Recombinant Shigella sonnei Magnesium transport protein CorA (corA)

What is the structural organization of CorA in Shigella sonnei compared to other bacterial species?

The CorA magnesium transport protein in Shigella sonnei shares significant homology with other enterobacteria, particularly E. coli and Salmonella typhimurium. Based on structural studies, CorA exists as an integral membrane protein with a unique topology that includes both periplasmic and cytoplasmic domains. The protein in related species consists of 316 amino acids with multiple membrane-spanning segments . The N-terminal region (approximately 235 amino acid residues) is located in the periplasmic space, forming a single periplasmic domain, while the C-terminal region contains three membrane-spanning segments, with the C-terminus deposited in the cytoplasm .

This topology suggests that CorA functions as an oligomer, as three membrane loops are likely insufficient for forming a membrane pore or channel independently . The protein likely exhibits both symmetric closed conformations and multiple asymmetric open conformations that dynamically change to facilitate magnesium transport . Researchers should note that while CorA is an integral membrane protein, it contains approximately 28% charged amino acids, which is unusual for membrane proteins .

How does CorA mediate magnesium transport in bacterial systems?

CorA functions as a dual-directional transporter, mediating both the influx and efflux of Mg²⁺ across bacterial membranes . In Salmonella typhimurium and E. coli, the CorA locus product alone is sufficient for Mg²⁺ influx, while products of the unlinked CorBCD loci enable CorA to mediate efflux in addition to influx .

The transport mechanism involves conformational transitions between symmetric closed states and asymmetric open states, which are influenced by intracellular Mg²⁺ levels . When magnesium 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 conformation and subsequent transport . Hydroxyl-bearing residues are critical for this function, as mutations in conserved residues such as S260 and T270 in S. typhimurium (corresponding to S299 and T309 in M. smegmatis) result in loss of measurable cation transport .

What experimental approaches can determine if recombinant S. sonnei CorA is properly folded and functional?

To assess proper folding and functionality of recombinant Shigella sonnei CorA:

• Membrane localization assays: Confirm membrane integration using subcellular fractionation techniques. The Sec pathway dependence observed in E. coli CorA can serve as a reference for protein targeting studies .

• Magnesium transport assays: Measure Mg²⁺ uptake and efflux in cells expressing recombinant CorA compared to deletion mutants. This can be achieved using radioactive ²⁸Mg²⁺ or fluorescent magnesium indicators.

• Complementation studies: Express recombinant CorA in CorA-deficient strains and assess restoration of phenotypes. For example, in M. smegmatis, complementation of CorA-deleted mutants with recombinant CorA restored susceptibility levels to various antibiotics .

• Site-directed mutagenesis: Generate mutations in conserved residues (similar to S299A and T309A in M. smegmatis CorA) and evaluate their impact on transport function . Loss of function in these mutants would confirm proper folding of the wild-type recombinant protein.

• Structural analysis: Employ circular dichroism, limited proteolysis, or thermal shift assays to assess protein stability and folding.

What are the optimal conditions for recombinant expression of S. sonnei CorA?

While the search results don't provide specific details for S. sonnei CorA expression, effective protocols can be adapted from successful approaches with related bacterial CorA proteins:

• Expression system selection: E. coli expression systems have been successfully used for CorA from M. smegmatis . Consider BL21(DE3) or specialized membrane protein expression strains like C41(DE3) or C43(DE3).

• Vector design: Include appropriate promoters (T7, tac, or arabinose-inducible) and fusion tags (His6, MBP) to facilitate detection and purification. The pMIND vector system with tetracycline-inducible expression has been effective for CorA complementation studies .

• Induction conditions: For membrane proteins like CorA, lower temperature induction (16-25°C) and reduced inducer concentrations often improve proper folding and membrane insertion.

• Media supplementation: Consider supplementing growth media with additional Mg²⁺ (e.g., 100 ppm) as this has been shown to affect CorA function in bacterial systems .

• Expression verification: Confirm expression using Western blotting with antibodies against CorA or fusion tags, and assess membrane localization through fractionation studies.

What challenges are associated with purifying recombinant CorA protein and how can they be addressed?

