Recombinant Salmonella paratyphi B Potassium-transporting ATPase C chain (kdpC)

What is the kdpFABC operon and what role does kdpC play within it?

The kdpFABC operon encodes a potassium transport channel in Salmonella enterica and represents one of the most highly induced functional groups during bacterial dehydration . Within this operon, kdpC functions as an essential component of the high-affinity K⁺ transport system. The KdpC protein serves as a regulatory subunit that interacts with both KdpB (the catalytic subunit) and KdpA (the K⁺ binding subunit) to facilitate potassium transport across the bacterial membrane. This system becomes particularly important under conditions of potassium limitation or osmotic stress, allowing Salmonella to maintain homeostasis in challenging environments .

How does S. Paratyphi B differ from other Salmonella serovars in terms of its potassium transport systems?

Salmonella Paratyphi B exists in two distinct biotypes - sensu stricto and Java - that cause different clinical manifestations (enteric fever and gastroenteritis, respectively) . While the kdpFABC operon is conserved across Salmonella serovars, genomic analysis has revealed subtle differences in potassium transport systems between these biotypes. The sensu stricto biotype, associated with invasive disease, may exhibit different expression patterns of the kdp genes compared to the Java biotype, which typically causes localized gastrointestinal infections . These differences potentially contribute to the distinct pathogenesis mechanisms of each biotype, although the specific variations in kdpC expression between biotypes require further investigation.

What experimental approaches are recommended for expressing recombinant Salmonella paratyphi B kdpC protein?

For expressing recombinant Salmonella paratyphi B kdpC protein, researchers should consider multiple expression systems based on the specific research requirements. Expression in E. coli is often preferred for initial studies due to high yield and ease of manipulation . For optimal expression, the kdpC gene should be cloned into an appropriate vector (such as pET series) with a histidine tag to facilitate purification. Expression conditions typically include induction with IPTG (0.1-1.0 mM) at 18-30°C to prevent inclusion body formation. Alternative expression systems include yeast, baculovirus, or mammalian cell systems, each offering advantages for specific applications . For structural studies requiring properly folded protein, insect cell expression using baculovirus vectors often provides superior results compared to bacterial systems.

How can researchers effectively distinguish between the functions of kdpC and other components of the kdpFABC operon in Salmonella pathogenesis?

To effectively distinguish between the functions of kdpC and other components of the kdpFABC operon, researchers should implement a comprehensive genetic and biochemical approach. First, create single gene knockout mutations for each component (kdpF, kdpA, kdpB, and kdpC) using lambda red recombinase technology following protocols established for Salmonella . Compare these with the wild-type strain and complete operon deletion mutants in various stress conditions, particularly focusing on potassium limitation, osmotic stress, and desiccation environments.

For more nuanced functional analysis, complementation studies should be performed where each mutant is transformed with a plasmid containing the corresponding wild-type gene under an inducible promoter. Additionally, site-directed mutagenesis of conserved residues in kdpC can reveal specific amino acids critical for its function. Protein-protein interaction studies using bacterial two-hybrid systems or co-immunoprecipitation can identify specific interactions between KdpC and other components.

The table below outlines a systematic approach to distinguish kdpC function:

These approaches collectively provide a comprehensive understanding of kdpC's specific role distinct from other operon components .

What are the optimal conditions for assessing kdpC function in Salmonella paratyphi B during dehydration stress?

The assessment of kdpC function during dehydration stress requires carefully controlled experimental conditions. Based on transcriptomic analysis of dehydrated Salmonella, the kdpFABC operon shows significant upregulation during this stress condition . To optimally assess kdpC function specifically, researchers should:

• Prepare bacterial cultures in logarithmic growth phase (OD₆₀₀ = 0.4-0.6) in low K⁺ medium (≤10 mM K⁺) to pre-induce the kdp system.

• Subject cultures to controlled dehydration on sterile filter paper at 25°C with 33% relative humidity in a desiccation chamber for time periods ranging from 1 to 72 hours.

• Compare wild-type strains with ΔkdpC mutants and complemented strains (ΔkdpC/pkdpC) for survival rates, measured by recovery and plating on non-selective media.

• Implement real-time quantitative PCR to monitor kdpC expression patterns throughout the dehydration process, with samples collected at 0, 1, 2, 6, 24, and 48 hours.

• Measure intracellular K⁺ concentrations using inductively coupled plasma mass spectrometry (ICP-MS) to correlate kdpC function with K⁺ homeostasis during dehydration stress.

The optimal temperature range for these experiments is 25-30°C, as this provides sufficient stress without completely inactivating cellular processes. Additionally, experiments should include control conditions testing cold storage tolerance, as mutations in kdp system components have been shown to significantly impair long-term persistence during cold storage .

