Recombinant Yersinia pseudotuberculosis serotype O:1b ATP synthase subunit c (atpE)

What is Yersinia pseudotuberculosis serotype O:1b and what is its significance in research?

Yersinia pseudotuberculosis serotype O:1b is one of the 21 recognized serotypes within the Y. pseudotuberculosis complex. This serotype has particular significance as genetic evidence indicates that Yersinia pestis, the causative agent of plague, emerged from a Y. pseudotuberculosis O:1b progenitor . The O:1b serotype is defined by its specific O-antigen polysaccharide structure, which forms part of the lipopolysaccharide (LPS) in the outer membrane of this gram-negative bacterium .

This serotype serves as a crucial model for understanding bacterial evolution and pathogenicity mechanisms. Y. pseudotuberculosis is primarily a zoonotic pathogen that resides in warm-blooded animals including mammals and birds, causing gastroenteritis in humans with symptoms that can mimic appendicitis . The bacterium appears microscopically as an ovoid-shaped cell (coccobacillus) that stains gram-negative, forms small translucent gray colonies, and displays temperature-dependent motility—being motile at lower temperatures but non-motile at human body temperature (35°C) .

What is the ATP synthase subunit c (atpE) and how does it function in bacterial systems?

ATP synthase subunit c, encoded by the atpE gene, is a critical component of the F0 portion of F0F1-ATP synthase, the enzyme complex responsible for ATP production during oxidative phosphorylation. In bacterial systems including Y. pseudotuberculosis, this protein forms a ring structure in the membrane that facilitates proton translocation across the membrane. This proton movement drives the rotary motion of the enzyme complex, which enables the catalytic synthesis of ATP from ADP and inorganic phosphate.

The c-ring consists of multiple copies of the subunit c protein (typically 10-15 in bacteria, depending on the species) arranged in a circle. Each c subunit contains two transmembrane helices connected by a small loop, with a conserved acidic residue (usually aspartate or glutamate) that is essential for proton binding and transport.

How is recombinant expression of Y. pseudotuberculosis proteins typically achieved?

Recombinant expression of Y. pseudotuberculosis proteins, including those from serotype O:1b, is typically achieved through molecular cloning techniques where the gene of interest is inserted into an expression vector. As demonstrated in research with other Y. pseudotuberculosis proteins, successful approaches include:

• Vector selection: Plasmids such as pYA5199 have been successfully used for recombinant protein expression in Y. pseudotuberculosis systems .

• Expression systems: Both homologous and heterologous expression systems can be employed:

  • Homologous expression using attenuated Y. pseudotuberculosis strains such as χ10069 with specific mutations (e.g., ΔyopK ΔyopJ Δasd)

  • Heterologous expression in E. coli using T7 promoter-based systems

• Secretion strategies: Type III secretion system (T3SS) has been effectively utilized for the secretion of recombinant proteins in Y. pseudotuberculosis, as demonstrated with the YopE-LcrV fusion protein .

• Induction conditions: For T3SS-dependent protein secretion, calcium-deprived conditions at 37°C have been shown to induce protein secretion in Y. pseudotuberculosis .

What evolutionary relationship exists between Y. pseudotuberculosis O:1b and Y. pestis, and how might this impact atpE research?

Y. pestis evolved from Y. pseudotuberculosis O:1b, as evidenced by genetic analysis showing that Y. pestis isolates carry genes for the O:1b serotype . This evolutionary relationship provides a valuable framework for comparative studies of essential proteins like ATP synthase subunit c (atpE).

While Y. pestis retains the genetic machinery for O:1b antigen synthesis, it has four inactivating mutations in O-antigen genes that prevent production of the O antigen . This represents a key evolutionary adaptation as Y. pestis transitioned from a gastrointestinal pathogen to a vector-borne pathogen causing systemic disease.

For atpE research, this close evolutionary relationship means:

• Comparative analysis of atpE sequences between these species can reveal conservation patterns essential for function versus regions that may have undergone adaptive evolution.

• Functional differences in ATP synthase between these closely related pathogens might represent adaptations to different host environments and transmission mechanisms.

• Molecular tools developed for one species may often be applicable to the other, facilitating research across both pathogens.

What are the challenges in isolating and purifying recombinant membrane proteins like atpE from Yersinia species?

Purification of recombinant membrane proteins such as ATP synthase subunit c presents several specific challenges:

• Hydrophobicity: The highly hydrophobic nature of atpE, with its transmembrane domains, makes it prone to aggregation and precipitation during purification.

• Native conformation: Maintaining the native structure of atpE during extraction from the membrane environment is difficult, as detergents used for solubilization may disrupt protein-protein interactions essential for function.

• Expression toxicity: Overexpression of membrane proteins often leads to toxicity in host cells, which can be addressed through:

  • Using tightly regulated inducible promoters

  • Expressing the protein as a fusion with solubility enhancers

  • Selecting appropriate host strains with enhanced membrane protein expression capabilities

• Extraction efficiency: The efficiency of membrane protein extraction varies with different detergents and solubilization conditions. A methodical approach testing multiple detergents is often necessary.

