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

What is the Potassium-transporting ATPase C chain (kdpC) in Salmonella dublin?

The Potassium-transporting ATPase C chain (kdpC) is a critical component of the KdpFABC complex in Salmonella dublin. This protein functions as part of the potassium transport system with the enzyme classification EC 3.6.3.12. It is also known by alternative names including ATP phosphohydrolase [potassium-transporting] C chain, Potassium-binding and translocating subunit C, and Potassium-translocating ATPase C chain. The KdpC subunit is encoded by the kdpC gene (locus name SeD_A0812) and comprises 194 amino acids in its full-length form .

How does the KdpC protein function within the larger KdpFABC complex?

The KdpC protein functions as an integral part of the KdpFABC complex, which is involved in potassium ion (K+) transport across the bacterial membrane. Recent research suggests that the mechanism of K+ transport is more complex than previously thought. While earlier assumptions indicated that the KdpA subunit alone was responsible for K+ translocation (similar to KtrB and TrkH proteins), newer evidence suggests that the entire complex works together to facilitate potassium transport. Cryo-EM structural studies have revealed that rather than KdpA functioning independently as a channel-like protein, the KdpFABC complex appears to operate through a mechanism involving two joined half-channels that cooperatively facilitate K+ movement .

What expression systems are recommended for producing recombinant Salmonella dublin kdpC protein?

For expressing recombinant Salmonella dublin kdpC protein, researchers should consider bacterial expression systems optimized for membrane proteins. The protein can be expressed with various tags determined during the production process. For storage stability, a Tris-based buffer with 50% glycerol is typically used, and the recombinant protein should be stored at -20°C, or at -80°C for extended storage. To maintain protein integrity, repeated freeze-thaw cycles should be avoided, and working aliquots are best maintained at 4°C for up to one week .

What are the optimal methods for detecting Salmonella dublin in experimental samples?

For detecting Salmonella dublin in experimental samples, real-time PCR assays have proven effective. The XP-Design Assay Salmonella Dublin uses a TaqMan-based approach for qualitative detection of specific DNA sequences unique to Salmonella Dublin. This method can be applied to both isolated colonies and food/environmental samples.

For colony confirmation:

• Grow Salmonella colonies on non-selective agar plates

• Extract DNA from isolated colonies following standard protocols

• Perform PCR using FAM-labeled probes specific to Salmonella Dublin

• Run the appropriate thermal cycling protocol

For food and environmental samples:

• Enrich samples according to established protocols (similar to iQ-Check Salmonella spp. II method)

• Extract DNA from enrichments using the Easy protocol

• Perform PCR detection with FAM-labeled probes

These methods show high specificity (verified on 94 strains representing various serotypes) and robustness to inhibition from matrices including chicken carcass rinses, turkey sponge swabs, poultry boot swabs, and beefsteak .

How can researchers differentiate Salmonella Dublin from other Salmonella serotypes in mixed samples?

To differentiate Salmonella Dublin from other Salmonella serotypes in mixed samples, researchers should implement a multi-faceted approach:

• Molecular detection: Utilize PCR-based methods with primers targeting unique genomic regions of Salmonella Dublin. The XP-Design Assay Salmonella Dublin offers specificity for detection of DNA sequences unique to this serotype.

• Serological techniques: Perform serotyping using specific antisera against O and H antigens characteristic of Salmonella Dublin.

• Growth characteristics: Monitor seasonal variations in prevalence, as studies show Salmonella Dublin accounts for different proportions of serotypes depending on season (43.8% in spring, 24.4% in summer, 28.3% in winter, but only 3.5% in fall) .

• Indicator microorganism patterns: Note that Salmonella Dublin shows an inverse association with indicator microorganism concentrations compared to other Salmonella serotypes in certain matrices like boneless beef .

• Genomic sequencing: For definitive identification, whole genome sequencing allows phylogenetic analysis to definitively identify Salmonella Dublin isolates within the broader Salmonella enterica family .

What are the genomic determinants of antimicrobial resistance in Salmonella Dublin isolates?

Antimicrobial resistance in Salmonella Dublin isolates is determined by specific genetic elements. Studies of clinical isolates from cattle in the United States (2014-2017) revealed extensive multidrug resistance patterns, with 98% of isolates resistant to more than four antimicrobials. The genomic basis for this resistance includes:

• Resistance genes: The most prevalent AMR genes in Salmonella Dublin isolates include:

  • sulf2 and tetA (98.6% of isolates) - conferring resistance to sulfonamides and tetracyclines

  • aph(6)-Id (97.9%) and aph(3'')-Ib (97.1%) - conferring aminoglycoside resistance

  • floR (94.3%) - conferring phenicol resistance

  • blaCMY-2 (85.7%) - conferring beta-lactam resistance

• Plasmid-mediated resistance: Ten plasmid types were identified among isolates, with IncA/C2, IncX1, and IncFII(S) being the most frequent carriers of resistance genes.