How can researchers effectively differentiate between the roles of potassium transport in S. Paratyphi B sensu stricto versus Java biotypes?

Differentiating the roles of potassium transport between S. Paratyphi B sensu stricto and Java biotypes requires a sophisticated experimental approach that accounts for their distinct pathogenesis mechanisms. Whole-genome sequencing analysis has established that these biotypes are genetically distinct despite sharing the same serotype classification .

To effectively compare kdpC function between these biotypes:

• First, select representative strains of both biotypes based on genomic verification: use CMF 6999 or similar strains for sensu stricto and CDC00-0301 for Java biotype, as these have been characterized in previous studies .

• Generate isogenic ΔkdpC mutants in both biotype backgrounds using identical genetic engineering protocols to ensure comparable mutations.

• Compare gene expression profiles of the kdpFABC operon between biotypes under various conditions using RNA-seq and qRT-PCR, focusing particularly on:

  • Growth in potassium-limited media (K⁺ < 0.1 mM)

  • Intracellular survival in macrophage cell lines

  • Response to osmotic stress (0.3M NaCl)

  • Conditions mimicking enteric fever versus gastroenteritis environments

• Perform infection models that reflect the different clinical manifestations: use intraperitoneal infection in BALB/c mice for sensu stricto (systemic infection model) and oral infection for Java (gastroenteritis model) .

• Implement comparative proteomics to identify biotype-specific interaction partners of KdpC that might contribute to the distinct pathogenesis mechanisms.

The expected differences include potentially higher dependency on functional kdpC in sensu stricto biotypes during systemic infection, versus relatively less critical role in Java biotypes, which primarily cause localized gastrointestinal infections .

What approaches should be used to analyze the contribution of kdpC to Salmonella virulence in animal models?

To rigorously analyze the contribution of kdpC to Salmonella virulence, researchers should implement a multi-faceted approach using complementary animal models. The experimental design should:

• Utilize both standard BALB/c mice and streptomycin-pretreated mice models to assess different aspects of Salmonella pathogenesis. BALB/c mice are susceptible to systemic infection and can be used to assess the role of kdpC in invasive disease caused by S. Paratyphi B sensu stricto .

• Compare wild-type, ΔkdpC mutant, and complemented strains (ΔkdpC/pkdpC) in both intraperitoneal (i.p.) and peroral (p.o.) infection routes. The i.p. route is preferred for studying systemic infection, as previous studies with S. Paratyphi B demonstrated greater lethality via this route .

• Determine the 50% lethal dose (LD₅₀) for each strain by challenging mice with bacterial doses ranging from 10³ to 10⁹ CFU. For S. Paratyphi B, previous attenuated strains showed LD₅₀ values 1-2 log₁₀ higher than wild-type strains .

• Monitor bacterial loads in key organs (liver, spleen, intestine, mesenteric lymph nodes) at 24h, 72h, and 7 days post-infection to assess tissue tropism and clearance kinetics.

• Evaluate immune responses by measuring cytokine profiles (particularly IL-6, IL-1β, TNF-α, and IFN-γ) in serum and tissue homogenates.

For more nuanced analysis, consider specialized models:

• Ligated ileal loop model to assess intestinal inflammation and fluid accumulation

• Ex vivo infection of primary intestinal epithelial cells to evaluate invasion capabilities

• Competitive index experiments where wild-type and ΔkdpC mutants are co-inoculated to directly compare fitness within the same animal

Statistical analysis should include at minimum 8-10 animals per group, with appropriate controls for each experiment and analysis using ANOVA with Tukey's post-hoc test for multiple comparisons .

How should researchers interpret contradictory data between kdpC mutants showing normal dehydration tolerance but impaired cold storage survival?

The seemingly contradictory observation that mutations in kdp system components show normal dehydration tolerance but impaired cold storage survival requires careful interpretation based on the underlying molecular mechanisms . To properly interpret such data:

• Recognize that potassium transport systems likely serve multiple functions in bacterial physiology beyond simple ion homeostasis. The kdpFABC operon is highly induced during dehydration, suggesting importance, yet kdpA mutants show normal dehydration tolerance initially .

• Consider temporal aspects of the stress response. Short-term dehydration tolerance may rely on redundant systems, while long-term persistence during cold storage reveals the essential nature of the kdp system. This suggests a time-dependent role shift for potassium transport channels.

• Analyze the data through the lens of compensatory mechanisms. During initial dehydration, other potassium transport systems (like Trk) may compensate for kdp mutation, but these systems may be insufficient during prolonged cold storage stress.

