Recombinant Salmonella gallinarum Potassium-transporting ATPase C chain (kdpC)

What is the basic structure and function of Salmonella gallinarum Potassium-transporting ATPase C chain (kdpC)?

Salmonella gallinarum Potassium-transporting ATPase C chain (kdpC) is a 194-amino acid membrane protein that forms part of the KdpFABC complex responsible for high-affinity potassium ion uptake. The protein has the following characteristics:

• Full amino acid sequence: MIGLRPAFSTMLFLLLLTGGVYPLLTTALGQWWFPWQANGSLI HKDNVIRGSALIGQSFTAAGYFHGRPSATADTPYNPLASGGSNLAASNPELDAQIQSRVAALRAANPQASSAVPVELATASASGLDNNLTPGAAAWQIPRVAAARQLPVEQVAQLVAEYTHRPLARFLGQPVVNIVELNLALDALQGHRAK

• Expression region: 1-194 amino acids

• UniProt accession: B5R669

• EC classification: 3.6.3.12 (ATP phosphohydrolase [potassium-transporting])

• Alternative names: ATP phosphohydrolase [potassium-transporting] C chain, Potassium-binding and translocating subunit C, Potassium-translocating ATPase C chain

Functionally, kdpC acts as the potassium-binding component of the complex, working in conjunction with kdpA (the transmembrane channel component), kdpB (the catalytic ATP-hydrolyzing subunit), and kdpF (a small regulatory peptide). Together, they form a P-type ATPase that transports K+ ions against their concentration gradient using energy from ATP hydrolysis.

What expression systems are optimal for recombinant production of Salmonella gallinarum kdpC?

For recombinant production of Salmonella gallinarum kdpC, Escherichia coli-based expression systems have demonstrated high efficiency. The methodology involves:

• Vector selection: pET-based expression vectors containing an N-terminal 10xHis tag facilitate efficient purification while maintaining protein functionality .

• Host strain selection: E. coli BL21(DE3) or similar strains optimized for membrane protein expression are recommended to minimize toxicity and increase yield.

• Induction conditions: Expression at lower temperatures (16-20°C) after induction with 0.1-0.5 mM IPTG helps prevent inclusion body formation.

• Media optimization: Supplementing growth media with 1% glucose helps repress basal expression before induction.

Table 1: Comparison of Expression Systems for Recombinant kdpC Production

The most successful approach involves expressing kdpC with an N-terminal 10xHis tag in E. coli, followed by detergent solubilization and purification via immobilized metal affinity chromatography (IMAC).

What are the critical parameters for purifying functional recombinant kdpC protein?

Purification of functional recombinant kdpC requires careful attention to multiple parameters:

• Membrane extraction: Gentle extraction using mild detergents (DDM, LDAO, or C12E8) at concentrations just above their critical micelle concentration preserves protein structure and function.

• Buffer composition: Purification buffers should contain:

  • 20-50 mM Tris or HEPES buffer (pH 7.5-8.0)

  • 100-300 mM NaCl

  • 5-10% glycerol as a stabilizer

  • 1-5 mM DTT or 2-ME to maintain reduced cysteines

  • Detergent concentration at 2-3× CMC

  • Optional: 1-5 mM MgCl₂ and 0.1-0.5 mM ATP

• Purification strategy:

  • IMAC using Ni-NTA resin with imidazole gradients (10-250 mM)

  • Size exclusion chromatography to remove aggregates

  • Optional ion exchange chromatography for higher purity

• Storage considerations: Store at -20°C/-80°C with 50% glycerol or lyophilized with 6% trehalose . Avoid repeated freeze-thaw cycles as they significantly reduce protein activity.

What established assays can be used to assess the functional activity of recombinant kdpC?

