Recombinant Salmonella enteritidis PT4 Potassium-transporting ATPase C chain (kdpC)

What is the kdpC protein and what is its role in Salmonella enteritidis PT4?

The kdpC protein is a component of the high-affinity ATP-driven K+ transport system (Kdp) in Salmonella enteritidis PT4, functioning as part of the KdpABC complex. This 194-amino acid protein chain serves as a peripheral membrane subunit that interacts with the catalytic KdpB component and plays a crucial role in potassium homeostasis under conditions of potassium limitation . The kdpC gene is co-transcribed with kdpAB and regulated by the KdpDE two-component system, with the entire complex being essential for bacterial adaptation to changing environmental potassium concentrations . Potassium transport in Salmonella is particularly important for type III secretion systems and pathogenesis, making kdpC a significant factor in the organism's virulence mechanisms .

How does the Kdp system relate to Salmonella pathogenicity?

The Kdp potassium transport system plays a multifaceted role in Salmonella pathogenicity through several mechanisms:

• Support of Type III Secretion Systems (T3SS): Potassium transport is critical for the proper functioning of T3SS, which are key virulence factors encoded by Salmonella Pathogenicity Islands (SPIs) .

• Adaptation to host environments: During infection, Salmonella encounters potassium-limited environments within host cells. The high-affinity Kdp system enables bacterial survival under these conditions .

• Gene expression regulation: The Kdp system influences the expression of various virulence-associated genes, with kdpC playing a regulatory role beyond mere potassium transport .

• Intracellular survival: Proper potassium homeostasis supports Salmonella's ability to survive and replicate within macrophages, a critical step in systemic infection .

Research has demonstrated that Salmonella enteritidis PT4 contains multiple pathogenicity islands, with SPI-1 and SPI-2 encoding T3SS showing high conservation and directly contributing to cell invasion, intestinal colonization, and intracellular survival .

What is the genetic context of kdpC in the Salmonella enteritidis PT4 genome?

The kdpC gene is part of the kdpFABC operon in Salmonella enteritidis PT4, with this organization being highly conserved among Enterobacteriaceae. Genome sequencing has revealed the following genetic arrangement:

• The kdpC gene is located downstream of kdpB and upstream of the kdpDE two-component regulatory system .

• The small membrane peptide KdpF is co-transcribed with kdpABC genes and appears to have regulatory functions beyond merely stabilizing the KdpABC complex .

• The entire kdp operon is regulated by the KdpDE two-component system, which responds to environmental potassium levels .

Comprehensive genome analysis of Salmonella enteritidis PT4 shows that this operon is part of the core genome maintained across various Salmonella serovars, reflecting its essential role in bacterial physiology .

What are the optimal conditions for expressing recombinant kdpC from Salmonella enteritidis PT4?

Successful expression of recombinant Salmonella enteritidis PT4 kdpC requires careful optimization of several parameters:

Expression System Selection:

• E. coli has been successfully used as an expression host for recombinant kdpC protein, providing good yields and proper folding .

• The addition of an N-terminal His-tag facilitates purification without significantly affecting protein function .

Expression Conditions:

• Induction parameters should be optimized at lower temperatures (16-25°C) to minimize inclusion body formation.

• IPTG concentration typically ranges from 0.1-0.5 mM for T7 promoter-based systems.

• Extended expression times (16-24 hours) at reduced temperatures often yield better results than shorter periods at 37°C.

Buffer Optimization:

• Purification is typically performed using Tris/PBS-based buffers at pH 8.0 .

• Addition of 6% trehalose as a stabilizing agent helps maintain protein integrity during storage .

• Working aliquots should be stored at 4°C for up to one week to avoid freeze-thaw damage .

Reconstitution Protocol:

• Lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Addition of 5-50% glycerol (final concentration) is recommended for long-term storage at -20°C/-80°C .

How can I assess the functional activity of recombinant kdpC in experimental systems?

