Recombinant Salmonella paratyphi A Magnesium transport protein CorA (corA)

What is the primary function of CorA in Salmonella paratyphi A?

The primary function of CorA in S. paratyphi A is magnesium transport across the bacterial cell membrane. Mg²⁺ cannot readily cross biological membranes due to its ionic nature, necessitating specialized transport proteins like CorA . This protein plays a crucial role in maintaining magnesium homeostasis, which is essential for numerous cellular processes including protein synthesis, nucleic acid synthesis, and membrane integrity .

The transport mechanism appears to be driven by membrane potential rather than proton gradient, as observed in other CorA family members . The protein exists in both symmetric closed conformations and multiple asymmetric open conformations that change dynamically to facilitate Mg²⁺ import, particularly when intracellular Mg²⁺ levels are low .

What cations can be transported by CorA proteins, and how is this selectivity determined?

CorA proteins demonstrate a specific but not exclusive transport profile for divalent cations. Fluorescence-based transport assays with reconstituted CorA proteins in proteoliposomes have shown that they readily transport:

What are the recommended methods for recombinant expression and purification of S. paratyphi A CorA?

For recombinant expression and purification of S. paratyphi A CorA, researchers have successfully utilized E. coli expression systems with N-terminal His-tags. The recommended protocol includes:

• Cloning the full-length corA gene (encoding amino acids 1-316) from S. paratyphi A into an appropriate expression vector with an N-terminal His-tag .

• Transforming the construct into E. coli expression strains.

• Inducing protein expression under optimized conditions.

• Lysing cells and purifying the protein using metal affinity chromatography.

• Further purification may be achieved through size exclusion chromatography to maintain the native pentameric structure.

• The purified protein should be stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

• For long-term storage, the addition of glycerol (5-50% final concentration) and aliquoting for storage at -20°C/-80°C is recommended to prevent protein degradation from freeze-thaw cycles .

For reconstitution, the lyophilized protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

What functional assays can be used to characterize the transport activity of recombinant CorA?

Several functional assays can be employed to characterize the transport activity of recombinant CorA:

• Fluorescence-based transport assays: Reconstitute purified CorA into proteoliposomes and measure the uptake of fluorescently labeled Mg²⁺ or other divalent cations. This approach has been successfully used to demonstrate that CorA proteins transport Mg²⁺, Co²⁺, Ni²⁺, and Zn²⁺ .

• Radioisotope flux assays: Using radioactive isotopes of magnesium (²⁸Mg) to directly measure transport rates across membranes containing reconstituted CorA.

• Membrane potential studies: Since CorA transport is stimulated by membrane potential rather than proton gradient (unlike the related ZntB proteins), assays measuring transport under various membrane potential conditions can characterize the electrophysiological properties of the channel .

• Antibiotic accumulation assays: Given CorA's potential role in antibiotic efflux, measuring the intracellular accumulation of antibiotics like norfloxacin and ofloxacin in cells expressing CorA versus controls can provide insights into its secondary functions .

• Biofilm formation assays: Quantitative assessment of biofilm formation in cells expressing recombinant CorA can elucidate its role in this physiological process .

How can researchers effectively study the conformational changes of CorA during ion transport?

Studying the conformational changes of CorA during ion transport requires a multidisciplinary approach:

• Cryo-electron microscopy (cryo-EM): This technique can capture the protein in different conformational states, revealing the symmetric closed conformation and multiple asymmetric open conformations that CorA adopts during transport cycles.

• X-ray crystallography: While challenging due to the dynamic nature of the protein, crystallography can provide high-resolution structural information of specific conformational states.

• Molecular dynamics simulations: Computational approaches can model the transitions between conformational states based on experimental structures .

• Site-directed mutagenesis: Introducing mutations at key residues hypothesized to be involved in conformational changes, followed by functional assays to assess the impact on transport.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can identify regions of the protein that undergo conformational changes upon binding of magnesium or other substrates.

• Fluorescence resonance energy transfer (FRET): By labeling specific domains with fluorophores, researchers can monitor conformational changes in real-time.

The research suggests that CorA transitions between symmetric and asymmetric states are influenced by intracellular Mg²⁺ levels. When Mg²⁺ levels are low, the closed state becomes less common, reducing the energy barrier to open states and increasing the dynamics of CorA, which facilitates the open state necessary for transport .

What role does CorA play in S. paratyphi A virulence and pathogenesis?

While direct evidence specifically for S. paratyphi A CorA's role in virulence is limited in the provided search results, studies on related species suggest that CorA likely contributes to pathogenesis through several mechanisms:

• Magnesium homeostasis: As magnesium is essential for numerous cellular processes, CorA's primary function in maintaining magnesium homeostasis is critical for bacterial survival and replication within the host .

• Potential contribution to antimicrobial resistance: Research on the CorA homolog in M. smegmatis has shown that expression of corA increases tolerance to various structurally unrelated antibiotics, suggesting a potential role in drug efflux .

• Biofilm formation: CorA has been shown to enhance biofilm-forming ability in cells expressing it, which could contribute to persistent infections and increased resistance to host immune defenses and antimicrobial agents .

