The atpE protein is highlighted as a potential vaccine candidate due to its conserved role in bacterial survival. Salmonella ATP synthase subunit c is critical for energy production, making it a viable target for disrupting pathogenic processes. Suppliers note its use in preclinical studies to elicit immune responses, though specific efficacy data remain unpublished .
Recombinant atpE serves as an antigen in enzyme-linked immunosorbent assays (ELISA) to detect anti-Salmonella antibodies. Its high purity ensures minimal cross-reactivity, enabling precise serological analysis .
Although no direct studies on S. enteritidis atpE are available, broader research on bacterial ATP synthases provides context:
Energy Metabolism: ATP synthase subunit c facilitates proton translocation, driving ATP synthesis. In Salmonella Pullorum, energy metabolism pathways (e.g., hydrogenases, succinate dehydrogenase) are downregulated under stress, highlighting the enzyme’s role in survival .
Virulence and Host Interaction: While atpE itself is not directly linked to virulence, ATP synthase activity supports intracellular replication and evasion of host immune responses. For example, Salmonella mutants deficient in energy metabolism genes exhibit reduced fitness in macrophages .
KEGG: set:SEN3684
ATP synthase subunit c is a building block of the membrane rotor within the F₀ complex of ATP synthase. It functions as part of the molecular turbine that uses transmembrane proton flow to generate ATP. In Salmonella enteritidis PT4, this protein forms a ring structure (c-ring) that rotates as protons pass through the membrane, driving the conformational changes necessary for ATP synthesis . The atpE gene is highly conserved across Salmonella serovars, reflecting its essential function in cellular bioenergetics. The amino acid sequence of atpE (position 1-79) contains the critical residues involved in proton binding and translocation that power the rotational mechanism of ATP synthase .
Subunit c interacts primarily with subunit a, which forms part of the membrane stator of the ATP synthase. This interaction is crucial for assembling the proton channel of ATP synthase. Research has demonstrated that subunit a can be molecularly fused with subunit c, and this fusion is capable of incorporating into the ATP synthase complex, providing a valuable structural model for studying the proton channel . The interaction between subunit a and monomeric subunit c is a key initial step in ATP synthase assembly, as it triggers the insertion of subunit a into the membrane and initiates formation of the a-c complex, which constitutes the ion-translocating module of ATP synthase . This interaction precedes the assembly of the c-ring, with the fused c subunit either incorporating into the c-ring or remaining on its periphery depending on the orientation of the transmembrane helices in the fusion protein .
Researchers can distinguish between functional and non-functional atpE mutations through several methodological approaches:
Complementation studies: Wild-type and mutant forms of atpE can be expressed in atpE-deficient strains to determine if function is restored.
Proton translocation assays: Measuring pH changes across membranes containing reconstituted ATP synthase complexes with different atpE variants.
ATP synthesis measurements: Quantifying ATP production in systems with various atpE mutations can directly assess functional impact.
Molecular fusion approaches: As demonstrated in research, creating fusion proteins between subunit a and subunit c can help determine if specific orientations and interactions are functional. For example, researchers have observed that a/c fusion proteins with correct orientation of transmembrane helices could be inserted into the membrane and co-incorporated into the F₀ complex, while fusions with incorrect orientation required wild-type subunit c for membrane insertion .
Motility assays: For Salmonella, which uses ATP synthase to power flagellar rotation, motility tests can indirectly assess atpE function, particularly in the context of type-III secretion systems which share evolutionary relationships with ATP synthase components .
The atpE gene in Salmonella enteritidis PT4 is part of a larger atp operon that encodes multiple subunits of the ATP synthase complex. Analysis of the complete genome of S. enteritidis PT4 (strain P125109, EMBL accession no. AM933172) reveals high conservation of this core metabolic machinery . The gene organization follows the typical arrangement found in most bacteria, with atpE positioned within a cluster of genes encoding other ATP synthase components.
Comparative genome analysis between S. enteritidis PT4 and S. Typhimurium LT2 shows extremely high nucleotide identity (98.98%) between shared orthologs, suggesting the ATP synthase genes are part of the extensive core gene-set that maintains colinearity between these serovars . This high conservation reflects the essential nature of ATP synthase genes for bacterial viability and energy metabolism across Salmonella species.
Comparative genomic analysis reveals several important evolutionary insights about atpE:
High conservation: The atpE gene shows remarkable sequence conservation across Salmonella serovars, including between S. enteritidis PT4 and S. Typhimurium LT2, which share >90% of their coding sequences as part of an extensive core gene-set .
Evolutionary relationship with type-III secretion systems: Research has demonstrated a strong homology between cytoplasmic components of type-III secretion systems and the F₀F₁ ATP synthase. This suggests that the bacterial flagellum, which includes components analogous to ATP synthase, may have evolved from a proto F₀F₁-ATP synthase .
Evolutionary sequence: Studies indicate that a proto ATPase may have been added to a primordial proton-powered type-III export system, with the evolutionary benefit of facilitating the export process. This is supported by findings that ATPase activity can be dispensable for type-III protein export in Salmonella under conditions of increased proton motive force .
Minimal pseudogenization: Unlike many virulence-associated genes that undergo pseudogenization in host-adapted strains, core metabolic genes like atpE typically maintain their functionality. For example, while S. Gallinarum (a highly host-adapted chicken pathogen) harbors a significantly higher number of pseudogenes compared to S. enteritidis PT4, essential metabolic genes remain functional .
This evolutionary perspective provides important context for understanding the fundamental role of atpE in bacterial physiology and its relationship to other bacterial systems.
