Recombinant Haemophilus influenzae Magnesium transport protein CorA (corA)

What is the fundamental role of CorA in Haemophilus influenzae?

CorA in Haemophilus influenzae functions primarily as a magnesium transport protein that plays a crucial role in maintaining magnesium homeostasis. As part of the 2-TM-GxN family of membrane proteins, it regulates the uptake and transport of magnesium ions (Mg²⁺), which are essential cofactors for numerous biochemical and physiological reactions including protein synthesis, cell membrane integrity, and nucleic acid synthesis. CorA represents a ubiquitous family of transport proteins extensively studied in bacteria like E. coli and Salmonella species, but less characterized in Haemophilus influenzae . Recent research indicates that beyond its role in magnesium transport, H. influenzae CorA may also influence antimicrobial susceptibility patterns, potentially through effects on membrane permeability or interaction with efflux systems, as suggested by analogous research in other bacterial species .

How does H. influenzae CorA structure compare to other bacterial CorA homologs?

H. influenzae CorA shares structural characteristics with other bacterial CorA homologs, following the canonical pentameric assembly pattern. The protein consists of:

• A pentameric structure with each monomer contributing to the formation of a central pore

• Two transmembrane α-helices per protomer, arranged as inner and outer pentamers

• A large cytoplasmic domain believed to play a regulatory function

• The signature motif GxN located in loops connecting the transmembrane helices

Comparative analyses with CorA proteins from other organisms, such as Thermotoga maritima (TmCorA) and Methanocaldococcus jannaschii (MjCorA), reveal important conserved features. The H. influenzae CorA belongs to subgroup B of CorA proteins, which are primarily Mg²⁺-selective transporters, while subgroup A (including TmCorA) shows preference for Co²⁺ . The key distinction between these subgroups appears to be the presence of threonine residues in subgroup A versus serine residues in subgroup B at specific positions in the first transmembrane domain (TM1), which influences substrate selectivity .

What expression systems have been successfully used for recombinant H. influenzae CorA production?

Recombinant expression of H. influenzae CorA has been achieved primarily in E. coli expression systems. Based on the available research:

• E. coli is the most common expression host, with successful expression reported using T7 promoter-based systems

• The protein can be expressed with or without its N-terminal lipid modification, depending on experimental requirements

• Codon optimization may be necessary for efficient expression due to differences in codon usage between H. influenzae and E. coli

A notable approach involves replacing the N-terminal lipid modification signal sequence with a standard protein secretion signal when high yields of purified protein are required. This strategy facilitates easier extraction from bacterial membranes while maintaining the protein's key functional characteristics . For expression in H. influenzae itself (particularly nontypeable strains), specialized conjugal expression systems have been developed that overcome the limitations of traditional transformation methods, allowing for the introduction of recombinant constructs into clinical isolates .

What are the optimal conditions for expressing and purifying recombinant H. influenzae CorA?

For optimal expression and purification of recombinant H. influenzae CorA, the following methodological approach is recommended:

Expression System Selection:

• Use E. coli strain C41(DE3) for membrane proteins, as it has shown superior yields for challenging membrane proteins

• Consider codon optimization of the corA gene sequence for expression in E. coli

• Employ T7 promoter-based vectors with IPTG induction for controlled expression

Culture Conditions:

• Grow cultures at 30°C rather than 37°C after induction to reduce formation of inclusion bodies

• Use LB medium supplemented with appropriate antibiotics for selection

• Induce expression at mid-log phase (OD600 ≈ 0.6) with 0.5-1.0 mM IPTG

Purification Strategy:

• Extract membrane proteins using mild detergents (n-dodecyl-β-D-maltoside or CHAPS)

• Apply immobilized metal affinity chromatography (IMAC) using an N- or C-terminal His-tag

• Perform size exclusion chromatography as a second purification step

This approach typically yields 20-40 mg/L of soluble and active recombinant protein, representing a significant improvement compared to expression in native H. influenzae or other bacterial systems . For functional studies requiring membrane-embedded CorA, the protein can be reconstituted into proteoliposomes following purification, as demonstrated in studies with other CorA homologs .

