vs. mutant strains under various pH conditions (pH 3.0-7.0)

  • Measure ATP production capacity in wild-type vs. mutant strains

  • Evaluate intracellular survival and replication within epithelial cell models

  • Quantify virulence using cell infection models and Sereny tests (for in vivo virulence)

• Integration with other virulence mechanisms:

  • Analyze the relationship between ATP synthase activity and type III secretion system (T3SS) function

  • Examine potential interactions between ATP metabolism and stress response systems

What techniques are most effective for studying the role of ATP synthase subunit b in Shigella acidic stress response?

To investigate the role of ATP synthase subunit b in acid tolerance:

• pH challenge assays:

  • Culture wild-type and ΔatpF mutant strains in media at varying pH values (3.0-7.0)

  • Monitor survival rates at defined time points using viable cell counting

  • Calculate survival percentages relative to initial inoculum

  • Compare growth curves under different pH conditions

• Gene expression analysis:

  • Use qRT-PCR to measure atpF expression under various pH conditions

  • Analyze expression correlation with pH values (similar to approaches used for sRNA regulators like Ssr1, which showed R = 0.785 correlation with pH)

  • Perform RNA-seq to identify genes co-regulated with atpF under acidic stress

• Protein-protein interaction studies:

  • Identify potential interaction partners of subunit b during acid stress

  • Use pull-down assays with tagged ATP synthase subunit b

  • Employ bacterial two-hybrid systems to verify specific interactions

How does ATP synthase subunit b contribute to Shigella dysenteriae metabolic adaptation during host infection?

ATP synthase subunit b likely plays a critical role in Shigella's metabolic adaptation during infection, particularly in:

• Energy harvesting during intracellular growth:

  • Shigella captures host cell metabolism outputs, primarily pyruvate, and efficiently converts them to energy

  • ATP synthase is essential for generating the ATP needed for rapid intracellular replication

  • The pathogen must balance energy production with maintaining host cell viability

• Adaptation to changing host environments:

  • During infection, Shigella encounters varying pH conditions in different host compartments

  • ATP synthase activity is modulated to maintain proton motive force under these conditions

  • Similar to other membrane components, ATP synthase likely participates in maintaining membrane integrity during stress

• Relationship with acetate metabolism pathway:

  • Shigella's unique high-flux nutrient acquisition strategy involves a three-step pathway converting pyruvate to acetate

  • ATP synthase function is linked to this central metabolic pathway that sustains rapid growth

  • Mutations in related metabolic genes (pta, ackA) reduce intracellular growth by 40-60%

What structural and functional insights can be gained by comparing ATP synthase subunit b across different Shigella species and E. coli?

Comparative analysis of ATP synthase subunit b across Shigella species and E. coli can reveal:

• Evolutionary adaptations:

  • Identify potential convergent mutations that may have occurred during Shigella evolution from E. coli

  • Analyze selection pressures on ATP synthase components during adaptation to intracellular lifestyle

  • Determine if mutations in ATP synthase correlate with virulence potential across species

• Functional specialization:

  • Compare ATP synthesis efficiency between pathogenic Shigella strains and commensal E. coli

  • Identify structural differences that may confer adaptation to intracellular environments

  • Assess whether specific mutations contribute to antimicrobial resistance phenotypes

• Species-specific variations:

  • Examine sequence conservation across the four Shigella species (S. dysenteriae, S. flexneri, S. boydii, S. sonnei)

  • Identify potential species-specific adaptations in ATP synthase structure and function

  • Determine if highly virulent strains like S. dysenteriae type 1 possess unique modifications

The recent findings on mutational convergence in Shigella evolution suggest that certain core genes, potentially including ATP synthase components, may have independently acquired similar adaptive mutations across different lineages .

What are the potential approaches for targeting ATP synthase subunit b in development of novel antimicrobial strategies against Shigella dysenteriae?

Given the essential role of ATP synthesis in Shigella pathogenesis, targeting ATP synthase subunit b offers promising avenues for antimicrobial development:

• Structure-based drug design:

  • Use high-resolution structural data of ATP synthase to identify binding pockets in subunit b

  • Design small molecule inhibitors that specifically target these sites

  • Employ molecular docking and dynamics simulations to optimize inhibitor binding

• Bacterial energy metabolism disruption:

  • Develop compounds that specifically disrupt proton translocation through the F₀ sector

  • Target the interface between subunit b and other ATP synthase components

  • Focus on inhibitors that selectively target bacterial ATP synthase over human homologs

• Combination therapy approaches:

  • Pair ATP synthase inhibitors with existing antibiotics to enhance efficacy

  • Target multiple components of Shigella energy metabolism (e.g., ATP synthase and acetate pathway)

  • Develop strategies that reduce the emergence of resistance mutations

• Experimental validation pipeline:

  Stage	Methodology	Outcome Measures
  In vitro screening	Biochemical assays with purified ATP synthase	Inhibition of ATP synthesis activity
  Cellular validation	Growth inhibition assays with Shigella cultures	MIC determination, bactericidal/bacteriostatic effects
  Intracellular activity	Infected cell culture models	Reduction in intracellular bacterial loads
  Specificity assessment	Comparative testing against human ATP synthase	Selectivity index calculation
  In vivo efficacy	Animal infection models	Reduction in bacterial burden, survival improvement

What are the major technical challenges in studying ATP synthase subunit b function in Shigella dysenteriae?

