Recombinant Shigella dysenteriae serotype 1 Magnesium transport protein CorA (corA)

What is the CorA magnesium transport protein in Shigella dysenteriae and what is its primary function?

The CorA protein in Shigella dysenteriae is a member of the ubiquitous CorA family of transport proteins responsible for maintaining magnesium homeostasis in bacterial cells. Similar to its homologs in other bacterial species, S. dysenteriae CorA plays a crucial role in magnesium uptake and regulation of cellular magnesium concentration. Magnesium is the most abundant divalent cation in living cells and is essential for various biochemical and physiological reactions, including protein synthesis, cell membrane integrity, and nucleic acid synthesis . The CorA protein helps bacterial cells achieve optimal intracellular magnesium concentration, which is particularly important in the context of host-pathogen interactions, where magnesium availability may be limited within host macrophages during infection .

The CorA transport system exhibits dynamic conformational changes between symmetric closed states and asymmetric open states that facilitate magnesium import. These conformational transitions are influenced by intracellular magnesium 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 and magnesium import .

What experimental approaches are used to study the expression and function of recombinant S. dysenteriae CorA protein?

Studying recombinant S. dysenteriae CorA typically employs a comprehensive experimental workflow that combines molecular cloning, protein expression, and functional characterization techniques:

Molecular Cloning and Recombinant Expression:

• PCR amplification of the corA gene from S. dysenteriae genomic DNA using gene-specific primers

• Restriction enzyme digestion of PCR products for cloning into appropriate expression vectors

• Transformation of expression vectors into suitable host cells (e.g., E. coli or native bacterial species)

• Optimization of protein expression conditions using inducible promoters

Based on established protocols for CorA proteins, the gene can be cloned into vectors such as pBAD18-Cam using restriction enzymes like NheI and HindIII, or into vectors like pMIND using BamHI and PstI . Expression can be optimized using inducers such as arabinose at 0.1% (w/v) concentration for pBAD-based vectors or tetracycline for pMIND-based vectors .

Functional Characterization Methods:

• Antibiotic susceptibility testing to assess the impact of CorA expression on drug resistance

• Intracellular dye accumulation assays to measure efflux activity

• Biofilm formation assays to evaluate the influence of CorA on biofilm development

• Growth curve analysis under varying magnesium concentrations

• Site-directed mutagenesis to study structure-function relationships

These methods provide complementary data that collectively reveal the various functions of the CorA protein in bacterial physiology and antimicrobial resistance.

How is CorA protein structure organized and what are its key functional domains?

The CorA protein exhibits a highly conserved architecture across bacterial species, with a pentameric structure that forms a selective ion channel in the bacterial membrane. While specific structural details for S. dysenteriae CorA have not been fully elucidated, homology-based modeling reveals several key features that are likely conserved:

Structural Components:

• Cytoplasmic N-terminal domain containing metal-sensing regions

• Transmembrane domain comprising two transmembrane helices

• A periplasmic loop that contributes to ion selectivity

• A hydrophobic gate that regulates ion conductance

The protein functions as a homopentamer with five-fold symmetry around a central pore that serves as the transport pathway for magnesium ions. Critical functional elements include:

• Magnesium-binding sites within the cytoplasmic domain that serve as sensors for intracellular magnesium concentration

• Conserved hydroxyl-bearing residues (similar to S260 and T270 in Salmonella, or S299 and T309 in M. smegmatis) that are crucial for transport activity

• Inter-subunit interfaces that may serve as binding sites for antibiotics and other compounds

• Transmembrane pores that facilitate ion passage across the membrane

The protein alternates between symmetric closed conformations and multiple asymmetric open conformations, enabling the transport of magnesium ions across the bacterial membrane .

What role does CorA play in antimicrobial resistance mechanisms in enteric pathogens?

Recent research reveals that CorA contributes to antimicrobial resistance through multiple mechanisms, extending beyond its primary function in magnesium transport:

Efflux Pump Activity:
Studies with CorA in Mycobacterium smegmatis have demonstrated that expression of corA increases the tolerance of host cells toward various structurally unrelated antibiotics, including fluoroquinolones (norfloxacin, ofloxacin, sparfloxacin, ciprofloxacin), aminoglycosides (amikacin, gentamicin, apramycin), and anti-tubercular drugs (isoniazid, rifampicin) . This broad substrate specificity suggests that CorA may function as a multidrug efflux system.