How can researchers verify the functional integrity of recombinant H. influenzae CorA?

Verifying functional integrity of recombinant H. influenzae CorA requires multiple complementary approaches:

Transport Assays:

• Fluorescence-based transport assays using reconstituted proteoliposomes loaded with magnesium-sensitive fluorescent dyes (e.g., Mag-Fura-2)

• Radiolabeled Mg²⁺ uptake assays (using ²⁸Mg²⁺) to measure transport kinetics

• Measurement of cation selectivity by competition assays with Co²⁺, Ni²⁺, and Zn²⁺

Biophysical Characterization:

• Circular dichroism spectroscopy to assess secondary structure integrity

• Size exclusion chromatography with multi-angle light scattering to confirm pentameric assembly

• Thermal stability assays to determine protein stability

Functional Complementation:

• Expression in CorA-deficient bacterial strains to assess restoration of magnesium transport

• Growth assays under magnesium-limited conditions

Key parameters to determine include the apparent Km and Vmax for Mg²⁺ transport, with functional CorA typically showing Km values in the micromolar range. For example, analogous bacterial FbpABC transporters demonstrate an apparent Km = 0.9 μM and Vmax = 1.8 pmol/10⁷ cells/min for similar transport processes . The effects of known transport inhibitors such as cobalt hexammine or ruthenium red can also be evaluated to confirm specificity.

What role do specific conserved residues play in H. influenzae CorA function?

The function of H. influenzae CorA depends critically on several conserved amino acid residues:

GxN Signature Motif:
The GxN motif, conserved across the CorA family, is essential for substrate selection and transport. This motif is located in the loops connecting the transmembrane helices and forms part of the ion permeation pathway.

Conserved Hydroxyl-Bearing Residues:
Studies on homologous CorA proteins reveal that specific hydroxyl-bearing residues are crucial for transport function. In Mycobacterium smegmatis CorA, mutations S299A and T309A (corresponding to S260 and T270 in Salmonella typhimurium) completely abolished transport function without affecting protein folding or assembly . These findings likely extend to H. influenzae CorA, where similar conserved residues would play comparable roles.

The impact of these mutations was demonstrated in functional assays where:

• Wild-type M. smegmatis CorA provided 2-8 fold increased tolerance to antibiotics including norfloxacin, ofloxacin, and rifampicin

• The S299A and T309A mutants showed complete loss of this resistance phenotype

• Intracellular accumulation of antibiotics in cells expressing mutated CorA was similar to CorA-deficient cells

These results suggest that the hydroxyl groups in these conserved residues are critical for the coordinated movement of metal ions through the channel, potentially by providing coordination sites for partially hydrated cations during transport.

How does the pentameric structure of CorA contribute to its transport mechanism?

The pentameric structure of CorA creates a unique transport architecture that is fundamental to its function:

Channel Architecture:

• Forms a central pore through which ions are transported

• Creates multiple symmetric and asymmetric conformational states necessary for transport

• Provides intersubunit interfaces that may participate in cation coordination

Conformational Dynamics:
Based on structural studies of CorA homologs, the pentamer transitions between symmetric closed conformations and multiple asymmetric open conformations. These transitions are dynamically 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 .

Gating Mechanism:
The pentameric assembly enables a unique gating mechanism involving a helical turn upon binding of Mg²⁺ to regulatory intracellular sites. This conformational change converts a polar ion passage into a narrow hydrophobic pore, effectively regulating ion flux . The table below summarizes structural parameters from a crystal structure of the CorA homolog from Thermotoga maritima:

This structure reveals the intricate pentameric arrangement that creates both the ion conduction pathway and the regulatory sites needed for transport function .

What is the proposed mechanism for CorA-mediated antibiotic resistance in bacteria?