Purification of membrane proteins like CorA presents several challenges:

• Detergent selection: CorA, as an integral membrane protein with three transmembrane segments, requires careful detergent selection for extraction from membranes. Screen multiple detergents (DDM, LMNG, CHAPS) at various concentrations for optimal solubilization while maintaining protein structure and function.

• Protein stability: CorA contains a high percentage of charged amino acids (28% in related species) , which may affect stability during purification. Include stabilizing agents such as glycerol, specific lipids, or magnesium ions in purification buffers.

• Oligomeric state preservation: Since CorA likely functions as an oligomer , conditions must be optimized to maintain native quaternary structure. Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can verify oligomeric state preservation.

• Functional verification: After purification, confirm that the protein retains magnesium binding capability using isothermal titration calorimetry (ITC) or fluorescence-based assays.

• Reconstitution strategies: For functional studies, reconstitute purified CorA into proteoliposomes or nanodiscs to provide a membrane-like environment that supports native conformation and activity.

How can site-directed mutagenesis be effectively applied to study S. sonnei CorA function?

Based on studies in related bacterial species, site-directed mutagenesis provides valuable insights into CorA function:

• Target selection: Focus on conserved hydroxyl-bearing residues similar to S260 and T270 in S. typhimurium (S299 and T309 in M. smegmatis), which are crucial for transport function . Sequence alignment can identify corresponding residues in S. sonnei CorA.

• Mutagenesis strategy: Employ PCR-based site-directed mutagenesis techniques, such as QuikChange, to introduce specific amino acid substitutions. Alanine substitutions (e.g., S299A, T309A) have been effective in M. smegmatis studies .

• Functional validation: After generating mutants, assess:

  • Magnesium transport capability

  • Antibiotic resistance profiles (as CorA affects drug efflux)

  • Biofilm formation capacity

  • Protein localization to confirm proper expression and membrane insertion

• Quantitative comparisons: Measure intracellular accumulation of antibiotics (e.g., norfloxacin, ofloxacin) in cells expressing wild-type versus mutant CorA using fluorescence-based assays .

• Structural impact assessment: Consider computational modeling to predict how mutations affect protein conformation, particularly at inter-subunit interfaces where ligand binding may occur .

How does CorA expression influence antimicrobial susceptibility profiles?

Recent research has revealed CorA's significant impact on antimicrobial resistance:

• Broad-spectrum resistance: Expression of corA increases bacterial tolerance to structurally unrelated classes of antibiotics, including fluoroquinolones (norfloxacin, ofloxacin, sparfloxacin, ciprofloxacin), aminoglycosides (amikacin, gentamicin, apramycin), and anti-tubercular drugs (isoniazid, rifampicin) .

• Quantifiable resistance changes: In M. smegmatis, deletion of corA resulted in 2-8 fold reduction in tolerance to various antibiotics, with rifampicin showing the highest fold change (8-fold) . This effect was reversed when the deletion was complemented with functional CorA expression .

• Magnesium-enhanced resistance: The presence of sub-inhibitory concentrations of magnesium (100 ppm) further decreased susceptibility of CorA-expressing cells by 4-16 fold, suggesting magnesium facilitates CorA-mediated resistance .

• CCCP sensitivity: The addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP), which disrupts membrane potential, increased susceptibility to some antibiotics (norfloxacin, ofloxacin, ciprofloxacin, gentamicin) by 2-fold in CorA-expressing cells, indicating energy-dependent mechanisms in CorA-mediated resistance .

• Mutation effects: Single amino acid substitutions S299A and T309A completely nullified CorA-mediated resistance to all tested antibiotics, highlighting these residues' critical role in the resistance mechanism .

What experimental approaches can quantify CorA's role in drug efflux?

To investigate CorA's contribution to drug efflux:

• Intracellular drug accumulation assays: Measure fluorescent antibiotic (e.g., norfloxacin, ofloxacin) accumulation in cells expressing wild-type CorA compared to CorA-deleted mutants or those expressing mutated CorA. Lower accumulation in CorA-expressing cells indicates enhanced efflux .