• Examine gene expression profiles comprehensively. While kdp genes are strongly induced during dehydration, their functional importance may manifest primarily during recovery or under combined stresses rather than during the initial dehydration event.

• Validate findings through complementation studies to confirm phenotype specificity to kdpC rather than polar effects or secondary mutations.

For clarification of contradictory results, implement experiments that:

• Test cold tolerance following dehydration versus cold tolerance alone

• Measure intracellular K⁺ concentrations during different stress phases

• Examine membrane integrity changes during stress transitions

• Monitor expression of alternative K⁺ transport systems to identify compensatory mechanisms

This approach acknowledges that stress response pathways are complex networks rather than linear pathways, with different components becoming critical under specific combinations of stressors or temporal phases .

What experimental protocols are recommended for analyzing the interaction between KdpC and other components of the Kdp transport system in Salmonella paratyphi B?

For analyzing protein interactions between KdpC and other components of the Kdp transport system in Salmonella paratyphi B, researchers should implement a multi-method approach that captures both physical interactions and functional relationships:

• Bacterial Two-Hybrid System Analysis:

  • Clone kdpC and potential interaction partners (particularly kdpA, kdpB, and kdpF) into appropriate vectors

  • Co-transform into reporter strains and quantify interaction strength by measuring reporter gene activity

  • Include positive controls (known interacting proteins) and negative controls (empty vectors)

• Co-Immunoprecipitation from Native Conditions:

  • Express epitope-tagged versions of KdpC (e.g., His-tag, FLAG-tag) in S. Paratyphi B

  • Perform gentle lysis under non-denaturing conditions using specialized detergents (DDM or LMNG at 1-2%)

  • Immunoprecipitate KdpC using appropriate antibodies or tag-specific matrices

  • Identify co-precipitated proteins by mass spectrometry

• FRET/BRET Analysis for Membrane Protein Interactions:

  • Generate fusion constructs of KdpC and other Kdp components with appropriate fluorescent/luminescent proteins

  • Express in Salmonella under native regulatory control

  • Measure energy transfer efficiency under various conditions (K⁺ limitation, osmotic stress)

• Cross-linking Mass Spectrometry:

  • Treat intact cells or membrane preparations with membrane-permeable cross-linkers (DSP, DTSSP)

  • Enrich for Kdp components using affinity purification

  • Identify cross-linked peptides by mass spectrometry to map interaction interfaces

• Functional Complementation Analysis:

  • Create chimeric proteins by swapping domains between KdpC and related proteins

  • Express these in appropriate knockout backgrounds

  • Assess functional restoration to identify critical interaction domains

The table below outlines key experimental conditions for protein interaction studies:

These methodologies collectively provide a comprehensive view of the interaction network within the Kdp transport system, allowing researchers to determine the specific role of KdpC in organizing the functional complex .

How might genetic engineering of the kdpC gene contribute to the development of live attenuated Salmonella paratyphi B vaccines?

Genetic engineering of the kdpC gene represents a promising avenue for developing live attenuated Salmonella paratyphi B vaccines. Building on established attenuated vaccine approaches, strategic modification of kdpC could provide novel attenuation mechanisms with several advantages:

• Balanced Attenuation Strategy:
  Unlike complete deletion of essential virulence factors that might over-attenuate the vaccine strain, targeted modifications to kdpC could create precisely calibrated attenuation. Since mutations in kdp components show a moderate increase in LD₅₀ values compared to wild-type strains, they could provide sufficient safety while maintaining immunogenicity .

• Dual-Purpose Attenuation:
  Engineering kdpC to function normally during initial host colonization but fail during systemic spread could create a vaccine strain that effectively stimulates mucosal immunity in the intestine while preventing potentially dangerous systemic infection. This approach takes advantage of the differential importance of potassium transport in various host environments.

• Complementary Attenuation System:
  Combining kdpC modifications with established attenuating mutations in guaBA or clpX genes could create multi-layered safety. Previous vaccine development for S. Paratyphi B demonstrated that strains with mutations in guaBA and clpX (CVD 2005) showed significantly increased LD₅₀ values (>2 log₁₀ higher than wild-type) .

• Cold-Chain Independence:
  Since kdp mutations affect cold storage survival , engineered kdpC variants could be designed to function normally at body temperature but lose viability during refrigeration, potentially creating temperature-sensitive vaccine strains that naturally attenuate outside the host.

