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

What is the fundamental role of kdpC in bacterial potassium transport?

The KdpC subunit plays a crucial role in high-affinity potassium uptake in bacteria. It functions as a catalytic chaperone within the ATP-driven KdpFABC complex, which mediates potassium transport across bacterial membranes. In this complex, while KdpB serves as the ATP-hydrolyzing subunit (classified as a type IA P-type ATPase), and KdpA promotes K+ transport (resembling a potassium channel), KdpC specifically enhances the efficiency of ATP binding and hydrolysis . This creates a unique chimera of ion pumps and ion channels in bacterial systems, establishing the KdpFABC complex as a distinctive bacterial transport mechanism essential for potassium homeostasis.

How can the KdpC subunit be manipulated in recombinant Salmonella choleraesuis to enhance vaccine delivery systems?

A methodological approach would involve:

• Identifying optimal insertion sites within or adjacent to kdpC that don't disrupt its critical domains

• Constructing expression vectors with regulated promoters to control KdpC-antigen fusion protein expression

• Evaluating the stability of the recombinant constructs through multiple passages (>50 as demonstrated with other S. choleraesuis recombinants)

• Assessing growth kinetics compared to wild-type strains to ensure viable vaccine production

The attenuated S. choleraesuis vector containing regulated delayed attenuation and exogenous synthesis systems (as seen in rSC0016) provides a promising platform for such manipulations .

What are the molecular mechanisms underlying the ATP-binding enhancement by KdpC, and how can these be experimentally verified?

The molecular mechanism of ATP-binding enhancement by KdpC involves the formation of a transient ternary complex between KdpB, KdpC, and ATP. This process relies heavily on the conserved glutamine residue in KdpC, which facilitates high-affinity nucleotide binding . To experimentally verify these mechanisms, researchers should employ a multi-faceted approach:

These methods would collectively elucidate how KdpC enhances ATP binding and influences the catalytic cycle of the KdpFABC complex .

What are the optimal conditions for expressing recombinant KdpC in Salmonella choleraesuis vectors?

Optimal expression of recombinant KdpC in S. choleraesuis vectors requires careful consideration of several parameters:

Growth curve analysis should be performed to compare recombinant strains with empty vector controls and wild-type S. choleraesuis, as differences in growth rates have been observed with other recombinant proteins in this system . Environmental factors such as pH, osmolarity, and nutrient availability should be optimized based on the specific strain and application.

How can researchers effectively purify and characterize recombinant KdpC for structural and functional studies?

Effective purification and characterization of recombinant KdpC requires a systematic approach:

• Expression Optimization:

  • Incorporate affinity tags (His6, FLAG, or Strep-tag II) at either N- or C-terminus

  • Test both periplasmic and cytoplasmic expression strategies

  • Evaluate codon optimization for enhanced expression

• Purification Protocol:

  • Membrane fraction isolation via differential centrifugation

  • Solubilization using mild detergents (DDM, LDAO, or CHAPS)

  • Affinity chromatography as initial purification step

  • Size exclusion chromatography for final polishing

  • Consider lipid nanodiscs for maintaining native-like environment

• Functional Characterization:

  • ATPase activity assays measuring Pi release

  • Nucleotide binding studies using fluorescent ATP analogs

  • Thermal stability assessments via differential scanning fluorimetry

  • Surface plasmon resonance for KdpB-KdpC interaction studies

• Structural Analysis:

  • Circular dichroism for secondary structure assessment

  • Hydrogen-deuterium exchange mass spectrometry for conformational analysis

  • X-ray crystallography or cryo-EM for high-resolution structural determination

The purified protein should be assessed for its ATP binding properties, with particular attention to the role of the conserved glutamine residue and the hydroxyl groups of the ATP ribose moiety, as these have been shown to significantly influence binding and hydrolysis activity .

What are the most effective approaches for generating knockout and complementation models to study KdpC function?