• Real-time efflux kinetics: Use fluorescent probes with real-time monitoring to measure efflux rates in the presence and absence of CorA expression.

• Efflux inhibitor studies: Assess the effect of proton motive force disruptors like CCCP on drug accumulation and resistance profiles to determine if CorA-mediated efflux is energy-dependent .

• Dose-response analysis: Generate comprehensive dose-response curves for multiple antibiotics in the presence and absence of CorA expression to calculate precise MIC (minimum inhibitory concentration) values and fold changes.

• Magnesium dependence: Systematically vary magnesium concentrations to establish dose-dependent relationships between magnesium levels and antibiotic resistance in CorA-expressing cells .

• Mutational analysis: Compare efflux activity between wild-type CorA and transport-deficient mutants (e.g., S299A, T309A) to establish structure-function relationships .

What is the hypothesized mechanism for CorA-mediated drug efflux?

Based on molecular docking and functional studies, researchers have proposed a model for CorA-mediated drug export:

• Antiporter hypothesis: CorA may function as an antiporter that imports Mg²⁺ and exports antibiotics simultaneously .

• Binding sites: Molecular docking studies suggest antibiotics may bind to sites at inter-subunit interfaces (designated C5 or C4) in the closed symmetric conformation of CorA . For example, isoniazid binding sites were primarily located at the inter-subunit interfaces of the cytoplasmic domain .

• Conformational changes: The transition between symmetric (closed) and asymmetric (open) conformations, which normally facilitates Mg²⁺ import, may simultaneously drive antibiotic export .

• Transport pathway: Antibiotics likely move through the inter-subunit interfaces and enter the transmembrane pores during conformational transitions .

• Mutation effects: Mutations in critical residues (e.g., T309 and S299 in M. smegmatis CorA, corresponding to T270 and S260 in S. typhimurium) may prevent proper conformational transitions, halting both Mg²⁺ import and antibiotic export .

What techniques are most effective for determining CorA structure-function relationships?

To elucidate CorA structure-function relationships:

• Membrane topology mapping: The topology of CorA can be determined by constructing deletion derivatives and genetically fusing them to reporter cassettes (e.g., BlaM or LacZ). Enzymatic activities of these hybrid proteins can identify periplasmic versus cytoplasmic domains .

• Hydropathy analysis: Computational hydropathy plots help predict transmembrane regions, though experimental validation is crucial as CorA has been shown to contain three membrane-spanning segments rather than the two suggested by hydropathy analysis alone .

• Mutational scanning: Systematic alanine scanning mutagenesis of conserved residues can identify amino acids critical for transport function, as demonstrated by the S299A and T309A mutations in M. smegmatis CorA .

• Homology modeling: When crystal structures are unavailable, homology modeling based on related bacterial CorA proteins can provide structural insights for further experimental design .

• Molecular docking: Docking analysis can predict potential binding sites for substrates and inhibitors at inter-subunit interfaces and within transmembrane regions .

• Functional assays: Correlate structural features with transport function through:

  • Magnesium transport measurements

  • Antibiotic susceptibility testing

  • Intracellular antibiotic accumulation assays

  • Biofilm formation quantification

How can conformational changes in CorA during transport be studied?

CorA transitions between symmetric closed and asymmetric open conformations during transport, which can be studied through:

• Disulfide crosslinking: Introduce cysteine residues at strategic positions and monitor disulfide bond formation to track relative movements of protein domains during transport.

• Fluorescence resonance energy transfer (FRET): Label specific domains with fluorescent probes to monitor conformational changes in real-time as magnesium or antibiotic transport occurs.

• Electron paramagnetic resonance (EPR) spectroscopy: Site-directed spin labeling at key residues can detect distance changes during conformational transitions.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Identify regions with altered solvent accessibility during transport cycles.

• Computational molecular dynamics: Simulate conformational transitions between closed and open states, particularly focusing on how magnesium binding affects these transitions .

• Cryo-electron microscopy: Capture CorA in different conformational states by varying magnesium concentrations or using transport-inhibited mutants like S299A and T309A .

What experimental evidence supports oligomerization of CorA and how does this affect function?