Implementation would require precise genetic engineering techniques:

• Site-directed mutagenesis of specific functional domains rather than whole gene deletion

• Temperature-sensitive variants created through directed evolution approaches

• Inducible expression systems where kdpC expression is controlled by in vivo signals

• Dual-plasmid systems where kdpC complementation is unstable in vivo

The development pathway should include comprehensive safety and immunogenicity testing, with particular attention to ensuring the strain remains attenuated even after multiple passages through hosts .

What research approaches would best elucidate the role of kdpC in conferring antimicrobial resistance in Salmonella paratyphi B?

Investigating the potential role of kdpC in antimicrobial resistance requires a comprehensive approach that integrates genomic, transcriptomic, and phenotypic analyses. Although the direct contribution of potassium transport to antimicrobial resistance is not fully understood, several methodological approaches can elucidate this relationship:

• Comparative Genomics Analysis:

  • Analyze genome sequences of antimicrobial-resistant and susceptible S. Paratyphi B isolates, focusing on variants in the kdpC gene and its regulatory regions

  • Correlate specific kdpC polymorphisms with resistance profiles using genome-wide association studies (GWAS)

  • Compare kdpC sequences between sensu stricto and Java biotypes to identify biotype-specific variations potentially related to different resistance patterns

• Transcriptional Response Studies:

  • Perform RNA-seq analysis comparing wild-type and ΔkdpC mutants exposed to sub-inhibitory concentrations of various antimicrobials

  • Focus particularly on aminoglycosides and cationic antimicrobial peptides, as these may interact with potassium homeostasis pathways

  • Monitor expression changes in efflux pump systems and membrane integrity genes that might be influenced by potassium transport disruption

• Minimum Inhibitory Concentration (MIC) Testing:

  • Determine MICs for various antimicrobial classes in wild-type, ΔkdpC, and complemented strains

  • Test antimicrobial efficacy under varying potassium concentrations to determine if K⁺ availability modulates resistance

  • Implement checkerboard assays to identify potential synergistic effects between potassium transport inhibitors and conventional antibiotics

• Membrane Potential and Permeability Studies:

  • Measure membrane potential changes using fluorescent probes (DiSC3(5)) in wild-type versus ΔkdpC strains during antimicrobial exposure

  • Assess outer membrane permeability using NPN uptake assays to determine if kdpC deletion affects barrier function

  • Visualize membrane integrity using electron microscopy to detect structural differences that may influence antimicrobial penetration

• Efflux Pump Activity Assessment:

  • Measure efflux activity using fluorescent substrates (ethidium bromide, Nile red) in the presence and absence of functional kdpC

  • Determine if potassium gradient disruption affects proton-motive force-dependent efflux systems

These approaches would collectively provide a comprehensive understanding of how KdpC-mediated potassium transport might contribute to antimicrobial resistance, potentially revealing new targets for combination therapies that disrupt resistance mechanisms .

How can structural biology approaches enhance our understanding of KdpC function in Salmonella paratyphi B?

Structural biology approaches offer powerful tools to elucidate the molecular mechanisms of KdpC function within the Kdp transport system. A comprehensive structural biology investigation would include:

• X-ray Crystallography or Cryo-EM Analysis:

  • Express and purify recombinant KdpC alone and as part of the KdpFABC complex

  • For successful membrane protein crystallization, use detergents like DDM, LMNG, or nanodiscs to maintain native conformation

  • Obtain structures at different states (ATP-bound, ADP-bound, potassium-bound) to capture the transport cycle

  • Compare structural differences between Salmonella paratyphi B KdpC and characterized KdpC proteins from model organisms like E. coli

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Map conformational dynamics and solvent accessibility of KdpC during interaction with other Kdp components

  • Identify regions with altered exchange rates upon complex formation or potassium binding

  • This technique is particularly valuable for membrane proteins that resist crystallization

• Molecular Dynamics Simulations:

  • Develop atomic-level simulations of KdpC within a membrane environment

  • Model potassium ion movement through the channel and conformational changes during transport

  • Predict effects of specific mutations on protein stability and function

  • Simulate interactions between KdpC and other components of the KdpFABC complex

• Site-Directed Spin Labeling and EPR Spectroscopy:

  • Introduce spin labels at specific residues in KdpC

  • Measure distances between labeled sites to track conformational changes during transport cycle

  • This approach provides dynamic information complementary to static crystal structures

• Cross-Linking Mass Spectrometry:

  • Use chemical cross-linkers with different spacer lengths to identify interaction interfaces

  • Map the spatial arrangement of KdpC relative to other components in the complex

The table below outlines the expected outcomes from various structural approaches:

These structural investigations would significantly enhance understanding of how KdpC contributes to potassium transport and regulatory functions within the Kdp system, potentially revealing novel mechanisms for targeting bacterial potassium homeostasis .