Generating effective knockout and complementation models for studying KdpC function requires precise genetic manipulation strategies:

Knockout Approaches:

• CRISPR-Cas9 System:

  • Design sgRNAs targeting conserved regions of kdpC

  • Use homology-directed repair to introduce premature stop codons

  • Confirm knockouts via PCR, sequencing, and Western blot analysis

• Lambda Red Recombineering:

  • Generate linear DNA fragments with antibiotic resistance cassettes flanked by homology regions

  • Transform into S. choleraesuis expressing Lambda Red proteins

  • Select for recombinants and verify gene disruption

• Suicide Vector Integration:

  • Clone internal fragments of kdpC into suicide vectors (e.g., pRE112)

  • Select for single-crossover integrants to disrupt gene function

  • Confirm integration and functional disruption

Complementation Strategies:

• Chromosomal Integration:

  • Integrate wild-type or modified kdpC at neutral sites using Tn7-based systems

  • Use inducible promoters to control expression levels

  • Verify expression levels comparable to wild-type

• Plasmid-Based Complementation:

  • Clone kdpC into balanced-lethal plasmid systems like the pYA3943 vector

  • Address potential copy number effects by using low-copy vectors

  • Include native promoter elements for physiological expression patterns

• Trans-Complementation Analysis:

  • Complement with kdpC variants containing specific mutations

  • Assess restoration of phenotypes (growth, potassium uptake, ATP binding)

  • Evaluate structural-functional relationships of specific domains

All genetic constructs should be verified by sequencing, and expression levels should be confirmed through qRT-PCR and Western blot analysis to ensure appropriate comparisons between wild-type, knockout, and complemented strains.

How should researchers interpret changes in potassium transport efficiency when KdpC is modified in recombinant systems?

Interpreting changes in potassium transport efficiency requires comprehensive analysis at multiple levels:

• Quantitative Transport Measurements:

  • Monitor 86Rb+ or 42K+ uptake rates under varying external K+ concentrations

  • Calculate kinetic parameters (Km, Vmax) to assess affinity and capacity changes

  • Compare transport rates under energy-limiting conditions to evaluate ATP utilization efficiency

• Growth Phenotype Analysis:

  • Evaluate growth curves in potassium-limited media (compare to results in )

  • Determine minimum K+ concentration required for growth

  • Assess growth recovery after potassium starvation

• ATP Binding and Hydrolysis Correlation:

  • Measure ATP hydrolysis rates in parallel with transport activity

  • Calculate coupling ratios (K+ transported per ATP hydrolyzed)

  • Analyze the impact of the conserved glutamine residue modifications on both activities

• Systematic Data Interpretation Framework:

Researchers should consider that modifications affecting the conserved glutamine residue in KdpC will likely have significant impacts on ATP binding affinity and subsequently on transport efficiency, as this residue is critical for high-affinity nucleotide binding .

What statistical approaches are most appropriate for analyzing immunological responses to recombinant Salmonella choleraesuis expressing modified KdpC?

When analyzing immunological responses to recombinant S. choleraesuis expressing modified KdpC, researchers should employ robust statistical approaches:

• Experimental Design Considerations:

  • Use appropriate sample sizes based on power analysis (typically n ≥ 8 per group)

  • Include positive controls (inactivated vaccines), negative controls (empty vectors), and placebo groups

  • Consider factorial designs to evaluate multiple variables simultaneously

• Statistical Methods for Different Immune Parameters:

• Advanced Analytical Approaches:

  • Multivariate analysis to identify correlates of protection

  • Principal component analysis to reduce dimensionality of complex immune datasets

  • Machine learning algorithms to identify immune signatures associated with protection

  • Longitudinal data analysis to track immune responses over time

• Reporting Standards:

  • Report exact p-values rather than thresholds

  • Include confidence intervals for all effect size estimates

  • Clearly state multiple testing corrections applied

  • Share raw data in public repositories when possible

As demonstrated in studies with other recombinant S. choleraesuis strains, comparing immune responses across multiple parameters (mucosal, humoral, and cellular) provides a comprehensive understanding of vaccine efficacy .

How can researchers distinguish between the effects of KdpC modification and vector-related factors in recombinant expression systems?

Distinguishing between KdpC-specific effects and vector-related factors requires systematic experimental design and appropriate controls:

• Essential Control Groups:

  • Wild-type S. choleraesuis (e.g., C78-3 strain)

  • Attenuated S. choleraesuis without any plasmid

  • Attenuated S. choleraesuis with empty vector (e.g., rSC0016(pYA3493))

  • Attenuated S. choleraesuis expressing unmodified KdpC

  • Attenuated S. choleraesuis expressing modified KdpC variants

• Key Parameters to Compare:

  • Growth kinetics under standard and stress conditions

  • Plasmid stability over multiple passages

  • Protein expression levels (Western blot quantification)

  • In vivo colonization patterns and persistence

  • Host immune responses to vector vs. insert antigens

• Analytical Framework for Attribution of Effects:

• Advanced Approaches:

  • Dose-response studies with regulated expression systems

  • Cross-complementation with different vector backbones

  • Temporal analysis of phenotype development

  • Transcriptomic profiling to identify global effects

As observed with the rSC0016(pS-PlpE) construct, foreign antigen expression can influence growth capacity compared to empty vector controls, highlighting the importance of these comparative analyses .