Recombinant S. enteritidis PT4 atpE protein has several potential applications in vaccine development:
As a component in attenuated live vaccines: Salmonella strains with modified atpE could serve as attenuated vaccine carriers. Research has demonstrated that Salmonella spp. can be genetically modified to create multivalent live carrier vaccines for simultaneous immunization against several unrelated pathogens .
Engineering acid resistance: The ATP synthase is linked to acid resistance mechanisms in Salmonella. Researchers have re-engineered acid resistance systems in Salmonella vaccine strains to enhance survival under acidic conditions, which could improve vaccine efficacy when administered orally . Similar approaches could potentially utilize atpE modifications.
Antigen presentation platform: As a component of the bacterial membrane, atpE could potentially be engineered to present foreign epitopes or antigens, leveraging Salmonella's ability to deliver effector proteins to host cells through type-III secretion systems .
Target for attenuating mutations: Introducing specific mutations in atpE that reduce ATP synthesis efficiency without abolishing it completely could create attenuated strains with reduced virulence but maintained immunogenicity.
When developing such applications, researchers must consider that recombinant Salmonella atpE protein products are strictly for research purposes and cannot be used directly on humans or animals without appropriate regulatory approval and clinical trials .
Purification of recombinant S. enteritidis PT4 atpE presents challenges due to its hydrophobic nature as a membrane protein. The following methodological approaches have proven effective:
Expression systems selection: Multiple expression systems can be utilized, including E. coli, yeast, baculovirus, or mammalian cells . E. coli systems are commonly preferred for initial screening due to their high yield and cost-effectiveness, though eukaryotic systems may provide better folding for structural studies.
Membrane protein extraction: Effective extraction requires careful solubilization using detergents such as n-dodecyl-β-D-maltoside (DDM) or digitonin that maintain protein structure while removing it from the lipid bilayer.
Affinity chromatography: His-tagged recombinant atpE can be purified using immobilized metal affinity chromatography (IMAC). For fusion constructs like the a/c fusion proteins described in the literature, appropriate tag placement is critical to ensure accessibility without interfering with structure .
Size exclusion chromatography: This technique helps separate monomeric atpE from aggregates or incomplete translation products, which is particularly important when studying the monomeric form's interaction with subunit a .
Reconstitution methods: For functional studies, purified atpE must often be reconstituted into liposomes or nanodiscs to restore its native membrane environment.
The choice of purification method should be guided by the intended experimental application, whether structural analysis, functional studies, or immunological investigation.
The interaction between proton motive force and atpE function represents a fundamental aspect of bioenergetics:
Primary energy source: The proton motive force (PMF) serves as the primary energy source driving ATP synthesis through the ATP synthase complex. Protons flowing through the a/c interface of ATP synthase cause rotation of the c-ring (composed of multiple copies of subunit c/atpE) .
Dispensability of ATPase activity: Research has revealed that under conditions of increased proton motive force, the requirement for ATPase activity can be bypassed in certain secretion systems related to ATP synthase. This finding has important implications for understanding the evolutionary relationship between ATP synthase and type-III secretion systems .
Experimental evidence: Studies with Salmonella have demonstrated that mutations increasing the proton motive force allowed formation of functional flagella even in the absence of type-III ATPase activity. This was further supported by observations that increased proton motive force could bypass the requirement of the Salmonella pathogenicity island 1 virulence-associated type-III ATPase for secretion .
Functional relationship: The role of subunit c (atpE) in this process involves forming the ion-binding sites that capture and release protons during rotation, converting the energy of the proton gradient into mechanical motion that drives ATP synthesis. The specific amino acid residues within atpE that participate in proton binding are critical for this function .
This fundamental relationship between proton motive force and atpE function underscores the central role of this protein in cellular bioenergetics and bacterial physiology.
Several structural features of atpE are essential for proper c-ring assembly and ATP synthase function:
Understanding these structural features has been enhanced through experimental approaches such as creating fusion proteins between subunits a and c, which have provided valuable insights into the organization of the proton channel and the assembly process of ATP synthase .
Comparative analysis of S. enteritidis PT4 atpE with homologs from other bacterial pathogens reveals important similarities and differences:
This high conservation of atpE across diverse bacterial species reflects its fundamental role in cellular bioenergetics. Comparative genomic analysis between S. enteritidis PT4 and S. Typhimurium LT2 shows extremely high nucleotide identity (98.98%) between shared orthologs , suggesting that ATP synthase genes like atpE are part of the extensive core gene-set that maintains evolutionary stability across Salmonella serovars.
Several lines of evidence support an evolutionary relationship between ATP synthase (which includes atpE) and type-III secretion systems:
Structural homology: The cytoplasmic components of the type-III secretion system share strong homology with the F₀F₁ ATP synthase, suggesting a common evolutionary origin .
Functional parallels: Both systems are involved in energy-dependent transport across membranes - ATP synthase transports protons for energy production, while type-III secretion systems transport proteins across membranes.
Experimental evidence of functional overlap: Research has demonstrated that the flagellar type-III secretion apparatus utilizes both the energy of the proton motive force and ATP hydrolysis to energize substrate translocation, similar to how ATP synthase utilizes the proton motive force .
Dispensability of ATPase: Studies have shown that functional flagella can form in the absence of type-III ATPase activity when mutations increase the proton motive force and flagellar substrate levels. This suggests that the proton motive force is the more fundamental energy source, with ATPase activity serving an enhancing role that may have been added later in evolutionary history .
Evolutionary model: The evidence supports a model where "a proto ATPase was added to a primordial proton-powered type-III export system with the evolutionary benefit of facilitating the export process" . This indicates that the original export system relied solely on the proton motive force, with the ATPase component being a later evolutionary addition that enhanced efficiency.
This evolutionary relationship provides important context for understanding both ATP synthase function and the mechanisms of bacterial protein secretion systems that are critical for pathogenicity.