Evidence supporting this role includes:

• Significantly lower accumulation of antibiotics (norfloxacin, ofloxacin) in cells expressing corA, indicating enhanced efflux pump activity

• 40-50% reduction in intracellular antibiotic concentration within 8-10 minutes in cells harboring CorA compared to deletion mutants

• Inhibition of this effect by CCCP (Carbonyl cyanide 3-chlorophenylhydrazone), a classic efflux pump inhibitor that disrupts membrane potential

Magnesium-Facilitated Resistance:
The presence of sub-inhibitory concentrations of Mg²⁺ further enhances antibiotic tolerance in CorA-expressing cells by 4-16 fold, suggesting that magnesium acts as a facilitator in the efflux process . This indicates a potential dual function where CorA may operate as an antiporter, importing magnesium while exporting antibiotics.

Impact on Biofilm Formation:
CorA expression enhances biofilm formation by 2-4 fold in bacterial cells, with CorA-expressing cells showing approximately 26% higher viability compared to CorA-deleted cells . This enhanced biofilm-forming ability contributes to antimicrobial resistance by creating physical barriers against antibiotic penetration and altering bacterial metabolism to promote survival under antibiotic stress.

How do site-directed mutations affect CorA protein function and what does this reveal about structure-function relationships?

Site-directed mutagenesis studies of CorA provide critical insights into the molecular basis of its transport mechanisms and multifunctional capabilities:

Impact of Conserved Hydroxyl Residue Mutations:
Substitution mutations of conserved hydroxyl-bearing residues (S299A and T309A in M. smegmatis CorA, comparable to S260 and T270 in Salmonella CorA) result in complete loss of antimicrobial efflux activity . These mutations:

• Nullify resistance to fluoroquinolones, aminoglycosides, and anti-tubercular drugs

• Eliminate the enhanced resistance typically observed in the presence of magnesium

• Abolish the reduced accumulation of antibiotics normally seen in CorA-expressing cells

Proposed Mechanistic Effects:
The molecular basis for these effects may involve:

• Increased affinity for metal ions, where cations bind too tightly for effective transport

• Disruption of conformational transitions between symmetric and asymmetric states

• Altered binding at inter-subunit interfaces that normally accommodate antibiotics

• Compromise of the selective transport pathway through the transmembrane domain

These findings suggest that the same structural elements and conformational dynamics that govern magnesium transport also facilitate antibiotic efflux, supporting a model where CorA functions as a multifunctional transport protein with interconnected activities .

What is the hypothesized mechanism for CorA-mediated antibiotic efflux compared to its magnesium transport function?

Based on homology modeling, molecular docking analyses, and functional studies, a dual-role model for CorA has been proposed:

Proposed Antibiotic Transport Mechanism:
CorA might function as an antiporter that imports Mg²⁺ while exporting antibiotics. In this model:

• In its closed symmetric conformation, antibiotics bind to sites at the inter-subunit interfaces of the cytoplasmic domain (likely at locations designated as C5 or C4)

• When the protein transitions to different asymmetric conformations (normally driven by low intracellular magnesium levels), these conformational changes facilitate the export of bound antibiotics

• The same conformational transitions that govern magnesium import also drive antibiotic export, explaining why mutations that affect magnesium transport also eliminate antibiotic efflux activity

This model is supported by molecular docking analyses showing that binding sites for antibiotics like isoniazid are predominantly located at the inter-subunit interfaces of the cytoplasmic domain, suggesting that antibiotics likely move through these interfaces before entering the transmembrane pores .

Magnesium as a Facilitator:
The observation that sub-inhibitory concentrations of magnesium enhance antibiotic resistance suggests that magnesium may play a direct role in the efflux process, possibly by:

• Inducing conformational changes that facilitate antibiotic binding or export

• Serving as a counter-ion for antibiotic efflux

• Modulating the electrostatic properties of the transport channel

The inhibitory effect of CCCP on both processes further supports the connection between energy-dependent magnesium transport and antibiotic efflux mechanisms .

