Recombinant Salmonella choleraesuis Magnesium transport protein CorA (corA)

What is the CorA magnesium transporter and what is its primary function in Salmonella?

CorA is the primary magnesium transporter in Salmonella enterica, responsible for maintaining magnesium homeostasis within bacterial cells. It functions as both an influx and efflux channel for Mg²⁺ ions across the bacterial membrane . While CorA primarily transports magnesium, it can also import cobalt when this metal is present at high concentrations in the growth medium . As a ubiquitous family of transport proteins, CorA is extensively studied in Salmonella and E. coli, where it serves as the main supply route for magnesium ions, which are essential cofactors for various biochemical and physiological processes .

How is CorA expression regulated in Salmonella?

CorA expression is regulated through multiple mechanisms. At the transcriptional level, CorA is regulated through both transcription initiation via the stringent response and transcription elongation through mechanisms that are not yet fully understood . Research has demonstrated that the stress sigma factor of RNA polymerase, σS/RpoS, influences corA expression in stationary phase Salmonella cells . This regulatory relationship creates an intricate connection between stress response and magnesium homeostasis. Unlike the other two magnesium transporters in Salmonella (MgtA and MgtB), which are expressed under magnesium starvation conditions through the PhoP-PhoQ regulatory system, CorA is expressed under various growth conditions .

What other magnesium transport systems exist in Salmonella, and how do they interact with CorA?

Salmonella imports magnesium via three known transporters: the widely conserved CorA transporter and the MgtA and MgtB P-type ATPases . While CorA is constitutively expressed under various growth conditions, MgtA and MgtB are specifically expressed under magnesium-limited conditions through the positive control of the PhoP-PhoQ regulatory system . Recent research has uncovered a regulatory crosstalk between the CorA and PhoP/MgtA systems that contributes to Salmonella resilience. Under magnesium-proficient conditions, the absence of CorA is sensed by the cell and compensated by both PhoP-independent production of MgtA and PhoP-dependent mechanisms . This regulatory network minimizes the impact of CorA deficiency on protein content, magnesium homeostasis, growth, and motility of Salmonella.

How does CorA contribute to Salmonella virulence and pathogenesis?

CorA significantly impacts Salmonella virulence through multiple mechanisms. Research has demonstrated that a Salmonella corA mutant exhibits attenuated virulence in mice, reduced epithelial cell invasion capabilities, and compromised macrophage survival . Interestingly, the virulence defect does not seem to be directly linked to magnesium deficiency, as mutant strains don't necessarily show reduced magnesium content .

A key finding in virulence research comes from studies showing that the regulation of CorA function, rather than just its presence, affects Salmonella virulence . When investigators complemented a corA mutation with corA alleles from various species, they found that the invasion defect was rescued only when the complementing allele was both functional and evolutionarily similar to S. enterica serovar Typhimurium CorA . This suggests that proper regulation of CorA function, likely involving species-specific protein-protein interactions or regulatory mechanisms, is critical for optimal virulence.

What experimental approaches are recommended for studying CorA transport kinetics?

When studying CorA transport kinetics, researchers should consider a multi-faceted approach:

• Transport Assays: Utilize radioactive isotopes (²⁸Mg²⁺) or fluorescent magnesium indicators to measure real-time transport rates across membranes.

• Electrophysiological Measurements: Patch-clamp techniques can be employed to measure ion currents through the CorA channel at different membrane potentials and ion concentrations.

• Inhibitor Studies: Selective inhibitors like Co(III) hexaammine can be used at various concentrations to block CorA function acutely or chronically, helping distinguish between direct transport effects and secondary regulatory responses .

• Expression Analysis: Monitoring corA transcription levels and CorA protein content under different conditions provides insight into regulatory mechanisms affecting transport .

• Intracellular Mg²⁺ Measurements: Total cellular magnesium content should be measured to correlate with transport activity under various experimental conditions .

For comprehensive kinetic analysis, researchers should examine both influx and efflux capabilities, as CorA has been shown to function bidirectionally depending on the concentration gradient .

What is the relationship between CorA and antimicrobial resistance?

Recent research has uncovered a surprising role for CorA in antimicrobial resistance. Expression of corA in both M. smegmatis and E. coli increases host cell tolerance toward various structurally unrelated antibiotics and anti-tubercular drugs . Mechanistic studies reveal that cells expressing corA show significantly lower accumulation of fluoroquinolones (norfloxacin and ofloxacin), suggesting that CorA enhances efflux pump activity .

The presence of sub-inhibitory concentrations of Mg²⁺ appears to enhance this low-level drug tolerance, indicating that magnesium might act as a facilitator in the drug efflux process . These findings suggest a novel mechanism by which bacteria may develop antimicrobial resistance through metal transporters, expanding our understanding beyond traditional resistance mechanisms.

