Recombinant Salmonella typhimurium ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) and what is its function in Salmonella typhimurium?

ATP synthase subunit c, encoded by the atpE gene, is a critical component of the F0 portion of the F0F1 ATP synthase complex. This membrane-integrated protein forms a ring structure in the F0 domain and is essential for proton translocation across the membrane during ATP synthesis. In Salmonella, the F0F1 ATPase complex plays a dual role: generating ATP through oxidative phosphorylation and utilizing ATP for cellular processes such as flagellar rotation . The atpE protein is relatively small (typically 79 amino acids in length) and highly hydrophobic, containing primarily transmembrane domains that contribute to the proton channel formation .

How can I express and purify recombinant Salmonella typhimurium atpE protein for in vitro studies?

The recombinant Salmonella atpE protein can be expressed using several heterologous systems:

• E. coli expression system: The most common approach involves cloning the atpE gene into an expression vector with an appropriate tag (typically His-tag) for purification. E. coli BL21(DE3) or similar strains are preferred host cells due to their high expression levels and reduced protease activity .

• Purification methodology:

  • Lyse cells under denaturing conditions due to the hydrophobic nature of atpE

  • Use immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Consider detergent solubilization (such as n-dodecyl β-D-maltoside) to maintain the native conformation

  • Dialyze against a buffer containing appropriate detergent concentrations

  • Verify protein purity via SDS-PAGE (>90% purity is typically achievable)

• Storage: Store as lyophilized powder or in solution with glycerol at -80°C to prevent repeated freeze-thaw cycles .

What are the general characteristics of atpE protein structure that impact experimental design?

The atpE protein has several structural characteristics that must be considered when designing experiments:

• Highly hydrophobic nature: The protein consists primarily of transmembrane domains with a predicted alpha-helical structure. This requires special consideration during solubilization and handling .

• Small size: At approximately 79 amino acids long, the protein has a molecular weight of approximately 8 kDa, which may require specialized electrophoresis techniques for visualization .

• Amino acid composition: The protein sequence (MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA) contains numerous hydrophobic residues that contribute to its membrane integration properties .

• Oligomerization potential: The native form of atpE functions as a multimeric complex (forming the c-ring of F0), which should be considered when studying structure-function relationships .

How does deletion of atpE or the entire ATP synthase operon affect Salmonella virulence and its potential as a vaccine candidate?

Deletion of the atpE gene or the entire ATP synthase operon (atpI-C) significantly impacts Salmonella virulence and creates potential vaccine candidates:

• Virulence attenuation: Studies have demonstrated that mutants lacking the complete F0F1 ATPase or individual F0/F1 subunits show reduced virulence in mouse infection models. Bacterial counts in the livers and spleens of intravenously infected mice are significantly decreased compared to wild-type strains .

• Cell-specific replication effects:

  • In epithelial cell lines (mICc12 and HeLa): ΔatpI-C mutants show moderate attenuation (reduced to 63% and 55% of wild-type replication, respectively) .

  • In macrophage cell lines (THP-1A and RAW 264.7): Replication is severely impaired (reduced to 10% and 18% of wild-type levels, respectively) .

• Vaccine potential: Following clearance of attenuated ATP synthase mutants from mouse organs, animals develop protective immunity against subsequent challenge with virulent wild-type strains. This indicates that ATP synthase-deficient Salmonella strains are promising live attenuated vaccine candidates .

• Complementation studies: The attenuated growth phenotype can be reversed by reintroducing the ATP synthase operon into the genome, confirming that the attenuation is specifically due to loss of ATP synthase function .

What are the metabolic adaptations of Salmonella typhimurium ATP synthase mutants during host cell infection?

Salmonella typhimurium adapts its metabolism significantly when ATP synthase function is compromised:

• Alternative ATP generation pathways:

  • In epithelial cells (mICc12 and HeLa), S. Typhimurium can partially compensate for ATP synthase deficiency, suggesting that substrate-level phosphorylation contributes significantly to ATP production in these environments .

  • In macrophage cells (THP-1A and RAW 264.7), oxidative phosphorylation via ATP synthase appears more critical, as evidenced by severe growth attenuation of ΔatpI-C mutants .

• Glycolysis dependency:

  • S. Typhimurium has a strict requirement for glycolysis in most host cells, as demonstrated by the inability of phosphofructokinase-deficient (ΔpfkAB) mutants to replicate within mICc12 cells and macrophages .

  • This suggests that glucose is a primary carbon source during intracellular growth.

• Role of fermentation and anaerobic respiration:

  • Experimental evidence indicates that neither fermentative metabolism nor anaerobic respiration plays a major role in energy generation during infection of the studied cell lines .

  • Instead, overflow metabolism resulting in acetate and lactate production appears to be the predominant route for ATP generation in ATP synthase-deficient strains .

How can recombinant Salmonella atpE be used as a component in vaccine development strategies?

Recombinant Salmonella atpE protein has potential applications in multiple vaccine development approaches:

• Live attenuated Salmonella vaccines:

  • ATP synthase mutants (including atpE deletions) can serve as vaccine vectors due to their attenuated virulence while maintaining immunogenicity .

  • These strains can be engineered to express heterologous antigens from other pathogens, creating multivalent vaccines .

• Balanced lethal systems:

  • Recombinant live-attenuated Salmonella vaccines (RASVs) can incorporate balanced lethal systems to ensure stability of plasmid vectors encoding protective antigens post-immunization .

  • These systems may utilize metabolic dependencies created by ATP synthase mutations to maintain selection pressure for antigen-expressing plasmids in vivo.