• Chromosomal mutations: Quinolone resistance was associated with mutations in the gyrA gene.

This genomic characterization explains the high resistance rates observed: 96% resistance to sulfonamides, 97% to tetracyclines, 95% to aminoglycosides, and 85% to beta-lactams. Notably, all isolates remained susceptible to azithromycin and meropenem .

How does the structure of the KdpFABC complex inform our understanding of potassium transport mechanisms?

Cryo-EM structural studies of the KdpFABC complex have substantially revised our understanding of potassium transport mechanisms in this system. While sequence alignments with similar transporters (KtrB and TrkH) initially suggested that KdpA alone might function as the K+-translocating subunit, experimental evidence has challenged this assumption.

Key structural insights include:

• The KdpFABC complex appears to function through a coordinated mechanism involving multiple subunits rather than through KdpA acting as an independent channel.

• Expression studies demonstrate that the KdpA subunit alone does not support potassium ion uptake, contrary to what would be expected if it retained independent channel-like functionality.

• Structural data suggests the complex operates through a mechanism involving "two joined half-channels" that collectively facilitate K+ translocation across the membrane.

These findings represent a paradigm shift in understanding how the KdpFABC complex functions, moving away from the model of a single subunit acting as a channel toward a more integrated, complex transport mechanism involving cooperative action among multiple protein components .

What methodological approaches can resolve contradictory findings regarding the role of kdpC in potassium transport?

To resolve contradictory findings regarding the role of kdpC in potassium transport, researchers should implement several methodological approaches:

These approaches can help reconcile the apparently contradictory observations that sequence homology suggests KdpA should function as a potassium channel, while experimental evidence indicates that KdpA alone cannot support potassium uptake and requires the coordinated action of the entire KdpFABC complex .

How does understanding kdpC function contribute to addressing Salmonella Dublin virulence and antimicrobial resistance?

Understanding the function of kdpC in the KdpFABC potassium transport system provides valuable insights into Salmonella Dublin virulence and antimicrobial resistance mechanisms. Potassium homeostasis is crucial for bacterial survival, particularly under osmotic stress conditions that bacteria encounter during infection and colonization.

The relationship between kdpC function and Salmonella Dublin's pathogenic capabilities can be approached through several research angles:

• Survival under stress conditions: Investigating how mutations in kdpC affect the ability of Salmonella Dublin to survive in potassium-limited environments encountered during infection.

• Virulence correlation: Examining whether strains with mutations in kdpC show altered virulence in animal models, as potassium transport systems are often essential for bacterial adaptation during infection.

• Antimicrobial resistance connections: Studies have shown that 98% of Salmonella Dublin isolates display multidrug resistance. Research should explore whether potassium transport systems provide physiological support for resistance mechanisms or influence the expression of resistance genes .

• Host adaptation: Salmonella Dublin infections in humans are significantly more severe than infections caused by other serotypes, with higher rates of sepsis, hospitalization, and mortality. Research into kdpC's role in the KdpFABC complex may reveal how this system contributes to the serotype's host adaptation and pathogenicity .

What are the epidemiological patterns of Salmonella Dublin infections, and how might potassium transport systems influence these patterns?

Salmonella Dublin demonstrates distinct epidemiological patterns that may be influenced by its potassium transport systems, including the KdpFABC complex containing kdpC:

• Demographic distribution: Salmonella Dublin infections show a unique age distribution, with higher incidence in adults older than 18 years and lower proportion in children aged 5-17 years compared to other Salmonella serotypes in the United States .

• Seasonal variation: Salmonella Dublin prevalence varies significantly by season, accounting for 43.8% of serotypes in spring, 24.4% in summer, 28.3% in winter, but only 3.5% in fall. This seasonal pattern may reflect environmental adaptations mediated by systems like the KdpFABC complex that help the bacteria survive changing environmental conditions .

• Severity of infection: Salmonella Dublin infections in humans are more likely to result in sepsis, hospitalization, and death compared to other serotypes. In a 2019 outbreak, 8 of 10 cases required hospitalization, and one died - a hospitalization rate far exceeding the typical 20% for non-Dublin Salmonella infections .

• Antimicrobial resistance trends: Salmonella Dublin displays high rates of antimicrobial resistance (up to 79% of isolates), which has increased significantly over the last 20 years. This trait complicates treatment and contributes to more serious illness outcomes .

• Geographical distribution: In the United States, Salmonella Dublin has been isolated from cattle in at least 21 states, with vendor-dependent prevalence ranging from 0% to 54.2% of isolates .

Understanding how potassium transport systems like KdpFABC contribute to these epidemiological patterns could provide insights into bacterial adaptability and inform targeted intervention strategies.

What novel experimental approaches could enhance our understanding of kdpC's role in bacterial physiology?

