Recombinant Salmonella choleraesuis ATP synthase subunit c (atpE)

What is the basic structure and function of ATP synthase subunit c (atpE) in Salmonella choleraesuis?

ATP synthase subunit c (atpE) in Salmonella choleraesuis is a membrane-spanning component of the F₀ sector of ATP synthase. It consists of 79 amino acids and forms an oligomeric structure that creates a channel for proton flow across the cytoplasmic membrane. The amino acid sequence of the protein is MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . This subunit is crucial for the proton flow that drives ATP synthesis, as it contains the transmembrane domains that allow protons to pass through the membrane, ultimately powering the catalytic activity of the F₁ portion of the ATP synthase complex.

What research methods are available to express and purify recombinant Salmonella choleraesuis atpE?

Recombinant Salmonella choleraesuis atpE can be expressed using several systems. The most common approach involves:

• Gene cloning: The atpE gene (typically expression region 1-79) is PCR-amplified and cloned into a suitable expression vector with an appropriate tag (His-tag is commonly used) .

• Expression system selection: E. coli BL21(DE3) is frequently used for expressing membrane proteins, though specialized strains designed for membrane protein expression may yield better results.

• Optimization of expression conditions: Induction parameters (temperature, IPTG concentration, time) must be optimized to increase protein yield while reducing formation of inclusion bodies.

• Membrane protein extraction: Detergent-based extraction (using DDM, LDAO, or similar detergents) is typically employed to solubilize the membrane-embedded atpE.

• Purification: Affinity chromatography (based on the tag used), followed by size exclusion chromatography to achieve high purity.

The purified protein is typically stored in a Tris-based buffer with 50% glycerol at -20°C or -80°C to maintain stability .

How does atpE contribute to Salmonella choleraesuis virulence and pathogenicity?

ATP synthase subunit c plays a critical role in Salmonella choleraesuis pathogenicity through several mechanisms:

• Energy provision: By facilitating ATP production, atpE supports the energy requirements for virulence factor expression and bacterial replication within host cells.

• pH homeostasis: ATP synthase, including the c subunit, contributes to pH regulation in Salmonella, which is crucial for survival in the acidic environment of phagosomes .

• Interaction with virulence proteins: The MgtC virulence protein in Salmonella interacts with the F₁F₀ ATP synthase complex (which includes atpE), inhibiting its activity to reduce ATP levels . This reduction prevents excessive cyclic diguanylate production, thereby repressing cellulose biosynthesis, which would otherwise interfere with intracellular replication.

• Adaptation to intraphagosomal environment: ATP synthase activity modulation helps Salmonella adapt to the nutrient-limited conditions inside phagosomes, supporting intracellular survival and replication.

Research shows that disruption of proper ATP synthase function can significantly alter virulence properties, indicating that atpE and other ATP synthase components are potential targets for anti-virulence strategies.

What is known about the relationship between ATP synthase activity and biofilm formation in Salmonella?

The relationship between ATP synthase activity and biofilm formation in Salmonella involves a complex regulatory network:

• ATP levels and c-di-GMP: ATP synthase activity directly influences intracellular ATP concentration, which affects cyclic diguanylate (c-di-GMP) levels, a second messenger that promotes biofilm formation .

• MgtC-mediated regulation: The virulence protein MgtC inhibits ATP synthase activity, thereby reducing ATP levels and preventing a rise in c-di-GMP . This mechanism represents a virulence strategy to repress biofilm formation during infection.

• Cellulose production: Elevated ATP levels in an mgtC mutant resulted in a sevenfold increase in bcsA mRNA (encoding cellulose synthase), leading to increased cellulose production . This was confirmed when expressing the α, β, and γ components of the F₁ subunit of ATP synthase prevented cellulose production in the mgtC mutant.

• Impact on virulence: Cellulose, a major structural component of Salmonella biofilms, interferes with replication inside macrophages and virulence in mice . By regulating ATP synthase activity, Salmonella can modulate biofilm formation to optimize its virulence.