The oligomeric nature of CorA is supported by several lines of evidence:

• Topology studies: The presence of only three membrane-spanning segments in the C-terminal region is likely insufficient for forming a membrane pore, suggesting CorA functions as an oligomer .

• Structural homology: Related bacterial CorA proteins form homopentamers, creating a central pore for ion conductance.

• Functional data: Mutational studies at inter-subunit interfaces affect transport function, suggesting these interfaces are critical for proper oligomerization and channel formation .

• Docking analysis: Molecular docking studies indicate that antibiotics bind at inter-subunit interfaces, further supporting the functional relevance of oligomerization in transport .

The oligomeric structure has several functional implications:

• Ion selectivity: The arrangement of subunits creates a selective filter for magnesium ions.

• Conformational coupling: Subunits likely undergo coordinated conformational changes during transport cycles.

• Multiple binding sites: Inter-subunit interfaces provide multiple binding sites for transported substrates, including antibiotics .

• Cooperative regulation: Magnesium binding to one subunit may influence the conformation of adjacent subunits, allowing for cooperative regulation of transport activity.

How does CorA expression influence biofilm formation?

Recent research has revealed important connections between CorA and biofilm development:

• Enhanced biofilm formation: Expression of corA significantly enhances biofilm formation in both E. coli and M. smegmatis, with biofilm formation index (BFI) increasing by 2-4 fold compared to CorA-deficient cells .

• Cell viability in biofilms: CorA-expressing M. smegmatis cells showed approximately 26% higher viability in biofilms compared to CorA-deleted strains, indicating CorA contributes to maintaining cellular fitness in biofilm communities .

• Energy dependence: CCCP, which disrupts membrane potential, exhibited inhibitory effects on biofilm formation in CorA-expressing cells, suggesting that the biofilm-enhancing effects of CorA are energy-dependent .

• Mechanistic connection: The dual role of CorA in both antibiotic efflux and biofilm enhancement suggests these phenomena may be mechanistically linked, potentially through magnesium homeostasis .

• Potential applications: Understanding CorA's role in biofilm formation could lead to novel strategies for disrupting bacterial biofilms, which are often associated with increased antimicrobial resistance and persistence.

What is the relationship between magnesium homeostasis and bacterial pathogenesis?

Magnesium homeostasis, regulated in part by CorA, plays multifaceted roles in bacterial pathogenesis:

• Biochemical cofactor: Magnesium serves as an essential cofactor for numerous enzymes involved in critical cellular processes including protein synthesis, cell membrane integrity, and nucleic acid synthesis .

• Stress response: Proper magnesium levels are crucial for bacterial stress responses, which are important during host colonization and infection.

• Antimicrobial resistance: Magnesium facilitates CorA-mediated resistance to multiple classes of antibiotics, with sub-inhibitory concentrations of Mg²⁺ (100 ppm) significantly enhancing resistance .

• Biofilm development: Magnesium homeostasis influences biofilm formation, with CorA-expressing cells showing enhanced biofilm capability . Biofilms contribute to bacterial persistence and reduced susceptibility to host defenses.

• Virulence gene regulation: Magnesium concentrations can influence the expression of virulence factors in pathogenic bacteria. In Shigella species, virulence plasmid stability and expression are critical for pathogenicity .

• Host-pathogen interactions: During infection, bacteria must adapt to varying magnesium concentrations in different host environments, making magnesium transporters like CorA important for successful colonization.

How might CorA function be leveraged for vaccine development against Shigella sonnei?

CorA's properties could potentially be exploited in S. sonnei vaccine development:

• Attenuated strain development: Knowledge of CorA's role in magnesium homeostasis and antimicrobial resistance could inform the creation of attenuated S. sonnei strains with modified corA genes for vaccine development, similar to how the virG deletion strategy was used to create the WRSS1 vaccine strain .

• Stability considerations: The stability of virulence phenotypes has been a challenge in S. sonnei vaccine development due to spontaneous loss of the invasion plasmid . Understanding how CorA influences bacterial fitness could help design more stable vaccine strains.