Assessing the functional activity of recombinant kdpC requires multiple complementary approaches:

Protein-Protein Interaction Assays:

• Co-immunoprecipitation with KdpA and KdpB to verify complex formation

• Surface plasmon resonance (SPR) to measure binding kinetics with other Kdp components

• Crosslinking studies to identify interaction domains

Functional Reconstitution:

• Liposome reconstitution with purified KdpA, KdpB, and kdpC

• ATP hydrolysis assays to measure the catalytic activity of the reconstituted complex

• Potassium transport assays using fluorescent indicators or radioisotopes

Complementation Studies:

• Introduction of recombinant kdpC into kdpC-deletion strains

• Assessment of growth restoration under potassium-limited conditions

• Evaluation of pathogenicity restoration in infection models

Expression Analysis:

• Quantitative reverse transcribed polymerase chain reaction (qRT-PCR) to measure effects on kdp operon expression

• Transcriptomic analysis to identify broader gene expression changes

• Western blotting to assess protein levels of Kdp components

The KdpF peptide has been shown to interact with the KdpD histidine kinase and influence kdp gene expression, suggesting similar interactions might occur with kdpC that could be explored using these methods .

What are the current challenges in structural analysis of kdpC and how can they be addressed?

Structural analysis of kdpC presents several challenges that can be addressed through specific methodological approaches:

Challenges:

• Membrane protein complex association makes isolation of functionally relevant kdpC difficult

• Dynamic interactions with KdpA and KdpB complicate structural determination

• Conformational changes during the transport cycle create heterogeneous samples

Methodological Solutions:

Recent advances have allowed visualization of K+ pathway in the KdpABC complex through identification of spherical densities representing K+ or water molecules . This approach provides a template for similar studies with recombinant Salmonella enteritidis PT4 kdpC. Importantly, structural studies should include the small regulatory peptide KdpF, which has been shown to remain static during transport cycles and plays a role in complex stability .

What sequence and structural conservation exists between kdpC from Salmonella enteritidis PT4 and other bacterial species?

Comparative genomic analysis reveals significant conservation patterns in kdpC across bacterial species, particularly within Enterobacteriaceae:

Sequence Conservation:

• The 194-amino acid kdpC from Salmonella enteritidis PT4 shows high sequence identity (>90%) with kdpC from Salmonella Typhimurium strains .

• Moderate sequence conservation (70-80%) exists with E. coli kdpC, reflecting evolutionary divergence.

• Key interface residues that interact with KdpB show higher conservation than peripheral regions.

Structural Conservation:

Evolutionary Implications:

• The high conservation of kdpC within the Salmonella genus reflects its essential role in potassium homeostasis.

• Genome comparisons between S. Enteritidis PT4 and S. Gallinarum 287/91 demonstrate that kdp genes are part of the core genome maintained during host adaptation .

• The kdpC gene has not undergone pseudogene formation in host-adapted Salmonella strains, unlike many other genes that show degradation during host specialization .

This conservation pattern underscores the fundamental importance of the Kdp system in bacterial physiology across diverse ecological niches and host environments.

What are common pitfalls in working with recombinant kdpC and how can they be avoided?

Working with recombinant kdpC presents several technical challenges that researchers should anticipate and address:

Expression Challenges:

• Problem: Low expression yields
  Solution: Optimize codon usage for the expression host; reduce expression temperature to 16-20°C; test different E. coli strains (BL21, Rosetta, C41/C43)

• Problem: Inclusion body formation
  Solution: Express as fusion with solubility tags (MBP, SUMO); add low concentrations (0.5-2%) of mild detergents; reduce IPTG concentration to 0.1 mM

• Problem: Protein degradation
  Solution: Add protease inhibitors immediately after cell lysis; work at 4°C throughout purification; minimize purification time

Purification Issues:

• Problem: Poor binding to Ni-NTA resin
  Solution: Verify His-tag accessibility; increase imidazole in binding buffer to 20-30 mM to reduce nonspecific binding; ensure proper pH (7.5-8.0)

• Problem: Protein aggregation
  Solution: Add 6% trehalose to storage buffer; include glycerol at 5-50% final concentration; avoid repeated freeze-thaw cycles

• Problem: Co-purification of contaminants
  Solution: Include additional purification steps (ion exchange, size exclusion); increase washing stringency with higher imidazole concentrations

Stability Concerns:

• Problem: Limited shelf-life
  Solution: Store working aliquots at 4°C for maximum one week; maintain stock at -80°C; lyophilize for long-term storage

• Problem: Activity loss during storage
  Solution: Add stabilizing agents (trehalose, glycerol); reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL concentration

Functional Analysis:

• Problem: Difficulty assessing function in isolation
  Solution: Co-express with KdpA and KdpB; use complementation assays in kdpC-deficient bacterial strains; develop in vitro reconstitution systems

How can I design experiments to investigate the role of kdpC in Salmonella virulence?