What experimental strategies can be employed to distinguish between CorA's roles in magnesium transport versus drug efflux?

Differentiating between CorA's dual functions requires sophisticated experimental approaches that can isolate specific aspects of its activity:

Differential Inhibition Studies:

• Using selective inhibitors that target either magnesium transport or drug efflux

• Employing varying concentrations of external magnesium to modulate transport direction

• Applying membrane potential modulators to distinguish energy-dependent versus independent processes

Genetic Approaches:

• Creating point mutations that specifically affect one function while preserving the other

• Developing chimeric proteins with domains from different CorA homologs to map functional regions

• Employing CRISPR-Cas9 genome editing to introduce subtle modifications to the endogenous corA gene

Advanced Biophysical Methods:

• Using fluorescently labeled substrates with real-time microscopy to track transport dynamics

• Employing patch-clamp electrophysiology to measure ion conductance under varying conditions

• Applying structural biology techniques (X-ray crystallography, cryo-EM) to capture different conformational states

Computational Approaches:

• Molecular dynamics simulations to model the transport of different substrates

• Binding affinity calculations for various antibiotics at potential binding sites

• In silico mutagenesis to predict the impact of specific amino acid changes

By combining these approaches, researchers can develop a comprehensive understanding of how CorA balances its dual roles and potentially identify ways to selectively target one function without affecting the other.

How might the CorA transporter be targeted for potential antimicrobial development against S. dysenteriae?

The emerging understanding of CorA's role in both magnesium homeostasis and antimicrobial resistance presents novel opportunities for therapeutic intervention:

Potential Targeting Strategies:

• Direct Inhibition of Transport Activity:

  • Developing small molecules that bind to critical residues like the conserved hydroxyl-bearing amino acids (equivalent to S299 and T309 in M. smegmatis)

  • Designing compounds that stabilize the closed conformation, preventing the conformational changes necessary for transport

• Targeting the Antibiotic Binding Sites:

  • Creating competitive inhibitors that occupy the inter-subunit interfaces where antibiotics bind

  • Developing allosteric modulators that alter the binding properties of these sites

• Exploiting the Magnesium-Dependent Enhancement:

  • Designing magnesium chelators that selectively reduce availability for CorA-mediated processes

  • Creating compounds that interfere with the magnesium-facilitated efflux mechanism

• Anti-Biofilm Approaches:

  • Targeting CorA's role in biofilm formation to enhance antibiotic effectiveness

  • Developing combination therapies that simultaneously inhibit CorA and disrupt biofilms

Advantages of CorA as a Drug Target:

• CorA is ubiquitous across bacterial species but structurally distinct from human magnesium transporters

• Its dual role in magnesium transport and drug efflux makes it a high-value target for overcoming resistance

• Inhibiting CorA may sensitize bacteria to existing antibiotics, potentially revitalizing current treatment options

Experimental Validation of Inhibitors:
Potential inhibitors could be evaluated using:

What are the optimal expression systems and purification strategies for recombinant S. dysenteriae CorA?

Successful expression and purification of functional CorA protein requires careful consideration of several technical factors:

Expression Systems:

• Prokaryotic Systems:

  • E. coli BL21(DE3) with pET-based vectors for high-yield expression

  • E. coli CS109 with arabinose-inducible vectors (pBAD18-Cam) for controlled expression

  • Mycobacterium smegmatis with tetracycline-inducible vectors (pMIND) for expression in a mycobacterial background

• Expression Optimization:

  • Induction parameters: 0.1% arabinose for pBAD systems or 20 ng/μL tetracycline for pMIND systems

  • Growth temperature: typically lowered to 25-30°C during induction to enhance protein folding

  • Media supplementation: including 5-10 mM MgCl₂ to stabilize the protein during expression

Purification Strategies:

• Membrane Protein Extraction:

  • Gentle cell lysis using enzymatic methods or mild detergents

  • Membrane fractionation by ultracentrifugation

  • Solubilization using detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG)

• Chromatography Approaches:

  • Immobilized metal affinity chromatography (IMAC) using His-tagged constructs

  • Size exclusion chromatography to isolate the pentameric assembly

  • Ion exchange chromatography for further purification

• Quality Assessment:

  • SDS-PAGE and Western blotting to confirm protein identity and purity

  • Circular dichroism spectroscopy to verify secondary structure

  • Dynamic light scattering to assess homogeneity

  • Functional assays using magnesium-sensitive fluorescent indicators to confirm transport activity

The choice between these systems depends on the specific research goals, with E. coli systems favored for structural studies and homologous expression preferred for functional characterization in a native-like environment.