To advance our understanding of kdpC's role in bacterial physiology, researchers should consider these innovative experimental approaches:

• Single-cell analysis techniques: Apply microfluidic platforms and single-cell imaging to monitor potassium transport in real-time at the individual bacterial cell level, revealing heterogeneity in kdpC-mediated transport functions across populations.

• CRISPR-Cas9 genome editing: Use precise genome editing to create specific mutations or regulatory alterations in kdpC to analyze phenotypic effects under various environmental conditions.

• Synthetic biology approaches: Construct synthetic potassium transport systems with modified kdpC components to identify minimal functional requirements and explore protein engineering possibilities.

• Multi-omics integration: Combine transcriptomics, proteomics, and metabolomics to build comprehensive models of how kdpC expression affects global cellular physiology under different growth and stress conditions.

• Bacterial two-hybrid systems: Develop specialized bacterial two-hybrid assays optimized for membrane proteins to map the interaction network of kdpC within the bacterial cell.

• In vivo potassium sensors: Develop genetically encoded potassium sensors that can be expressed in bacteria to directly monitor intracellular potassium levels in relation to kdpC activity.

• Cryo-electron tomography: Apply this technique to visualize the native configuration of KdpFABC complexes within the bacterial membrane, providing structural context for functional studies .

How might targeting the KdpFABC complex lead to novel antimicrobial strategies against multidrug-resistant Salmonella Dublin?

Targeting the KdpFABC complex represents a promising avenue for developing novel antimicrobial strategies against multidrug-resistant Salmonella Dublin, with several potential approaches:

• Small molecule inhibitors: Design compounds that specifically bind to critical regions of the KdpFABC complex, particularly at the interface between subunits or at functional domains within kdpC, disrupting potassium transport.

• Peptide-based inhibitors: Develop peptides mimicking essential interaction domains of kdpC that can competitively inhibit complex formation or function.

• Combination therapy approaches: Investigate synergistic effects between conventional antibiotics and KdpFABC inhibitors, potentially restoring sensitivity to antibiotics in resistant strains.

• Allosteric modulators: Identify compounds that bind to regulatory sites on the KdpFABC complex, locking it in non-functional conformational states.

• Immunological targeting: Develop antibodies or immunotherapeutic approaches targeting exposed epitopes of the KdpFABC complex on the bacterial surface.

This approach is particularly promising given the high prevalence of antimicrobial resistance in Salmonella Dublin, with 98% of isolates showing resistance to multiple antimicrobials. Studies have documented resistance rates of 96% to sulfonamides, 97% to tetracyclines, 95% to aminoglycosides, and 85% to beta-lactams . By targeting essential physiological processes distinct from those affected by conventional antibiotics, KdpFABC-directed therapeutics might overcome existing resistance mechanisms.

How does the kdpC protein in Salmonella Dublin compare structurally and functionally to homologous proteins in other pathogenic bacteria?

The kdpC protein in Salmonella Dublin shares structural and functional similarities with homologous proteins in other pathogenic bacteria, but also displays important differences:

What data suggests a relationship between kdpC function and the clinical severity of Salmonella Dublin infections?

Several lines of evidence suggest potential relationships between kdpC function as part of the KdpFABC complex and the distinctive clinical severity of Salmonella Dublin infections:

• Enhanced systemic spread: Salmonella Dublin infections are significantly more severe than infections caused by other serotypes, with higher rates of sepsis, hospitalization, and mortality. This suggests potential roles for potassium homeostasis systems in supporting bacterial survival during systemic infection .

• Age-dependent susceptibility: Salmonella Dublin shows an unusual age distribution pattern, with higher incidence in adults over 18 years compared to other serotypes. This demographic pattern might reflect physiological adaptation mechanisms involving potassium transport systems .

• Antimicrobial resistance: Studies have found 98% of Salmonella Dublin isolates are resistant to more than 4 antimicrobials, with high resistance rates to sulfonamides (96%), tetracyclines (97%), aminoglycosides (95%), and beta-lactams (85%). This extensive resistance profile complicates treatment and contributes to more serious illness outcomes .

• Environmental persistence: Salmonella Dublin's seasonal prevalence patterns suggest sophisticated environmental adaptation mechanisms that may involve potassium transport systems like KdpFABC. The ability to regulate potassium homeostasis under varying environmental conditions could contribute to survival and subsequent virulence .

• Host adaptation: Salmonella Dublin is a host-adapted serotype in cattle, associated with both enteritis and systemic disease. In cattle, it primarily manifests as respiratory disease, particularly in calves. This specialized host adaptation may involve specific adaptations in essential physiological systems like potassium transport .

While direct experimental evidence linking kdpC function to clinical outcomes remains limited, these epidemiological and clinical patterns suggest important areas for future research into how potassium transport systems contribute to Salmonella Dublin's distinctive pathogenicity profile.