• Biofilm modification: Since CorA influences biofilm formation , manipulating corA expression or function might help control biofilm formation in vaccine production processes or affect vaccine strain colonization properties.

• Adjuvant effects: Magnesium homeostasis affects multiple immune-relevant processes. Modified CorA proteins could potentially serve as adjuvants or immunomodulators in vaccine formulations.

• Immune response targeting: If CorA is surface-exposed, it could potentially serve as an antigen target in subunit or recombinant vaccine approaches, though this would require confirmation of surface accessibility.

• Resistance mechanisms: Understanding CorA's role in antimicrobial resistance could help design vaccine strains with appropriate antibiotic susceptibility profiles for safety and regulatory compliance.

How can computational approaches enhance CorA research?

Computational methods offer powerful tools for studying CorA:

• Homology modeling: Generate structural models of S. sonnei CorA based on known structures of related bacterial CorA proteins. These models can guide experimental design for mutational studies and functional analyses .

• Molecular docking: Predict binding sites for substrates, antibiotics, and potential inhibitors. Docking studies have already revealed that antibiotics like isoniazid likely bind at inter-subunit interfaces in the cytoplasmic domain of CorA .

• Molecular dynamics simulations: Model conformational transitions between symmetric (closed) and asymmetric (open) states to understand the transport mechanism at atomic resolution .

• Machine learning approaches: Analyze sequence-structure-function relationships across diverse bacterial CorA proteins to identify novel functional regions or predict effects of mutations.

• Systems biology modeling: Integrate CorA function into larger models of bacterial magnesium homeostasis, antibiotic resistance, and biofilm formation to predict system-level effects of CorA perturbations.

• Virtual screening: Identify potential inhibitors of CorA function through in silico screening of chemical libraries, targeting key binding sites identified through structural analysis and molecular docking .

What emerging technologies might advance CorA functional studies?

Several cutting-edge technologies hold promise for CorA research:

• Cryo-electron microscopy: Capture CorA in different conformational states at near-atomic resolution, particularly focusing on magnesium-bound versus unbound states and the structural effects of key mutations.

• Single-molecule techniques: Track individual CorA proteins during transport cycles using techniques like single-molecule FRET or atomic force microscopy to resolve conformational dynamics.

• Nanopore recording: Reconstitute purified CorA into lipid bilayers for electrical recording of individual transport events, providing insights into transport kinetics and ion selectivity.

• CRISPR-based approaches: Utilize CRISPR interference or activation to precisely modulate corA expression levels, allowing for dose-dependent studies of CorA function in native contexts.

• Microfluidic systems: Develop microfluidic platforms to rapidly assess CorA function under precisely controlled environmental conditions, including varying magnesium concentrations and antibiotic gradients.

• Mass spectrometry imaging: Map spatial distributions of magnesium and antibiotics in bacterial communities with and without CorA expression to visualize transport effects at the population level.

How might CorA be targeted for antimicrobial development?

CorA's dual role in magnesium homeostasis and antibiotic resistance makes it a promising target for novel antimicrobial strategies:

• Direct inhibition: Develop small molecule inhibitors targeting the magnesium transport function of CorA, potentially disrupting essential cellular processes dependent on proper magnesium levels.

• Efflux inhibition: Design compounds that specifically block CorA-mediated antibiotic efflux without affecting magnesium import, thereby enhancing the efficacy of existing antibiotics .

• Conformational targeting: Target compounds to stabilize CorA in specific conformational states, preventing the transitions between symmetric and asymmetric states necessary for transport function .

• Oligomerization disruption: Develop peptides or small molecules that interfere with CorA oligomerization, potentially disrupting channel formation and function.

• Biofilm disruption: Exploit CorA's role in biofilm formation by developing strategies that interfere with this function, potentially increasing bacterial susceptibility to existing antibiotics and host defenses.

• Combination therapies: Design therapeutic approaches that combine CorA inhibitors with conventional antibiotics to overcome resistance mechanisms and enhance antimicrobial efficacy.

• Magnesium modulation: Develop strategies to locally deplete or sequester magnesium to reduce CorA-mediated antibiotic resistance and biofilm formation .