Researchers face several technical challenges when investigating ATP synthase subunit b:

• Protein purification difficulties:

  • Membrane proteins like ATP synthase subunits are inherently challenging to purify in their native conformation

  • Maintaining the proper folding and activity of isolated subunit b requires specialized techniques

  • Reconstitution into functional complexes for mechanistic studies presents additional challenges

• Genetic manipulation considerations:

  • Complete deletion of essential ATP synthase components may be lethal, requiring conditional knockouts

  • Complementation studies must ensure physiologically relevant expression levels

  • Precise point mutations must be designed based on structural predictions to avoid complete loss of function

• Functional assay limitations:

  • Distinguishing the specific contribution of subunit b from other ATP synthase components

  • Accurately measuring subtle effects on ATP synthesis in complex bacterial environments

  • Correlating biochemical findings with in vivo relevance during infection

How might recent advances in structural biology techniques contribute to our understanding of ATP synthase subunit b in Shigella?

Recent structural biology advances offer new opportunities for ATP synthase research:

• Cryo-electron microscopy applications:

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

  • Provides insights into conformational changes during catalytic cycles

  • Allows structural determination without crystallization, overcoming limitations faced in traditional crystallography

• Integrative structural approaches:

  • Combining X-ray crystallography, NMR, and computational modeling to build comprehensive structural models

  • Using hydrogen-deuterium exchange mass spectrometry to map protein-protein interactions within the complex

  • Employing cross-linking mass spectrometry to identify spatial relationships between subunits

• Single-molecule techniques:

  • Super-resolution fluorescence microscopy to visualize ATP synthase distribution and dynamics in live bacteria

  • Atomic force microscopy to measure mechanical properties of ATP synthase complexes

  • Single-molecule FRET to detect conformational changes during ATP synthesis

What are the promising new research directions for understanding the role of ATP synthase in Shigella virulence and pathogenesis?

Several emerging research directions show particular promise:

• Systems biology approaches:

  • Multi-omics integration (genomics, transcriptomics, proteomics, metabolomics) to understand ATP synthase in the context of global metabolic networks

  • Constraint-based modeling of Shigella metabolism to predict the effects of ATP synthase perturbations

  • Network analysis to identify critical nodes connecting energy metabolism to virulence mechanisms

• Host-pathogen interaction studies:

  • Investigation of how ATP synthase activity affects Shigella's ability to manipulate host cell metabolism

  • Analysis of potential host immune recognition of bacterial ATP synthase components

  • Understanding how ATP synthase function relates to persistence and antimicrobial tolerance

• Therapeutic targeting strategies:

  • Development of ATP synthase inhibitors as novel antibacterials

  • Exploration of ATP synthase as a potential vaccine antigen

  • Investigation of combination therapies targeting energy metabolism and type III secretion systems

• Evolutionary considerations:

  • Comprehensive analysis of ATP synthase sequence variations across Shigella isolates

  • Investigation of parallel evolution in ATP synthase components across independent Shigella lineages

  • Understanding how ATP synthase adaptations contribute to the emergence of highly virulent clones

What are the most significant recent findings regarding ATP synthase in Shigella pathogenesis?

Recent research has revealed several important insights about ATP synthase in Shigella pathogenesis:

• Metabolic exploitation strategy:

  • Shigella employs a remarkable strategy to capture host cell metabolic outputs while preserving host cell viability

  • The ATP synthase complex is essential for energy production supporting the pathogen's exceptionally rapid intracellular growth

  • This metabolic strategy allows Shigella to maintain prolonged exploitation of host resources

• Integration with virulence systems:

  • Energy metabolism through ATP synthase is tightly linked to the functionality of virulence mechanisms

  • Studies on related ATPases like Spa47 demonstrate that ATP hydrolysis is essential for type III secretion system assembly and function

  • The proper secretion of virulence factors depends on adequate ATP supply

• Adaptive evolution insights:

  • Evidence suggests that mutational convergence has played a significant role in Shigella evolution

  • ATP synthase components may have undergone parallel adaptive changes across independent Shigella lineages

  • These adaptations potentially contribute to increased virulence and antimicrobial resistance