• Protein stability: Once extracted from the membrane, atpE may exhibit reduced stability. Addition of stabilizing agents like glycerol or specific lipids might be necessary during purification.

What expression systems are most effective for producing functional recombinant Y. pseudotuberculosis atpE?

Several expression systems have been evaluated for the production of functional bacterial membrane proteins like atpE:

• E. coli-based systems:

  • BL21(DE3) with pET vectors: Provides high-level expression but may lead to inclusion body formation

  • C41(DE3) and C43(DE3): Specialized strains for membrane protein expression with reduced toxicity

  • Lemo21(DE3): Allows tunable expression through rhamnose-inducible control of T7 RNA polymerase levels

• Yersinia-based homologous expression:

  • Attenuated Y. pseudotuberculosis strains like χ10069 with specific mutations (ΔyopK ΔyopJ Δasd) have shown success for recombinant protein expression

  • Advantage of native post-translational processing and membrane insertion

• Cell-free expression systems:

  • Allow direct synthesis of membrane proteins in the presence of detergents or lipid nanodiscs

  • Avoid cellular toxicity issues but may have lower yields

For optimal expression of functional atpE, critical factors include:

• Induction conditions (temperature, inducer concentration, duration)

• Growth media composition

• Co-expression of chaperones to assist proper folding

• Fusion tags that enhance expression and/or solubility

What techniques can be used to verify the structure and function of recombinant Y. pseudotuberculosis atpE?

Verification of proper structure and function of recombinant atpE requires multiple complementary approaches:

• Structural Integrity Assessment:

  • Circular Dichroism (CD) spectroscopy to verify secondary structure content and proper folding

  • NMR spectroscopy for detailed structural analysis of the purified protein

  • Cross-linking studies to verify proper oligomerization into c-rings

• Functional Assays:

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • ATP synthesis activity measurement when reconstituted with other ATP synthase subunits

  • Membrane potential measurements using voltage-sensitive dyes

• Binding Studies:

  • Isothermal Titration Calorimetry (ITC) to measure binding of known ATP synthase inhibitors

  • Surface Plasmon Resonance (SPR) to assess interaction with other ATP synthase subunits

• Reconstitution Studies:

  • Proteoliposome reconstitution to verify integration into lipid bilayers

  • Co-reconstitution with other ATP synthase subunits to assess complex assembly

How can researchers interpret contradictory results in atpE studies across different Yersinia strains?

When confronted with contradictory results in atpE studies across different Yersinia strains, researchers should implement a systematic approach to reconcile these findings:

• Strain-specific genetic differences:

  • Compare the atpE sequences between strains to identify amino acid differences

  • Analyze the genomic context of atpE, including promoters and regulatory elements

  • Consider horizontal gene transfer events that might have occurred, as Y. pseudotuberculosis has shown evidence of genetic exchange within MLST clusters

• Experimental condition variations:

  • Standardize growth conditions (media, temperature, pH, oxygen levels)

  • Control for expression levels when using recombinant systems

  • Verify that protein purification methods maintain similar structural integrity

• Functional context differences:

  • Assess whether strain-specific differences in membrane composition affect atpE function

  • Examine the ATP synthase complex as a whole, as variations in other subunits may influence atpE behavior

  • Consider serotype-specific factors, as O-antigen structures can influence membrane properties

• Statistical validation:

  • Perform sufficient biological and technical replicates

  • Use appropriate statistical tests to determine if differences are significant

  • Consider meta-analysis approaches when combining data from multiple studies

What are the implications of ATP synthase as a potential antibiotic target in Yersinia species?

ATP synthase represents a promising antibiotic target in bacterial pathogens including Yersinia species for several reasons:

For Y. pseudotuberculosis specifically, targeting ATP synthase offers unique considerations:

• Metabolic versatility: Y. pseudotuberculosis can adapt to different environments (environmental reservoirs, mammalian hosts), potentially relying on ATP synthase to different degrees depending on the growth condition.

• Temperature-dependent regulation: Like other Y. pseudotuberculosis proteins that show temperature-dependent regulation , ATP synthase expression or activity might be differentially regulated at environmental versus host temperatures.

• Growth phase considerations: ATP synthase inhibition might be particularly effective during specific growth phases or in particular host environments.

• Resistance development: The essential nature of ATP synthase may constrain resistance mutations, as changes that reduce drug binding might also impair enzymatic function.

How might structural studies of Y. pseudotuberculosis atpE contribute to understanding virulence mechanisms?

Structural studies of Y. pseudotuberculosis atpE could reveal unexpected connections to virulence mechanisms through several avenues:

• Energy-dependent virulence factor expression: Many virulence factors in Y. pseudotuberculosis, including the Type III Secretion System (T3SS) components like YopE and LcrV , require significant energy for expression, assembly, and function. Understanding how atpE contributes to ATP production during infection could reveal how energy metabolism is linked to virulence.