• Immune response characteristics:

  • RASVs effectively elicit long-lasting mucosal, systemic, and cellular immune responses when administered via various routes (oral, nasal, subcutaneous, intramuscular) .

  • The attenuation of virulence factors prevents unwanted side effects such as fever and diarrhea while maintaining immunogenicity .

• Molecular mechanisms of immune stimulation:

  • Attenuated Salmonella strains effectively cross the epithelial barrier and reach antigen-presenting cells in mucosal-associated lymphoid tissue (MALT) .

  • They establish limited infection, delivering in vivo synthesized antigens directly to B and T lymphocytes in gut-associated lymphoid tissue (GALT) .

  • Antigens are processed and presented via MHC class I and II molecules, stimulating T cell responses .

What methodological approaches are most effective for studying atpE function in the context of Salmonella pathogenesis?

Several methodological approaches have proven effective for investigating atpE function in Salmonella pathogenesis:

• Precise genetic manipulation techniques:

  • Targeted deletion of atpE or the entire ATP synthase operon using lambda Red recombinase system

  • Complementation studies to confirm phenotypic effects are due to the targeted mutations

  • Site-directed mutagenesis of specific residues to study structure-function relationships

• Cell infection models:

  • Epithelial cell lines: mICc12 (murine colon enterocyte) and HeLa cells

  • Macrophage cell lines: THP-1A (human) and RAW 264.7 (murine)

  • Measurement of intracellular bacterial replication via gentamicin protection assays and CFU enumeration

• Exometabolomic analysis:

  • Analysis of metabolites secreted by intracellular bacteria to identify alternative energy-generating pathways

  • Combination with mutational approaches to determine metabolic adaptations in response to ATP synthase deficiency

• Animal infection models:

  • Intravenous infection of mice followed by quantification of bacterial loads in liver and spleen

  • Protection studies to assess vaccine potential by challenging immunized mice with virulent wild-type strains

What are the structural and functional differences between atpE in Salmonella and its homologs in other bacteria?

While the search results don't provide detailed information specific to this comparison, research approaches to address this question would include:

• Sequence conservation analysis:

  • ATP synthase subunit c is highly conserved across bacterial species, with significant sequence similarity

  • Key differences often occur in the hydrophobic transmembrane regions that may affect proton translocation efficiency

  • Variations in the number of c-subunits per ring structure can impact the ATP/proton ratio and energy transduction efficiency

• Functional complementation studies:

  • Replacing Salmonella atpE with homologs from other species to assess functional complementation

  • Analyzing chimeric proteins containing domains from different bacterial atpE proteins to identify critical regions

• Expression and regulation differences:

  • Analyzing promoter structures and transcriptional regulation

  • Examining post-translational modifications that might differ between species

How can recombinant Salmonella expressing modified atpE be utilized in heterologous antigen delivery systems?

Recombinant attenuated Salmonella vaccines (RASVs) offer versatile platforms for heterologous antigen delivery:

• Vector construction strategies:

  • Attenuation through controlled modification of atpE or other ATP synthase components

  • Expression of heterologous antigens from bacterial, viral, or parasitic pathogens under appropriate promoters

  • Creation of balanced lethal systems to ensure plasmid stability during immunization

• Antigen delivery mechanisms:

  • Salmonella efficiently targets mucosal-associated lymphoid tissue (MALT) and induces local and systemic immunity

  • Recombinant proteins are produced under SPI-2-regulated conditions and translocated into the cytosol via the SPI-2 T3SS

  • Secreted peptides are processed and presented to MHC class I and II molecules to stimulate T cell responses

• Administration routes and dosing:

  • RASVs can be administered via various routes: oral, nasal, subcutaneous, and intramuscular

  • Each route demonstrates different efficacy for specific target pathogens

  • Multiple dosing regimens have been tested to optimize immune responses while minimizing bacterial shedding

What are the challenges in maintaining stability of atpE-based attenuated Salmonella vaccine strains?

Several challenges exist in developing stable atpE-modified vaccine strains:

• Genetic stability concerns:

  • Potential for reversion to virulence through compensatory mutations

  • Need for multiple attenuation mechanisms to ensure safety

  • Balance between attenuation and immunogenicity

• Plasmid stability issues:

  • Loss of expression plasmids in vivo without selective pressure

  • Development of balanced lethal systems that maintain selection pressure for antigen-expressing plasmids

  • Consideration of chromosomal integration of antigen genes as an alternative to plasmid-based expression

• Biocontainment considerations:

  • Variable levels of fecal shedding observed with different vectors

  • Need to test different vectors to achieve optimal balance among immunogenicity, stability, and biocontainment

  • Environmental release concerns and regulatory requirements for live attenuated vaccines

What are the future research priorities for recombinant Salmonella typhimurium atpE studies?

Based on current knowledge, several priority areas for future research emerge:

• Refinement of attenuation strategies:

  • Development of precisely engineered atpE mutations that balance attenuation with immunogenicity

  • Combination with other attenuating mutations for optimal vaccine safety and efficacy

  • Exploration of regulatory control mechanisms for in vivo atpE expression

• Advanced delivery systems:

  • Optimization of heterologous antigen expression and delivery via Salmonella vectors

  • Development of novel antigen presentation strategies using the ATP synthase platform

  • Investigation of tissue-specific targeting approaches

• Host-pathogen interactions:

  • Further characterization of metabolic adaptations in response to ATP synthase deficiency

  • Investigation of host immune responses to ATP synthase components

  • Exploration of potential cross-protection mechanisms

• Clinical translation:

  • Assessment of safety and efficacy in larger animal models

  • Development of manufacturing processes suitable for clinical-grade production

  • Regulatory considerations for live attenuated vaccine approval