What are the most reliable methods for assessing CorA-mediated antibiotic resistance?

Evaluating CorA's contribution to antibiotic resistance requires robust methodologies that can distinguish its effects from other resistance mechanisms:

Quantitative Susceptibility Testing:

• Broth Microdilution Method:

  • Determining minimum inhibitory concentrations (MICs) for various antibiotics

  • Comparing wild-type, CorA-deleted, and CorA-complemented strains

  • Including controls with and without magnesium supplementation (typically 100 ppm)

  Antibiotic	Wild-type	ΔmsCorA	ΔmsCorA::CorA	Wild-type + Mg²⁺	ΔmsCorA + Mg²⁺	ΔmsCorA::CorA + Mg²⁺
  Norfloxacin	16 μg/mL	4 μg/mL	16 μg/mL	64 μg/mL	8 μg/mL	64 μg/mL
  Rifampicin	32 μg/mL	4 μg/mL	32 μg/mL	128 μg/mL	8 μg/mL	128 μg/mL
  Isoniazid	8 μg/mL	2 μg/mL	8 μg/mL	32 μg/mL	4 μg/mL	32 μg/mL

  (Table values are representative based on reported fold-changes in the literature)

• Efflux Inhibitor Studies:

  • Including proton gradient uncouplers like CCCP (typically at 10-20 μM)

  • Measuring changes in MIC values with and without inhibitors

  • Comparing the inhibitor effect across different genetic backgrounds

Intracellular Accumulation Assays:

• Fluorescent Dye Accumulation:

  • Using ethidium bromide (EtBr) as a fluorescent substrate

  • Measuring fluorescence intensity over time (typically 0-30 minutes)

  • Comparing wild-type, deletion mutant, and complemented strains

• Antibiotic Accumulation:

  • Using fluorescent antibiotics (e.g., fluoroquinolones) or radiolabeled compounds

  • Quantifying intracellular concentrations at different time points

  • Evaluating the effect of magnesium supplementation on accumulation levels

• Real-time Efflux Measurements:

  • Loading cells with dyes or antibiotics, then measuring efflux kinetics

  • Calculating efflux rates under various conditions

  • Determining the effect of energy inhibitors on efflux activity

These methodologies provide complementary data that collectively establish the role of CorA in antibiotic resistance, with the intracellular accumulation assays providing particularly strong evidence for efflux pump activity.

How can researchers distinguish between direct and indirect effects of CorA on antibiotic susceptibility?

Differentiating direct efflux activity from indirect effects requires carefully designed experimental approaches:

Control Experiments:

• Genetic Controls:

  • Comparing multiple independently constructed deletion mutants

  • Using point mutants that specifically affect transport (e.g., S299A and T309A mutations)

  • Creating strains with varying levels of CorA expression using inducible promoters

• Physiological Controls:

  • Measuring membrane potential to rule out general membrane disruption

  • Assessing growth rates to control for growth phase-dependent effects

  • Monitoring intracellular pH to account for potential proton gradient alterations

Mechanistic Approaches:

• Transport Specificity Analysis:

  • Testing structurally diverse antibiotics to establish substrate range

  • Conducting competition assays between magnesium and antibiotics

  • Performing isothermal titration calorimetry to detect direct binding

• Proteoliposome Reconstitution:

  • Incorporating purified CorA into artificial membrane vesicles

  • Measuring direct transport of labeled substrates in a defined system

  • Evaluating the effect of membrane potential and ion gradients in isolation

• Protein Interaction Studies:

  • Conducting pull-down assays to identify potential interacting partners

  • Using bacterial two-hybrid systems to screen for protein-protein interactions

  • Performing co-immunoprecipitation to detect associations with known efflux components

By systematically addressing these aspects, researchers can build a convincing case for direct versus indirect mechanisms, providing a stronger foundation for therapeutic targeting strategies.