Several complementary approaches can evaluate the functional activity of recombinant kdpC:

• ATP hydrolysis assays: Measure the ATPase activity of reconstituted KdpFABC complex containing the recombinant kdpC using:

  • Malachite green phosphate detection assay

  • Coupled enzyme assays (pyruvate kinase/lactate dehydrogenase)

• Potassium transport assays:

  • Reconstitution into proteoliposomes with fluorescent K⁺ indicators

  • Rubidium-86 uptake assays in complementation studies

• Binding assays:

  • Isothermal titration calorimetry (ITC) to measure K⁺ binding affinities

  • Surface plasmon resonance (SPR) to study interactions with other Kdp complex components

• Complementation studies: Transform kdpC-deficient bacterial strains with recombinant kdpC and assess growth restoration under K⁺-limiting conditions.

The most definitive assessment combines multiple approaches, particularly measuring ATPase activity in reconstituted systems alongside potassium transport functionality.

How can researchers investigate the interaction between kdpC and other components of the KdpFABC complex?

Investigating interactions between kdpC and other KdpFABC components requires specialized techniques:

• Co-immunoprecipitation: Using antibodies against tagged versions of kdpC to pull down interacting partners.

• Crosslinking studies: Chemical crosslinkers (DSS, BS3, or glutaraldehyde) can capture transient interactions before mass spectrometry analysis.

• FRET/BRET analyses: Fluorescent or bioluminescent tags on different subunits can reveal proximity and conformational changes during transport cycles.

• Cryo-EM analysis: For structural characterization of the entire complex, with particular attention to:

  • Interaction interfaces between kdpC and kdpA/kdpB

  • Conformational changes associated with different states of the transport cycle

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Identifies regions of kdpC that become protected upon complex formation.

• Molecular docking and simulation: In silico approaches using homology models can predict interaction sites that can then be validated experimentally.

Table 2: Key Interaction Sites Between kdpC and Other Kdp Complex Components

How does kdpC contribute to Salmonella gallinarum virulence and pathogenicity?

The role of kdpC in Salmonella gallinarum virulence is linked to potassium homeostasis during infection:

• Survival in K⁺-limited environments: The KdpFABC system allows bacteria to survive in the potassium-restricted environments found within host cells and tissues.

• Osmotic regulation: By maintaining proper K⁺ levels, the bacteria can adapt to osmotic challenges encountered during infection.

• Gene regulation: K⁺ levels influence the expression of virulence genes through various regulatory systems.

• Host interaction: While not directly studied for kdpC in Salmonella gallinarum, research on related pathogens suggests the KdpFABC complex may influence:

  • Intracellular survival within macrophages

  • Biofilm formation

  • Resistance to host antimicrobial peptides

Experimental approaches to study this relationship include:

• Creating kdpC knockout mutants and assessing virulence in chicken models

• Measuring bacterial survival in potassium-limited conditions that mimic host environments

• Transcriptomic analysis to identify virulence genes regulated by potassium limitation

How does the function of kdpC in Salmonella gallinarum compare with other Salmonella species?

Comparative analysis of kdpC across Salmonella species reveals important insights:

• Sequence conservation: Multiple sequence alignment shows high conservation of kdpC across Salmonella species, with specific differences that may correlate with host specificity.

• Functional differences: While the core potassium transport function is conserved, subtle differences in:

  • Regulation of kdpC expression

  • Protein-protein interactions within the complex

  • Transport kinetics and efficiency

• Host adaptation: Salmonella gallinarum is host-restricted to avian species, unlike broader host-range Salmonella species, which may be reflected in adaptations of its potassium transport systems.

While not directly studying kdpC, recent research on Salmonella gallinarum has demonstrated that genetic modifications affecting fundamental metabolic processes can significantly attenuate virulence. For example, deletion of the purB gene resulted in a strain with zero mortality in chicken models compared to 80% mortality with wild-type strains , suggesting that disruption of essential physiological processes (like potassium transport) could similarly affect pathogenicity.

What potential does recombinant kdpC have as a component in vaccine development against Salmonella gallinarum?