This data suggests that ATP synthase subunit c, as part of the ATP synthase complex, plays an indirect but significant role in biofilm regulation through its primary function in ATP synthesis.

How does atpE function relate to type-III protein secretion systems in Salmonella?

The relationship between atpE function and type-III protein secretion systems (T3SS) in Salmonella involves energy coupling mechanisms:

• Shared energy requirements: Both ATP synthase and T3SS utilize the proton motive force (PMF) across the bacterial membrane. ATP synthase uses PMF to generate ATP, while T3SS uses it directly for protein export .

• ATP-independent secretion: Research has shown that type-III protein secretion can occur in the absence of type-III ATPase activity when the proton motive force is increased . This suggests that ATP hydrolysis is not absolutely required for T3SS function if sufficient PMF is available.

• Evolutionary implications: The finding that T3SS can function without ATP hydrolysis suggests that a proto-ATPase was likely added to a primordial proton-powered export system during evolution to enhance secretion efficiency . This has implications for understanding both ATP synthase and T3SS evolution.

• Metabolic coordination: Both systems must be metabolically coordinated during infection, as energy utilization must be balanced between virulence factor secretion and cellular energy maintenance.

Understanding the interplay between these systems provides insights into bacterial adaptation mechanisms during infection and may reveal potential targets for therapeutic intervention.

What are the best experimental approaches to study the interaction between atpE and potential inhibitors?

The study of interactions between atpE and potential inhibitors can be approached through multiple complementary methods:

• Biochemical assays:

  • ATP synthesis assays using inverted membrane vesicles to measure IC₅₀ values of inhibitors

  • ATP hydrolysis assays to determine the effect on the reverse reaction

  • Proton translocation assays to directly assess the impact on c-ring function

• Structural biology approaches:

  • X-ray crystallography or cryo-EM of the ATP synthase c-ring in complex with inhibitors

  • NMR studies of labeled recombinant atpE with various inhibitors

  • Molecular docking and in silico modeling to predict binding sites

• Resistance mutation analysis:

  • Selection of resistant mutants at 5× and 50× MIC concentrations

  • Whole-genome sequencing to identify mutations in the atpE gene

  • Site-directed mutagenesis to confirm the role of specific residues in inhibitor binding

• Cellular assays:

  • Membrane potential measurements using fluorescent probes

  • Determination of intracellular ATP levels following inhibitor treatment

  • Growth inhibition assays under various metabolic conditions

Research has shown that compounds targeting ATP synthase subunit c typically show IC₅₀ values between 1-3 μg/ml in biochemical assays, which correlate well with MIC values in whole-cell screening assays .

What methods can be used to study the role of atpE in bacterial persistence and antibiotic tolerance?

To investigate atpE's role in bacterial persistence and antibiotic tolerance, researchers can employ the following methods:

• Genetic approaches:

  • Construction of atpE conditional expression strains using inducible promoters

  • Site-directed mutagenesis of key residues to create partially functional variants

  • Gene knockdown studies using antisense RNA or CRISPR interference

• Persistence assays:

  • Time-kill curves in the presence of bactericidal antibiotics

  • Determination of persister cell frequencies after antibiotic treatment

  • Microscopy-based single-cell analysis of bacterial subpopulations

• Metabolic analysis:

  • Measurement of ATP/ADP ratios in persister vs. non-persister populations

  • Membrane potential analysis using voltage-sensitive dyes

  • Oxygen consumption and proton translocation rates in different growth phases

• Transcriptomic and proteomic studies:

  • RNA-seq analysis comparing wild-type and atpE variant strains

  • Proteomics to identify interaction partners during persistence

  • ChIP-seq to identify regulators of atpE expression

• In vivo models:

  • Animal infection models to assess bacterial persistence

  • Tissue colonization studies comparing wild-type and atpE variants

  • Antibiotic treatment efficacy in established infections

These approaches help elucidate how ATP synthase activity modulation contributes to bacterial survival under stress conditions and antibiotic exposure.