• Stress response: Proper magnesium levels regulated by CorA may help bacteria cope with various stresses encountered within the host environment.

Understanding CorA's role in S. paratyphi A pathogenesis could potentially inform the development of novel therapeutic strategies or vaccine candidates targeting this protein.

How does CorA contribute to antimicrobial resistance in S. paratyphi A and related bacteria?

Recent research has revealed that CorA may play an unexpected role in antimicrobial resistance:

• Enhanced drug efflux: Expression of corA in both M. smegmatis and E. coli increased tolerance toward various structurally unrelated antibiotics and anti-tubercular drugs, suggesting that CorA may facilitate multi-drug efflux activity .

• Reduced drug accumulation: Cells expressing corA showed significantly lower accumulation of fluoroquinolones (norfloxacin and ofloxacin), providing further evidence for enhanced efflux pump activity .

• Magnesium-mediated resistance: The presence of sub-inhibitory concentrations of Mg²⁺ resulted in increased low-level tolerance toward tested drugs, suggesting that Mg²⁺ might act as a facilitator in the efflux process .

• Potential antiporter mechanism: Based on molecular modeling and in vivo analysis, CorA might function as an antiporter that imports Mg²⁺ and exports antibiotics. In its closed symmetric conformation, antibiotics may bind at the inter-subunit interfaces, and when the protein transitions to different asymmetric conformations, it may allow for the export of these bound antibiotics .

This dual functionality of CorA as both a magnesium importer and a potential contributor to drug efflux represents an important area for further research, particularly in the context of increasing antimicrobial resistance in enteric pathogens.

What is the relationship between CorA function and biofilm formation in pathogenic bacteria?

Research has established a connection between CorA function and biofilm formation in bacteria:

• Enhanced biofilm formation: CorA has been demonstrated to enhance the biofilm-forming ability of cells expressing it, as observed in both M. smegmatis and E. coli models .

• Magnesium-dependent mechanism: The effect on biofilm formation is likely related to CorA's role in magnesium homeostasis, as proper magnesium levels are critical for various cellular processes that contribute to biofilm development.

• Structural contributions: The protein's ability to undergo conformational changes and its pentameric structure at the cell membrane may influence cell surface properties relevant to biofilm formation.

• Potential regulatory interactions: CorA may interact with regulatory networks that control biofilm formation in response to environmental conditions, including magnesium availability.

This relationship between CorA and biofilm formation has significant implications for bacterial persistence and resistance to both host immune responses and antimicrobial treatments, as biofilms provide a protective environment for bacteria.

How can CorA be utilized in vaccine development strategies against S. paratyphi A?

While CorA itself is not a primary vaccine antigen in the current literature, understanding its function can contribute to vaccine development strategies:

• Attenuated vaccine strains: Modifying CorA function could potentially be used to develop attenuated S. paratyphi A strains for live vaccine candidates. The relationship between magnesium homeostasis and virulence means that carefully engineered CorA variants might reduce pathogenicity while maintaining immunogenicity.

• Complementary approach to Vi-based vaccines: Current research focuses on engineering S. paratyphi A to express the Vi capsular polysaccharide (normally found in S. Typhi) to create bivalent vaccines against enteric fever . Understanding CorA's role in bacterial physiology could help optimize these genetically modified vaccine strains.

• Target for adjuvant development: Knowledge of CorA's role in magnesium homeostasis and potential contribution to antimicrobial resistance could inform the development of adjuvants that enhance vaccine efficacy by modulating these pathways.

• Biomarker for vaccine efficacy: Given CorA's role in bacterial stress response and survival, monitoring changes in CorA expression or activity could potentially serve as a biomarker for evaluating vaccine-induced immune responses.

Current vaccine development for S. paratyphi A is focused on creating bivalent vaccines that protect against both S. Typhi and S. paratyphi A, as existing vaccines like Ty21a, Vi subunit vaccine, and Vi-conjugate vaccine provide inadequate cross-protection against S. paratyphi A infection .

What does molecular docking analysis reveal about potential binding sites for antibiotics in the CorA protein structure?

Molecular docking analyses have provided insights into how antibiotics might interact with CorA:

• Binding site locations: Most binding sites for antibiotics like isoniazid are located at the inter-subunit interfaces of the cytoplasmic domain of CorA .

• Transport pathway hypothesis: Based on docking analysis, antibiotics likely move through the inter-subunit interfaces and enter the transmembrane pores during export .

• Conformational dependency: The binding and transport of antibiotics appear to be dependent on the conformational transitions of CorA between symmetric closed and asymmetric open states .

• Specificity determinants: The same structural features that determine cation selectivity in CorA may also influence which antibiotics can be transported.

• Key residues: Mutations in residues T270 and S260 (as studied in S. typhimurium CorA) that hinder ion transport may also affect antibiotic export, suggesting these residues are critical for the conformational changes necessary for transport .

This molecular understanding of antibiotic interactions with CorA provides a foundation for potential future drug development targeting this transporter to overcome resistance mechanisms.