For researchers investigating this phenomenon, experimental approaches should include:

• Drug accumulation assays with fluorescent antibiotics

• Minimum inhibitory concentration (MIC) determinations in the presence and absence of magnesium

• Gene expression analyses of known efflux pumps when CorA is present versus absent

• Transport studies using radiolabeled antibiotics to directly measure efflux rates

How can researchers effectively work with recombinant CorA protein for in vitro studies?

Working with recombinant CorA protein requires careful attention to protein stability and functionality. Based on available information for recombinant Salmonella choleraesuis CorA protein :

Recommended Storage and Handling Protocol:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Briefly centrifuge vials before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended 50%) and aliquot for long-term storage

• Avoid repeated freeze-thaw cycles

• For working aliquots, store at 4°C for up to one week

Experimental Considerations:

• Functional Assays: Incorporate the recombinant protein into liposomes to measure transport activity

• Structural Studies: Use purified His-tagged protein for crystallography or cryo-EM structural analysis

• Protein-Protein Interaction Studies: Employ the His-tag for pull-down assays to identify interaction partners

• In vitro Regulation Studies: Test the effects of various metal ions and potential inhibitors on protein conformation and activity

When designing experiments, researchers should consider that the His-tag might affect protein function in some assays and include appropriate controls.

What is known about the role of CorA in bacterial biofilm formation?

CorA has recently been implicated in enhancing biofilm formation in bacterial cells. Research demonstrates that CorA expression significantly enhances the biofilm-forming ability of both E. coli and Mycobacterium smegmatis . This finding adds a new dimension to our understanding of CorA's role beyond simple magnesium transport.

The mechanism linking CorA to biofilm formation remains under investigation, but several hypotheses can guide research:

• Magnesium-Dependent Pathway: CorA may influence biofilm formation by maintaining optimal intracellular magnesium levels, which could affect the expression of genes involved in biofilm production.

• Cell Surface Modifications: CorA activity might alter the bacterial cell surface properties, affecting initial attachment and biofilm development.

• Stress Response Connection: Given the regulatory connection between CorA and stress response factors like σS in Salmonella , biofilm formation might be part of a coordinated stress response.

For Salmonella specifically, research has shown that σS is required for biofilm formation , and the recently discovered relationship between σS and CorA expression suggests a potential regulatory pathway connecting these phenomena.

What genetic approaches are most effective for studying CorA function in Salmonella?

For genetic manipulation studies targeting CorA in Salmonella, researchers should consider:

• Gene Deletion Strategies: Complete deletion of corA using lambda Red recombination system or CRISPR-Cas9 approaches to generate clean knockouts .

• Complementation Analysis: As demonstrated in previous research, complementing corA mutations with corA alleles from various species can provide insights into functional conservation and specificity . When designing complementation experiments, consider:

  • Using inducible promoters to control expression levels

  • Including evolutionary similar and distant corA homologs

  • Engineering specific mutations in functional domains

• Transcriptional Fusions: corA-lacZ transcriptional fusions have been successfully used to monitor corA expression levels under various conditions .

• Point Mutations: Site-directed mutagenesis of conserved residues to identify amino acids critical for:

  • Magnesium transport

  • Cobalt transport

  • Channel gating

  • Protein-protein interactions

• Dual-System Analysis: Since there's crosstalk between CorA and the PhoP/MgtA systems, dual genetic manipulation of these systems can reveal compensatory mechanisms .

How can researchers accurately measure magnesium transport and content in bacterial cells?

Accurate measurement of magnesium transport and cellular content requires specialized techniques:

For Magnesium Transport Measurement:

• Radioisotope Uptake: Using ²⁸Mg²⁺ to track real-time uptake rates in intact cells

• Fluorescent Probes: Mag-fura-2 or other magnesium-sensitive fluorophores can monitor intracellular free Mg²⁺ concentrations

• Electrophysiology: For direct measurement of ion currents through CorA channels

For Cellular Magnesium Content Determination:

• Atomic Absorption Spectroscopy: Provides total cellular magnesium measurements with high sensitivity

• ICP-MS (Inductively Coupled Plasma Mass Spectrometry): Offers multi-element analysis with high precision

• Magnesium-Specific Colorimetric Assays: For routine quantification in cell lysates

When designing experiments, researchers should differentiate between:

• Total magnesium content (bound and free)

• Free ionic magnesium concentration

• Compartmentalized magnesium distribution

Previous studies have shown that differences in membrane composition, ribosome content, and ATP levels can affect magnesium reservoirs in cells , highlighting the importance of considering these factors when interpreting results.