What are the technical challenges in developing atpE-targeting antimicrobials with specificity for Salmonella?

Developing atpE-targeting antimicrobials specific for Salmonella faces several technical challenges:

• Selectivity issues:

  • ATP synthase is highly conserved across bacterial species and mitochondria

  • Achieving specificity for Salmonella requires targeting unique structural features

  • Studies show that diarylquinolines targeting ATP synthase have selectivity indices (SI) >10 for S. aureus vs. mitochondria, but designing Salmonella-specific compounds is more challenging

• Membrane penetration:

  • Gram-negative bacteria like Salmonella have an outer membrane that limits compound entry

  • E. coli studies suggest that limited uptake through the Gram-negative cell membrane contributes to resistance to ATP synthase inhibitors

  • Compounds need specific physicochemical properties to penetrate both membranes

• Efflux mechanisms:

  • Salmonella possesses multiple efflux pumps that can expel antibiotics

  • Drug efflux mechanisms contribute to resistance against ATP synthase inhibitors

  • Inhibitor design must account for retention within the bacterial cell

• Polymorphisms in target:

  • Amino acid variations at positions equivalent to V48 and V60 in the atpE gene can affect inhibitor binding

  • These polymorphisms may differ between Salmonella serovars

  • Inhibitor design must accommodate or specifically target these variations

• Testing limitations:

  • Standard antimicrobial susceptibility testing may not reflect in vivo efficacy

  • Metabolic dependency on ATP synthase varies with growth conditions

  • Specialized assays are needed to evaluate compound efficacy in relevant conditions

These challenges highlight the need for structure-based rational drug design approaches and combination strategies to develop effective atpE-targeting antimicrobials for Salmonella.

How can recombinant atpE be utilized in vaccine development against Salmonella choleraesuis?

Recombinant atpE can be employed in vaccine development against Salmonella choleraesuis through several strategic approaches:

• Subunit vaccine development:

  • Recombinant atpE can be formulated with appropriate adjuvants to stimulate specific immune responses

  • The conserved nature of atpE may provide cross-protection against multiple Salmonella serovars

  • Epitope mapping can identify immunogenic regions for focused immune responses

• Live attenuated vaccine vectors:

  • Attenuated Salmonella strains can be engineered to overexpress atpE to enhance immune recognition

  • This approach can be combined with other virulence-associated antigens for broader protection

  • Research with attenuated S. Typhimurium ΔznuABC has shown promise in protecting against S. Choleraesuis

• Prime-boost strategies:

  • Initial priming with attenuated Salmonella expressing recombinant atpE followed by boosting with purified protein

  • This approach has been successful with other antigens, as demonstrated by studies using attenuated S. Typhimurium ΔznuABC and inactivated S. Choleraesuis vaccine

  • The strategy enhances both cellular and humoral immune responses

• DNA vaccine approaches:

  • Plasmid DNA encoding atpE can stimulate both antibody production and cell-mediated immunity

  • DNA vaccines can be followed by protein boosts to enhance immunogenicity

  • The technique allows for co-delivery of immunostimulatory molecules

• Immunological evaluation:

  • Assessment of both humoral and cell-mediated immune responses is essential

  • Protection evaluation through challenge studies in appropriate animal models

  • Correlates of protection should be established to predict vaccine efficacy

The utilization of recombinant atpE in vaccine approaches must consider factors such as protein conformation, stability, and presentation to the immune system for optimal efficacy.

What is the significance of ATP synthase inhibition in developing new antimicrobial strategies against multidrug-resistant Salmonella?