What are the most promising approaches for elucidating the complete structural mechanism of CorA-mediated antibiotic efflux?

Resolving the structural basis of CorA's dual transport functions requires integrating cutting-edge technologies:

Advanced Structural Biology Approaches:

• Cryo-Electron Microscopy (Cryo-EM):

  • Capturing CorA in different conformational states with and without bound antibiotics

  • Using particle classification to identify heterogeneous conformations

  • Achieving high-resolution structures (better than 3.5 Å) to visualize ligand interactions

• X-ray Crystallography with New Strategies:

  • Co-crystallization with antibiotics and transport inhibitors

  • Using lipidic cubic phase crystallization for membrane protein structures

  • Employing nanobodies or crystallization chaperones to stabilize specific conformations

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Mapping conformational changes upon binding of magnesium versus antibiotics

  • Identifying regions with altered solvent accessibility during transport

  • Detecting allosteric networks connecting binding and transport events

Integrated Computational Approaches:

• Molecular Dynamics Simulations:

  • Simulating complete transport cycles for magnesium and antibiotics

  • Modeling the effect of mutations on transport pathways

  • Calculating energy barriers for different conformational transitions

• Quantum Mechanics/Molecular Mechanics (QM/MM):

  • Modeling electronic interactions at binding sites with high precision

  • Calculating transition states for ion and antibiotic transport

  • Evaluating the energetics of competing transport processes

• Machine Learning Applications:

  • Predicting antibiotic binding modes from structural data

  • Identifying novel binding sites based on surface properties

  • Developing models to predict substrate specificity across CorA homologs

These approaches, when combined, could yield a comprehensive model of how CorA balances its roles in magnesium transport and antibiotic efflux, potentially revealing new opportunities for selective targeting.

How might environmental factors affect CorA expression and function during S. dysenteriae infection?

Understanding the contextual regulation of CorA during infection could reveal new therapeutic opportunities:

Host-Pathogen Interface Factors:

• Magnesium Limitation:

  • Investigating how macrophage-imposed magnesium restriction affects CorA expression

  • Determining whether magnesium limitation enhances CorA's role in antibiotic resistance

  • Exploring potential compensatory mechanisms when magnesium is scarce

• pH Adaptation:

  • Examining CorA function across the pH range encountered during infection (pH 4.5-7.4)

  • Determining whether acidification alters substrate specificity or transport kinetics

  • Investigating pH-dependent changes in CorA expression or localization

• Oxidative Stress:

  • Assessing how reactive oxygen species affect CorA structure and function

  • Determining whether oxidative modifications alter transport properties

  • Exploring potential protective roles of CorA during oxidative stress

Systems Biology Approaches:

• Transcriptomic Analysis:

  • Profiling corA expression patterns during different stages of infection

  • Identifying co-regulated genes that may function with CorA

  • Mapping the regulatory networks controlling corA expression

• Metabolomic Studies:

  • Measuring changes in magnesium-dependent metabolic pathways during infection

  • Identifying metabolic signatures associated with CorA-mediated resistance

  • Exploring connections between magnesium homeostasis and central metabolism

• In vivo Infection Models:

  • Using fluorescent reporters to track CorA expression during infection

  • Comparing virulence of wild-type versus CorA mutants in animal models

  • Evaluating antibiotic efficacy against CorA mutants during active infection

These investigations could reveal how S. dysenteriae adapts CorA function to the changing host environment, potentially identifying intervention points that are specifically relevant during infection.

How does the CorA protein from S. dysenteriae compare to homologs from other pathogenic bacteria?