• Adaptation to host environments: Structural adaptations in atpE might reflect specialization for function in different conditions encountered during infection. Comparing atpE structures between environmental and pathogenic Yersinia species could reveal adaptation signatures.

• Interaction with host factors: ATP synthase components in some bacteria have been shown to interact with host proteins. Structural studies might identify potential interaction interfaces on atpE that could contribute to host-pathogen interactions.

• Stress response mechanisms: ATP synthase function is often modulated during stress responses. Structural features that enable rapid adaptation to changing environmental conditions might be important for surviving host defense mechanisms.

• Evolutionary insights: Comparing atpE structures between Y. pseudotuberculosis O:1b and Y. pestis could reveal how evolution from a gastrointestinal pathogen to a vector-borne pathogen affected this essential protein.

What methodological approaches can help investigate the role of atpE in Y. pseudotuberculosis metabolic adaptation?

To investigate atpE's role in Y. pseudotuberculosis metabolic adaptation, researchers should consider these methodological approaches:

• Conditional expression systems:

  • Temperature-sensitive promoters to mimic environmental versus host conditions

  • Inducible systems for controlled atpE expression levels

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

• In vivo imaging techniques:

  • ATP biosensors to monitor ATP levels in real-time during infection

  • Fluorescently tagged atpE to track localization under different conditions

  • FRET-based approaches to monitor interactions with other proteins

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and atpE mutants

  • Correlate ATP synthase activity with global metabolic profiles

  • Use flux balance analysis to model the impact of altered ATP synthase function

• Infection models with real-time monitoring:

  • Mouse infection models tracking bacterial burden and metabolism

  • Cell culture systems with metabolic analysis capability

  • Ex vivo tissue models that better recapitulate in vivo conditions

• Environmental simulation:

  • Microfluidic devices that can rapidly alter environmental conditions

  • Chemostat cultures to maintain defined metabolic states

  • Co-culture systems to assess competitive fitness

How can recombinant Y. pseudotuberculosis atpE be utilized in drug discovery platforms?

Recombinant Y. pseudotuberculosis atpE can be incorporated into drug discovery platforms through several approaches:

• High-throughput screening (HTS) systems:

  • Purified atpE incorporated into liposomes for proton translocation assays

  • Whole-cell ATP synthesis assays with recombinant expression systems

  • Competition binding assays with known ATP synthase inhibitors

• Structure-based virtual screening:

  • Homology models or experimentally determined structures of Y. pseudotuberculosis atpE

  • Molecular docking of compound libraries to identify potential binding sites

  • Molecular dynamics simulations to assess binding stability and conformational changes

• Fragment-based drug discovery:

  • NMR-based fragment screening against purified atpE

  • X-ray crystallography to identify fragment binding sites

  • Fragment growing and linking strategies to develop high-affinity compounds

• Target-based biosensors:

  • atpE-based biosensors that report binding through conformational changes

  • Surface plasmon resonance arrays for rapid screening of binding compounds

  • Thermal shift assays to identify stabilizing ligands

• Comparative studies with other bacterial atpE:

  • Parallel screening against Y. pseudotuberculosis, Y. pestis, and human ATP synthase

  • Selectivity profiling to identify compounds with specificity for bacterial targets

  • Cross-species activity assessment to develop broad-spectrum antimicrobials

What potential exists for using Y. pseudotuberculosis O:1b atpE in diagnostic or vaccine development?

The potential applications of Y. pseudotuberculosis O:1b atpE in diagnostics and vaccine development include:

• Diagnostic applications:

  • Recombinant atpE as a capture antigen in ELISA or lateral flow assays

  • PCR primers targeting strain-specific regions of the atpE gene

  • Antibody-based detection systems using anti-atpE antibodies

• Vaccine development approaches:

  • Attenuated Y. pseudotuberculosis strains with modified atpE could serve as live vaccines

  • Similar to the approach used with other Y. pseudotuberculosis proteins, attenuated strains like χ10069 with ΔyopK ΔyopJ Δasd mutations have shown promise as vaccine vehicles

  • Recombinant atpE could be included in subunit vaccine formulations

• Serological monitoring:

  • atpE-specific antibody responses could be used to monitor exposure or vaccine efficacy

  • Differentiation between Y. pseudotuberculosis and Y. pestis infections based on immune response patterns

• Cross-protective potential:

  • Given the evolutionary relationship between Y. pseudotuberculosis O:1b and Y. pestis , immune responses against conserved epitopes in atpE might provide cross-protection

  • Similar approaches with other proteins have shown protection against multiple Yersinia species, as seen with the YopE-LcrV fusion that protected against Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis

As demonstrated with other Y. pseudotuberculosis proteins, recombinant antigens can induce strong immune responses with single-dose oral immunization, including both systemic antibody responses and mucosal immunity through secretory IgA .