ATP synthase inhibition represents a promising strategy against multidrug-resistant Salmonella for several key reasons:

• Novel mechanism of action:

  • ATP synthase inhibitors act through a mechanism distinct from conventional antibiotics

  • This reduces the likelihood of cross-resistance with existing antimicrobials

  • ATP synthase function is essential under many growth conditions, making it a vulnerability

• Metabolic vulnerability:

  • ATP synthesis inhibition leads to energy depletion and bacterial growth arrest

  • Studies have shown that blocking respiratory ATP synthesis causes depletion of cellular ATP levels and bacterial killing

  • The central role of ATP synthase in energy metabolism creates a high barrier to resistance development

• Synergistic potential:

  • ATP synthase inhibitors can potentially sensitize resistant bacteria to traditional antibiotics

  • Energy depletion may compromise efflux pump function, reducing the expulsion of other antibiotics

  • Combination therapy approaches may reduce resistance development

• Target validation:

  • Genetic studies support the importance of ATP synthase for Salmonella virulence

  • The MgtC virulence protein naturally targets ATP synthase during infection

  • Downregulation of atpE gene transcription strongly decreases bacterial viability

• Challenges to consider:

  • Selectivity over mammalian ATP synthase requires careful inhibitor design

  • Salmonella's Gram-negative cell envelope may limit inhibitor access to the target

  • Potential metabolic adaptation mechanisms must be addressed

This data indicates that selective ATP synthase inhibitors can be developed with sufficient therapeutic windows compared to typical non-selective inhibitors like DCCD and oligomycin .

How does atpE function in the context of ATPase-independent type-III protein secretion in Salmonella?

ATP synthase subunit c (atpE) functions in a broader energetic context that relates to ATPase-independent type-III protein secretion through several mechanisms:

• Proton motive force generation and utilization:

  • ATP synthase (including atpE) traditionally uses proton motive force (PMF) to generate ATP

  • Research shows that type-III secretion systems can function using only PMF, without ATP hydrolysis

  • Under conditions of increased PMF, the requirement for type-III ATPase activity can be bypassed entirely

• Evolutionary relationships:

  • The cytoplasmic components of the type-III secretion system share strong homology with F₀F₁ ATP synthase

  • It is proposed that the flagellum was derived from a proto F₀F₁-ATP synthase

  • The finding that ATPase activity is dispensable for type-III secretion suggests that a proto-ATPase was added to a primordial proton-powered export system during evolution

• Energetic adaptation during infection:

  • During infection, Salmonella must balance energy production and utilization

  • ATP synthase and type-III secretion systems must be coordinately regulated

  • The ability to use PMF directly for protein secretion may conserve ATP for other essential processes

• Experimental evidence:

  • Functional flagella can form in the absence of type-III ATPase activity when mutations increase PMF and flagellar substrate levels

  • Increased PMF can bypass the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion

  • This indicates that type-III ATPases primarily enhance secretion efficiency under limited substrate concentrations

This research reveals an important principle: ATP synthase and type-III secretion systems share fundamental mechanisms of energy coupling to membrane transport, with important implications for understanding bacterial physiology and developing targeted interventions.

What emerging technologies could advance our understanding of atpE structure-function relationships in Salmonella?

Several cutting-edge technologies are poised to revolutionize our understanding of atpE structure-function relationships:

• Cryo-electron microscopy advancements:

  • High-resolution structures of the complete ATP synthase complex with bound inhibitors

  • Visualization of conformational changes during the catalytic cycle

  • Structural comparison of wild-type and mutant forms associated with inhibitor resistance

• Single-molecule techniques:

  • Real-time visualization of proton translocation through the c-ring

  • Direct measurement of c-ring rotation during ATP synthesis

  • Force measurements to determine the energetics of the process

• Advanced computational methods:

  • Molecular dynamics simulations of the entire ATP synthase in a lipid bilayer environment

  • Machine learning approaches to predict functional consequences of atpE mutations

  • Quantum mechanical calculations to understand proton transfer mechanisms

• Genetic tools:

  • CRISPR-Cas9 base editing for precise modification of key atpE residues

  • In vivo deep mutational scanning to comprehensively map structure-function relationships

  • Synthetic biology approaches to construct minimal or modified ATP synthase complexes

• Imaging technologies:

  • Super-resolution microscopy to visualize ATP synthase distribution in bacterial membranes

  • Correlative light and electron microscopy to connect structure and function

  • Label-free imaging techniques to study ATP synthase in native environments

These technologies will provide unprecedented insights into how atpE contributes to ATP synthase function and how this can be targeted for antimicrobial development.