Understanding the similarities and differences among CorA proteins across bacterial species provides insights into functional conservation and specialization:

Sequence and Structural Comparisons:
The CorA protein family shows considerable sequence conservation across diverse bacterial species, with particularly high conservation in the transmembrane domains and the GMN motif (a signature sequence in CorA proteins). Comparative analysis reveals:

• Conserved Functional Elements:

  • The magnesium-sensing domain in the N-terminal region

  • Critical hydroxyl-bearing residues in the transport pathway (e.g., S299 and T309 in M. smegmatis, equivalent to S260 and T270 in Salmonella)

  • The pentameric assembly with a central ion conduction pathway

• Variable Regions:

  • The cytoplasmic domains showing greater sequence divergence

  • Species-specific insertions or deletions that may influence substrate specificity

  • Surface-exposed loops that interact with the specific membrane environment

Functional Divergence:
Different bacterial species show variations in CorA-associated phenotypes that may reflect adaptation to specific ecological niches:

• Antibiotic Resistance Profiles:

  • Variations in the range of antibiotics affected by CorA expression

  • Differences in the magnitude of resistance conferred

  • Species-specific interactions with other resistance mechanisms

• Biofilm Contributions:

  • Variable effects on biofilm formation (2-4 fold enhancement in M. smegmatis and E. coli)

  • Differences in biofilm architecture and composition

  • Species-specific relationships between magnesium homeostasis and biofilm development

Evolutionary Implications:
The dual role of CorA in magnesium transport and antibiotic resistance suggests evolutionary pathways that may have selected for multifunctional transport proteins:

• Ancestral Function:

  • Primary selection for efficient magnesium acquisition

  • Conservation of core transport mechanism across diverse species

• Functional Expansion:

  • Secondary adaptation for antibiotic efflux as a protective mechanism

  • Potential co-evolution with intrinsic antibiotic resistance mechanisms

• Specialized Adaptations:

  • Species-specific refinements for particular host environments

  • Optimizations for pathogen-specific challenges (e.g., macrophage survival)

These comparative insights highlight both the fundamental conservation of CorA function and the specialized adaptations that may make S. dysenteriae CorA a unique target for species-specific therapeutic approaches.

What are the most effective experimental designs for evaluating potential CorA inhibitors as antibiotic adjuvants?

Developing and testing CorA inhibitors requires robust experimental paradigms that can identify truly effective compounds:

Primary Screening Approaches:

• High-Throughput Growth Inhibition:

  • Measuring synergistic effects of candidate inhibitors with various antibiotics

  • Using checkerboard assays to determine fractional inhibitory concentration indices

  • Including CorA deletion mutants as controls to confirm target specificity

  Experimental Design:

  Condition	No Inhibitor	Inhibitor Conc. 1	Inhibitor Conc. 2	Inhibitor Conc. 3
  No Antibiotic	Growth control	Toxicity control	Toxicity control	Toxicity control
  Antibiotic Conc. 1	Resistance baseline	Synergy test	Synergy test	Synergy test
  Antibiotic Conc. 2	Resistance baseline	Synergy test	Synergy test	Synergy test
  Antibiotic Conc. 3	Resistance baseline	Synergy test	Synergy test	Synergy test

• Functional Transport Assays:

  • Measuring inhibition of magnesium uptake using fluorescent indicators

  • Assessing effects on antibiotic accumulation using the methods described in section 3.2

  • Determining IC50 values for both magnesium transport and antibiotic efflux

Secondary Validation Studies:

• Binding Confirmation:

  • Surface plasmon resonance (SPR) to measure direct binding kinetics

  • Thermal shift assays to detect stabilization upon inhibitor binding

  • Competitive binding assays with known substrates

• Structural Validation:

  • X-ray crystallography or cryo-EM of CorA-inhibitor complexes

  • HDX-MS to map inhibitor-induced conformational changes

  • NMR studies to identify binding interfaces in solution

• Specificity Assessment:

  • Testing effects on human magnesium transporters to evaluate selectivity

  • Profiling activity against CorA homologs from different bacterial species

  • Assessing off-target effects using proteomic approaches

In Vivo Validation:

• Infection Model Testing:

  • Evaluating antibiotic efficacy in the presence of CorA inhibitors

  • Measuring bacterial burden in animal models

  • Assessing impact on emergence of resistance during treatment

• Pharmacokinetic/Pharmacodynamic Studies:

  • Determining inhibitor distribution in relevant tissues

  • Measuring duration of synergistic effects in vivo

  • Optimizing dosing regimens for maximal efficacy

These experimental approaches provide a comprehensive evaluation pipeline for developing CorA inhibitors as potential adjuvants to enhance antibiotic efficacy against S. dysenteriae and other pathogens.