Designing experiments to investigate kdpC's role in Salmonella virulence requires multilevel approaches spanning genomics, molecular biology, and infection models:

Genetic Manipulation Strategies:

• Generate precise kdpC deletion mutants using CRISPR-Cas9 or lambda Red recombination systems

• Create point mutations in key functional domains to distinguish transport vs. regulatory functions

• Develop complementation strains with wild-type or mutant kdpC variants

• Construct reporter fusions to monitor kdpC expression during infection

In Vitro Virulence Assays:

• Cell Invasion Assays: Compare invasion efficiency of wild-type vs. kdpC mutants in epithelial cell lines

• Intracellular Survival: Measure bacterial replication within macrophages over time

• T3SS Function: Assess secretion of effector proteins using reporter systems

• Biofilm Formation: Quantify biofilm development and expression of the red, dry, and rough (rdar) morphotype

Gene Expression Analysis:

• Perform RNA-Seq under infection-relevant conditions to identify kdpC-dependent gene networks

• Use qRT-PCR to validate expression changes in specific virulence genes

• Map protein-protein interactions using bacterial two-hybrid or co-immunoprecipitation approaches

In Vivo Infection Models:

• Mouse infection models to assess systemic spread and colonization

• Chicken infection models to evaluate host-specific interactions (particularly relevant for Salmonella Enteritidis PT4)

• Competition assays between wild-type and kdpC mutants to measure relative fitness in vivo

Experimental Controls:

• Include KdpF overexpression controls, as this related peptide has been shown to reduce intramacrophage replication

• Compare results with other Salmonella serovars (e.g., Typhimurium) to identify serovar-specific effects

• Use complementation with kdpC from other bacterial species to assess functional conservation

These approaches will help dissect the specific contributions of kdpC to Salmonella virulence, distinguishing its role in basic bacterial physiology from specialized virulence functions.

How has the kdpC gene evolved in Salmonella enteritidis PT4 compared to other Salmonella serovars?

The evolutionary trajectory of kdpC in Salmonella enteritidis PT4 reveals important insights about functional conservation and adaptation:

Genomic Conservation Patterns:

• Comparative genome analysis between S. Enteritidis PT4 and S. Gallinarum 287/91 (a host-restricted serovar) shows that kdpC is part of the extensive core gene set maintained during host specialization .

• Unlike many genes that undergo pseudogene formation during host adaptation (S. Gallinarum has 309 pseudogenes compared to 116 in S. Enteritidis), the kdpC gene remains functionally intact, indicating strong selective pressure for its maintenance .

• The average nucleotide identity between shared orthologous genes in S. Enteritidis PT4 and S. Typhimurium LT2 is 98.98%, with kdpC following this general conservation pattern .

Functional Evolution:

• The maintenance of kdpC across both host-generalist (S. Enteritidis PT4) and host-restricted (S. Gallinarum) serovars suggests its primary role in core bacterial physiology rather than host-specific adaptation .

• Unlike regions of genome variability (such as ROD40, which encodes Type I restriction/modification systems), the kdp operon shows high conservation in gene order and coding sequence .

• While the coding sequence is conserved, regulatory elements controlling kdpC expression may show more variation, potentially contributing to serovar-specific expression patterns.

Evolutionary Implications:

• The conservation of kdpC across Salmonella serovars with diverse host ranges supports its fundamental role in potassium homeostasis across different host environments.

• The retention of a functional kdpC gene in host-adapted serovars like S. Gallinarum contrasts with the genome degradation seen in other systems, suggesting that potassium transport remains critical even in specialized host niches .

• Experimental analysis in different host models (chickens and mice) using S. Enteritidis with mutations in genes that become pseudogenes in host-adapted strains could provide insights into adaptation mechanisms .

What functional relationships exist between kdpC and other potassium transport systems in Salmonella?

Salmonella possesses multiple potassium transport systems that function in a complementary manner, with kdpC and the KdpABC complex showing specific roles within this network:

Potassium Transport Systems in Salmonella:

• Kdp system (including kdpC): High-affinity, ATP-driven K+ transporter induced under severe K+ limitation

• Trk system: Moderate-affinity, constitutively expressed K+ transporter using proton motive force

• Kup system: Low-affinity K+ transporter active at low pH conditions

Functional Relationships:

• Expression Regulation:

  • The Kdp system (including kdpC) is strictly regulated by the KdpDE two-component system responding to K+ limitation

  • Other K+ transporters show different regulatory patterns, creating a hierarchical response to changing K+ availability

• Complementary Functions:

  • The KdpABC complex (containing kdpC) functions primarily under severe potassium limitation (<0.1 mM K+)

  • Trk becomes the dominant system at moderate K+ concentrations

  • This functional differentiation allows fine-tuned adaptation to various environmental conditions

• Virulence Connections:

  • The Kdp system has been specifically linked to type III secretion and pathogenesis

  • Other K+ transport systems show less direct involvement in virulence mechanisms

Regulatory Interactions:

• The small membrane peptide KdpF, which is co-transcribed with kdpABC, can interact with the KdpD histidine kinase and modulate expression of kdp genes

• This suggests potential regulatory cross-talk between different potassium sensing and transport systems

• The KdpD/KdpE two-component system may integrate multiple signals beyond just K+ availability, connecting K+ homeostasis to broader stress responses

Understanding these relationships provides important context for interpreting kdpC function within the broader framework of bacterial ion homeostasis and stress adaptation.

How does kdpC contribute to Salmonella enteritidis PT4's ability to infect multiple hosts?

The kdpC protein contributes to Salmonella enteritidis PT4's host promiscuity through several mechanisms related to potassium homeostasis and virulence regulation:

Adaptation to Diverse Host Environments:

• Salmonella enteritidis PT4 is classified as a host-generalist capable of infecting humans, poultry, and other animals .

• The kdpC-containing high-affinity potassium transport system enables adaptation to varying potassium concentrations encountered across different host tissues and cell types.

• This adaptability is particularly important given that S. Enteritidis PT4 must transition between intestinal and systemic infection sites with differing ionic compositions.

Support of Core Virulence Mechanisms:

• The genome of S. Enteritidis PT4 contains twelve Salmonella pathogenicity islands (SPIs) with SPI-1 and SPI-2 encoding type III secretion systems (T3SS) showing high conservation .

• Potassium transport via the KdpABC complex is critical for proper T3SS function, with kdpC playing an essential role in this process .

• Approximately 3.66% (165 genes) of the S. Enteritidis PT4 genome encodes virulence factors associated with cell invasion, intestinal colonization, and intracellular survival, many of which depend on proper ionic balance maintained in part by the Kdp system .

Gene Regulatory Networks:

• Beyond direct potassium transport, the KdpABC complex and associated regulatory elements influence expression of virulence genes.

• Similar to findings with the KdpF peptide, which can modulate expression of kdp genes and intramacrophage growth when overexpressed, kdpC likely participates in regulatory networks affecting virulence gene expression .

• This regulatory function may enable fine-tuned adaptation to different host environments through coordinated expression of appropriate virulence factors.

Comparative analysis between host-generalist S. Enteritidis PT4 and the chicken-restricted S. Gallinarum provides evidence that potassium transport systems remain critical even during host specialization, with the kdpC gene maintaining functional integrity despite extensive pseudogene formation in other systems .

What therapeutic potential exists in targeting kdpC for antimicrobial development?

The kdpC protein represents a promising antimicrobial target with several advantageous characteristics:

Target Validation:

• The high conservation of kdpC across Salmonella serovars indicates its fundamental importance .

• Unlike many virulence factors that become dispensable in certain hosts, potassium transport systems remain essential across diverse infection scenarios .

• The link between potassium transport and virulence mechanisms, particularly T3SS function, suggests inhibiting kdpC could simultaneously impact multiple virulence systems .

Therapeutic Approaches:

• Small Molecule Inhibitors:

  • Design compounds targeting the interface between kdpC and KdpB to disrupt complex formation

  • Develop allosteric modulators that alter conformational changes associated with transport cycles

  • Create peptide mimetics based on natural interaction domains between Kdp components

• Peptide-Based Strategies:

  • Engineer synthetic peptides similar to KdpF, which has been shown to modulate kdp gene expression and reduce intramacrophage growth when overexpressed

  • Design peptides targeting specific domains of kdpC involved in protein-protein interactions

  • Create cell-penetrating peptides to deliver inhibitory molecules intracellularly

• Alternative Approaches:

  • Develop RNA-based therapeutics targeting kdpC mRNA

  • Explore CRISPR-Cas delivery systems for targeted gene disruption

  • Investigate immunological approaches targeting surface-exposed domains of the Kdp complex

Advantages and Challenges:

The therapeutic potential of targeting kdpC is further supported by the observation that Salmonella Enteritidis infections are often related to consumption of chicken meat and eggs, providing a clear transmission route that could be interrupted through targeted antimicrobial strategies .