What are the recommended controls for CorA inhibition studies?

When conducting CorA inhibition studies, appropriate controls are crucial for result interpretation:

• Positive Control: Include a known CorA inhibitor like Co(III) hexaammine at high concentrations, which has been shown to effectively block CorA function .

• Concentration Gradient: Test inhibitors at multiple concentrations to distinguish between acute and chronic effects, as demonstrated in epithelial cell invasion studies .

• Genetic Controls:

  • ΔcorA mutant strain (complete knockout)

  • Strains with point mutations affecting transport but not expression

  • Complemented strains with various corA alleles

• Transport Specificity Controls:

  • Measure transport of other ions (Ca²⁺, Ni²⁺) to confirm inhibitor specificity

  • Include strains with functional MgtA/MgtB but lacking CorA

• Phenotypic Readouts:

  • Growth curves in magnesium-limited media

  • Virulence assays (epithelial cell invasion)

  • Biofilm formation assays

Research has shown that inhibiting CorA acutely or chronically with high concentrations of Co(III) hexaammine had no effect on Salmonella invasion of Caco-2 epithelial cells, while genetic deletion of corA did impair invasion . This discrepancy highlights the importance of using multiple approaches to validate findings in CorA function studies.

How does CorA function in stress response pathways during Salmonella infection?

Recent research has revealed an intricate relationship between CorA and stress response in Salmonella. The stress sigma factor σS/RpoS, which remodels global gene expression during stationary phase and under stress conditions, has been shown to influence corA expression . This relationship creates a potential regulatory loop where magnesium homeostasis and stress response are coordinated.

For researchers investigating this area, key experimental approaches should include:

• Transcriptomics: RNA-seq comparison of wild-type and ΔcorA strains under various stress conditions

• Proteomics: Global and analytical proteomic analyses to identify proteins differentially expressed in CorA-deficient strains

• Stress Survival Assays: Testing survival of wild-type versus ΔcorA strains under:

  • Oxidative stress

  • Acid stress

  • Antimicrobial peptide exposure

  • Nutrient limitation

The identified crosstalk between CorA and PhoP/MgtA systems suggests a sophisticated regulatory network that helps Salmonella adapt to changing environmental conditions, particularly during infection . Understanding this network could reveal new approaches for targeting bacterial resilience mechanisms.

What is the potential of CorA as a drug target for novel antimicrobial development?

CorA presents several characteristics that make it a potentially attractive drug target:

• Essential Function: As the primary magnesium transporter in many bacteria, CorA plays a crucial role in maintaining proper cellular magnesium levels .

• Virulence Contribution: Research demonstrates that CorA affects Salmonella virulence in mice and invasion capabilities in epithelial cells .

• Structural Uniqueness: The CorA protein structure differs from human magnesium transporters, potentially allowing for selective targeting.

• Resistance Connection: The recently discovered role of CorA in antimicrobial resistance suggests that targeting this protein might enhance the efficacy of existing antibiotics .

For researchers exploring CorA as a drug target, promising approaches include:

• High-throughput Screening: Developing assays to identify small molecules that specifically inhibit CorA transport function

• Structure-Based Drug Design: Using the known crystal structure of CorA to design inhibitors that block the channel

• Combination Therapy: Testing potential CorA inhibitors in combination with existing antibiotics to overcome resistance

• Attenuated Vaccine Development: Exploring ΔcorA strains as potential live attenuated vaccine candidates

The complex interplay between CorA and other cellular systems suggests that inhibiting this transporter could disrupt multiple bacterial processes simultaneously, potentially increasing therapeutic efficacy.

How do environmental factors influence CorA expression and function during infection?

The expression and function of CorA during infection are likely influenced by multiple environmental factors that bacteria encounter in the host:

• Magnesium Availability: Different host niches have varying magnesium concentrations, potentially affecting CorA expression and activity. For example, the PhoP-PhoQ system responds to low magnesium by activating mgtA and mgtB expression .

• Stress Conditions: The connection between stress sigma factor σS and corA expression suggests that various stressors encountered during infection modulate CorA levels .

• Acidic pH: As Salmonella encounters acidic environments in the stomach and within phagosomes, pH changes may affect CorA conformation and transport capability.

• Antimicrobial Peptides: Host defense peptides might interact with or influence CorA function, particularly given CorA's role in membrane integrity.

• Nutrient Competition: During infection, competition with the host and other microbes for magnesium could drive changes in magnesium transport systems.

Research investigating these factors should employ in vitro conditions that mimic specific host environments, as well as in vivo infection models that allow for spatial and temporal tracking of CorA expression and activity.