How do the functional differences between CorA and related transporters like ZntB inform our understanding of metal ion transport mechanisms?

Comparative studies between CorA and related transporters like ZntB have revealed important distinctions in their transport mechanisms:

• Energetic coupling differences: While both CorA and ZntB belong to the 2-TM-GxN family and can transport similar cations, they differ in their energetic coupling mechanisms. CorA transport is stimulated by membrane potential, whereas ZntB transport is stimulated by proton gradient .

• Evolutionary divergence: This functional difference suggests that CorA and ZntB proteins diverged to different transport mechanisms within the same protein scaffold during evolution .

• Structural implications: Despite their similar substrate specificity, the distinct energy coupling mechanisms suggest differences in how conformational changes are triggered and regulated in these protein families.

• Physiological contexts: The different energy coupling mechanisms may reflect adaptations to different physiological contexts or bacterial compartments with varying electrochemical gradients.

This comparative analysis highlights how related transport proteins can evolve distinct mechanisms while maintaining similar substrate specificities, providing insights into the evolution and diversification of metal ion transport systems in prokaryotes.

What are promising approaches for targeting CorA function to develop novel antimicrobials against S. paratyphi A?

Given CorA's essential role in magnesium homeostasis and potential contribution to antimicrobial resistance, several approaches show promise for therapeutic development:

• Small molecule inhibitors: Designing compounds that specifically bind to CorA and block its magnesium transport function could potentially disrupt bacterial viability. The inter-subunit interfaces identified in molecular docking studies as binding sites for antibiotics could serve as targets for rational drug design .

• Conformational state stabilizers: Developing molecules that lock CorA in its closed conformation could prevent magnesium uptake. Since conformational transitions between symmetric and asymmetric states are essential for transport function, stabilizing one state could effectively inhibit the protein .

• Combination therapies: Given CorA's potential role in antibiotic efflux, combining existing antibiotics with CorA inhibitors could potentially overcome resistance mechanisms and enhance therapeutic efficacy.

• Allosteric modulators: Targeting the cytoplasmic regulatory domains of CorA with allosteric modulators could potentially disrupt its function without directly competing with magnesium at the transport site.

• Peptide-based inhibitors: Designing peptides that mimic the interfaces between CorA subunits could potentially disrupt oligomerization or conformational changes necessary for function.

These approaches require further structural and functional characterization of S. paratyphi A CorA, particularly regarding its potential dual role in magnesium import and antibiotic export.

How might genetic variations in CorA across different S. paratyphi A strains impact virulence and drug resistance profiles?

Genetic variations in CorA across different S. paratyphi A strains could have significant implications for bacterial physiology and clinical outcomes:

• Transport efficiency variations: Amino acid substitutions in the GxN motif or other functional regions could alter magnesium transport efficiency, affecting growth rates and stress responses.

• Differential antibiotic efflux capabilities: If CorA indeed functions in antibiotic efflux as suggested by recent research, variations in its sequence could potentially influence the spectrum and efficiency of antibiotic export, contributing to strain-specific resistance profiles .

• Altered regulatory responses: Mutations in the cytoplasmic domains could affect how CorA responds to intracellular magnesium levels, potentially impacting the regulation of virulence factors controlled by magnesium-dependent pathways.

• Host adaptation: Variants of CorA might represent adaptations to specific host environments or niches with varying magnesium availability.

• Vaccine and therapeutic implications: Understanding the conservation and variability of CorA across clinical isolates would be crucial for evaluating its potential as a therapeutic target or vaccine component.

Comparative genomic and functional studies across clinical isolates with varying virulence and resistance profiles would help elucidate the relationship between CorA sequence variation and pathogenicity.

What is the interplay between CorA and other virulence factors in S. paratyphi A pathogenesis?

The relationship between CorA function and other virulence determinants in S. paratyphi A likely involves complex regulatory networks:

• Interaction with two-component systems: CorA may interact with two-component regulatory systems like PhoP/PhoQ, which control the expression of multiple virulence genes in Salmonella . The deletion of phoPQ has been explored in the development of attenuated S. paratyphi A vaccine strains .

• Coordination with Vi capsular expression: In engineered bivalent vaccine strains, the viaB locus (containing 10 genes responsible for Vi polysaccharide biosynthesis) has been introduced into S. paratyphi A . Understanding how CorA function impacts the expression and stability of introduced virulence factors like Vi would be important for vaccine development.

• Influence on stress response pathways: As a magnesium transporter, CorA likely impacts various stress response pathways that are crucial for survival within host environments and during infection.

• Biofilm regulation networks: Given CorA's role in enhancing biofilm formation, it likely intersects with established biofilm regulatory networks that control this important virulence trait .

• Metabolic integration: Magnesium is required as a cofactor for numerous enzymatic reactions, suggesting that CorA function is integrated with central metabolism and thereby influences the expression and function of various metabolically-linked virulence factors.

Further research using systems biology approaches would help elucidate these complex interactions and their implications for S. paratyphi A pathogenesis and vaccine development.