What are the potential implications of atpE research for developing cross-species Salmonella vaccines?

Research on atpE holds several important implications for cross-species Salmonella vaccine development:

• Conserved epitope identification:

  • Comparative analysis of atpE sequences across Salmonella serovars can identify highly conserved regions

  • These conserved epitopes may serve as targets for broadly protective immune responses

  • Structural analysis can determine which epitopes are accessible to the immune system

• Integration with attenuated vaccine strains:

  • Attenuated Salmonella strains (e.g., S. Typhimurium ΔznuABC) show promise as vaccine vectors

  • These strains could be engineered to express modified atpE proteins with enhanced immunogenicity

  • Prime-boost protocols combining attenuated live vaccines with inactivated vaccines have shown efficacy against S. Choleraesuis

• Host-pathogen interaction insights:

  • Understanding how atpE contributes to Salmonella survival in different host species

  • Identifying atpE modifications that optimize immune recognition while maintaining immunogenicity

  • Correlating immune responses to atpE with protection across different host species

• Adjuvant development:

  • ATP synthase components might serve as natural adjuvants due to their conserved nature

  • Specific fragments of atpE could enhance immune responses to co-administered antigens

  • Novel formulations combining atpE with other immunomodulators may enhance vaccine efficacy

• One Health applications:

  • Cross-species vaccines could reduce Salmonella prevalence in animal reservoirs

  • This approach aligns with One Health initiatives to control zoonotic infections

  • Vaccines effective across species barriers could have significant public health impact

The research on attenuated Salmonella strains as vaccines demonstrates significant progress in this direction, with prime-boost vaccination protocols showing protection against challenge infection in piglets .

How might research on atpE contribute to understanding bacterial adaptation to environmental stresses?

Research on ATP synthase subunit c (atpE) provides valuable insights into bacterial adaptation mechanisms:

• Energy conservation strategies:

  • ATP synthase modulation is a key mechanism for adapting to energy-limited environments

  • Studies show that MgtC-mediated inhibition of ATP synthase helps Salmonella adapt to magnesium-limited conditions

  • Understanding how bacteria regulate ATP synthase activity could reveal new aspects of stress adaptation

• pH homeostasis mechanisms:

  • ATP synthase plays a role in pH homeostasis in Salmonella and other bacteria

  • atpE, as part of the proton-translocating machinery, is central to this function

  • Research on atpE mutations could reveal how bacteria maintain pH balance under acidic stress

• Biofilm regulation:

  • ATP levels influence cyclic diguanylate production and cellulose synthesis

  • This connection between energy metabolism and biofilm formation represents an adaptation strategy

  • Studying how atpE variants affect this relationship could reveal new regulatory mechanisms

• Antimicrobial resistance development:

  • Mutations in atpE can confer resistance to ATP synthase inhibitors

  • Understanding the structural basis for these mutations provides insights into bacterial adaptation

  • This knowledge could help predict and counter resistance development to new antimicrobials

• Cross-talk with stress response systems:

  • ATP synthase activity likely interfaces with general stress response pathways

  • Energy status serves as a key input for global regulatory networks

  • Exploring this cross-talk could reveal how bacteria integrate multiple stress signals

By studying atpE and ATP synthase in the context of stress adaptation, researchers can gain fundamental insights into bacterial physiology that extend beyond energy production to encompass broader aspects of bacterial survival strategies.