The potential of recombinant kdpC in vaccine development can be assessed through several research angles:

• Antigenicity assessment: Evaluating the immunogenicity of purified recombinant kdpC through:

  • Antibody production in immunized animals

  • T-cell response profiling

  • Epitope mapping to identify immunodominant regions

• Subunit vaccine approaches: Incorporating recombinant kdpC into vaccine formulations:

  • As a single antigen with appropriate adjuvants

  • In combination with other Salmonella antigens

  • Delivered via nanoparticles or liposomes

• Attenuated strain development: Similar to the approach with the purB gene , creating attenuated Salmonella gallinarum strains with modified kdpC expression that maintain immunogenicity while reducing virulence.

• Vectored vaccine platforms: Using other attenuated bacterial or viral vectors to express kdpC as part of multi-component vaccines.

Research with other Salmonella gallinarum strains has shown that targeted genetic modification can create effective vaccine candidates. For example, the SG ΔpurB mutant demonstrated complete attenuation (0% mortality vs. 80% with wild-type) while maintaining immunogenicity , suggesting that similar approaches targeting the potassium transport system could be viable.

How can researchers utilize recombinant kdpC for studying membrane protein dynamics and transport mechanisms?

Recombinant kdpC offers opportunities for fundamental research on membrane protein dynamics:

• Site-directed spin labeling: Introducing spin labels at specific residues in kdpC allows electron paramagnetic resonance (EPR) spectroscopy to track conformational changes during transport cycles.

• Single-molecule FRET: Fluorescent labeling at key positions enables observation of real-time conformational dynamics at the single-molecule level.

• Nanodiscs and lipid bilayer systems: Reconstituting kdpC and the full KdpFABC complex into defined membrane environments allows precise control over lipid composition and investigation of lipid-protein interactions.

• Molecular dynamics simulations: In silico approaches can model conformational changes and ion movement, generating hypotheses that can be tested experimentally.

• Cryo-EM analysis: Capturing different conformational states of the complex can reveal the structural basis of the transport mechanism.

What are the current challenges in working with recombinant kdpC and how can they be addressed?

Researchers face several challenges when working with recombinant kdpC:

• Membrane protein instability:

  • Challenge: Membrane proteins often aggregate or denature during purification

  • Solution: Use of stabilizing additives (glycerol, specific lipids), amphipols, or nanodiscs to maintain native structure

• Low expression yields:

  • Challenge: Membrane proteins typically express at lower levels than soluble proteins

  • Solution: Optimization of expression conditions, use of specialized E. coli strains (C41/C43), or insect cell expression systems

• Functional reconstitution:

  • Challenge: Maintaining activity after purification requires proper reconstitution

  • Solution: Careful selection of detergents and lipids that mimic the native membrane environment

• Structural heterogeneity:

  • Challenge: Multiple conformational states complicate structural studies

  • Solution: Use of conformation-specific antibodies or nanobodies, or stabilizing mutations

• Complex assembly:

  • Challenge: Studying kdpC function often requires reconstitution of the entire KdpFABC complex

  • Solution: Co-expression systems or sequential reconstitution protocols with purified components

What are effective quality control measures to ensure the integrity and functionality of purified recombinant kdpC?

Quality control for recombinant kdpC should include:

• Purity assessment:

  • SDS-PAGE with Coomassie or silver staining (>95% purity ideal)

  • Western blot using anti-His tag or specific anti-kdpC antibodies

  • Mass spectrometry to confirm protein identity and detect modifications

• Structural integrity:

  • Circular dichroism (CD) spectroscopy to verify secondary structure content

  • Fluorescence spectroscopy to assess tertiary structure

  • Dynamic light scattering (DLS) to detect aggregation

• Functional validation:

  • Binding assays with known interaction partners

  • ATPase activity when reconstituted with other Kdp complex components

  • Potassium transport assays in proteoliposomes

• Storage stability:

  • Accelerated stability studies at different temperatures

  • Freeze-thaw cycle testing to establish optimal aliquoting protocols

  • Activity measurements after various storage conditions

A systematic quality control workflow combining these approaches ensures that experimental results are obtained with functionally relevant protein preparations.

Table 3: Troubleshooting Guide for Common Issues with Recombinant kdpC