What emerging technologies could advance our understanding of KdpC function in recombinant Salmonella choleraesuis systems?

Several emerging technologies show promise for deepening our understanding of KdpC function:

• Single-Molecule Techniques:

  • Single-molecule FRET to visualize conformational changes during the transport cycle

  • Magnetic tweezers to measure force generation during transport

  • Nanopore recording to capture individual transport events

• Advanced Structural Biology Approaches:

  • Time-resolved cryo-EM to capture intermediates in the transport cycle

  • Integrative structural biology combining multiple data types (X-ray, NMR, SAXS)

  • Computational structure prediction using AlphaFold2 for variant analysis

• Systems Biology Integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and metabolomics

  • Flux balance analysis to model potassium homeostasis in the context of cellular metabolism

  • Network analysis to identify interactions between KdpC and other cellular components

• In vivo Imaging Advances:

  • Genetically encoded potassium sensors for real-time monitoring

  • Intravital microscopy to track recombinant S. choleraesuis in animal models

  • Multiplexed ion beam imaging to visualize potassium distribution at cellular resolution

• Synthetic Biology Approaches:

  • Minimal recombinant systems reconstituted in liposomes or nanodiscs

  • Engineered chimeric transporters to dissect domain-specific functions

  • Optogenetic control of KdpC expression or activity

These technologies would allow researchers to move beyond the current understanding of KdpC as a catalytic chaperone and explore its dynamic interactions within the complex and in the context of the whole cell.

How might KdpC research contribute to the development of novel antimicrobial strategies targeting bacterial potassium transport?

KdpC research offers several promising avenues for novel antimicrobial development:

• Direct Inhibition Strategies:

  • Target the KdpC-KdpB interaction interface to disrupt ATP binding enhancement

  • Design compounds that compete for binding at the conserved glutamine residue

  • Develop allosteric inhibitors that lock KdpC in non-productive conformations

• Combination Therapy Approaches:

  • Pair KdpC inhibitors with conventional antibiotics to enhance efficacy

  • Target multiple components of potassium homeostasis simultaneously

  • Develop adjuvants that sensitize bacteria to potassium limitation

• Vaccine Development Applications:

  • Utilize recombinant S. choleraesuis expressing modified KdpC as live attenuated vaccines

  • Develop subunit vaccines targeting conserved epitopes of KdpC

  • Combine KdpC with other antigens in multivalent vaccine formulations

• Potential Advantages and Challenges:

• Translational Research Priorities:

  • In vitro testing against diverse bacterial pathogens

  • Optimization of targeting specificity

  • In vivo efficacy and safety studies

  • Resistance development monitoring

The unique role of KdpC as a catalytic chaperone in the ATP-driven KdpFABC complex offers a distinctive target that differs from conventional antibiotic approaches .

What are the implications of KdpC research for understanding broader aspects of bacterial physiology and host-pathogen interactions?

KdpC research has far-reaching implications for understanding bacterial physiology and host-pathogen interactions:

• Bacterial Stress Responses and Adaptation:

  • Elucidation of potassium's role as a second messenger in stress responses

  • Understanding adaptation to potassium-limited environments during infection

  • Insights into bacterial osmoadaptation mechanisms

• Evolutionary Perspectives:

  • Comparative analysis of KdpC across bacterial species reveals evolutionary adaptations

  • Understanding the chimeric nature of the KdpFABC complex as a product of molecular evolution

  • Insights into the convergent evolution of ATP-binding mechanisms

• Host-Pathogen Interaction Dynamics:

  • Role of potassium homeostasis in survival within host environments

  • Impact of host potassium sequestration as an antimicrobial strategy

  • Influence of potassium transport on expression of virulence factors

• Broader Physiological Integration:

  • Connections between potassium homeostasis and other cellular processes

  • Influence on bacterial biofilm formation and persistence

  • Links between ion homeostasis and antibiotic tolerance

• Translational Research Applications:

  • Development of attenuated vaccine vectors based on KdpC modulation

  • Creation of biosensors for environmental potassium monitoring

  • Engineering of probiotic bacteria with enhanced stress tolerance

The unique mechanism of KdpC as a catalytic chaperone that enhances ATP binding through an ABC transporter-like mechanism represents a fascinating example of molecular adaptation that may inform our understanding of other bacterial systems and provide new approaches for intervention in infectious diseases.