Recent research has uncovered a surprising role for CorA in mediating antibiotic resistance, particularly in Mycobacterium smegmatis. The proposed mechanisms include:

Enhanced Efflux Activity:

• Expression of corA increases tolerance to structurally unrelated antibiotics and anti-tuberculosis drugs

• CorA-expressing cells show significantly lower accumulation of antibiotics like norfloxacin and ofloxacin

• This suggests CorA enhances the activity of existing efflux systems rather than directly transporting antibiotics

Magnesium-Facilitated Resistance:

• The presence of sub-inhibitory concentrations of Mg²⁺ further increases antibiotic tolerance in CorA-expressing cells

• Magnesium may act as a facilitator in the efflux process

• Mutations in conserved hydroxyl residues (S299A, T309A) that disrupt Mg²⁺ transport also eliminate antibiotic resistance

Biofilm Enhancement:

• CorA expression enhances biofilm formation by 2-4 fold

• CorA-expressing cells show approximately 26% higher viability than CorA-deleted cells

• The proton gradient uncoupler CCCP inhibits both multi-drug efflux and biofilm formation, suggesting a mechanistic link

A hypothetical antiporter model has been proposed wherein CorA might function as an antiporter that imports Mg²⁺ and exports antibiotics. Molecular docking analyses suggest that antibiotics may bind to sites at the inter-subunit interfaces of the cytoplasmic domain, and then move through these interfaces to enter the transmembrane pores during conformational transitions of the protein .

What are the common challenges in expressing recombinant H. influenzae proteins and how can they be overcome?

Expressing recombinant H. influenzae proteins, including CorA, presents several technical challenges:

Transformation Difficulties:

• Many clinical isolates of nontypeable H. influenzae (NTHi) take up plasmids by transformation very inefficiently

• Clinical isolates can be refractory to introduction of shuttle vectors via electroporation

• Solution: Use intergeneric conjugation with E. coli strains carrying chromosomally-encoded transfer functions

Protein Expression Verification:

• Determining protein expression can be difficult without specific antisera

• Solution: Design vectors with appropriate epitope tags or reporter systems that enable easy detection

Codon Usage Bias:

• Differences in codon preference between H. influenzae and expression hosts can limit protein production

• Solution: Optimize codons for the expression host or use specialized strains with rare tRNAs

Membrane Protein Solubility:

• Membrane proteins like CorA often aggregate during overexpression

• Solution: Express with secretion signals rather than lipid modification signals when appropriate; use specialized E. coli strains like C41(DE3) designed for membrane protein expression

Practical approaches include designing broad host range vectors that are transferable via intergeneric conjugation, providing sites for cloning promoter regions, and carrying genes encoding resistance to different antibiotics for selection . Expression in E. coli has been optimized to yield 20-40 mg/L of soluble and active recombinant H. influenzae proteins, representing a significant improvement over expression in native hosts .

How can researchers overcome issues with functional reconstitution of CorA in artificial membrane systems?

Functional reconstitution of CorA in artificial membrane systems is essential for transport studies but presents specific challenges:

Protein Orientation:

• Challenge: Random insertion can result in mixed orientations

• Solution: Use reconstitution methods that favor unidirectional insertion, such as stepwise solubilization and detergent removal, or pH gradients during reconstitution

Lipid Composition Effects:

• Challenge: Lipid environment significantly affects CorA function

• Solution: Screen multiple lipid compositions; typically, a mixture of POPC:POPG (3:1) works well for bacterial transporters

Proteoliposome Size and Stability:

• Challenge: Heterogeneous liposome sizes affect measurement reproducibility

• Solution: Extrude proteoliposomes through defined pore-size filters and verify size distribution by dynamic light scattering

Assay Background and Sensitivity:

• Challenge: High background signals in fluorescence-based assays

• Solution: Include proper controls with protein-free liposomes and use impermeant quenchers to reduce external fluorescence

For functional studies mimicking the approach used with other CorA homologs, researchers have successfully used the following reconstitution protocol:

• Solubilize purified protein in mild detergent (0.1% n-dodecyl-β-D-maltoside)

• Mix with preformed liposomes at protein:lipid ratio of 1:200 (w/w)

• Remove detergent by adsorption to Bio-Beads SM-2

• Separate proteoliposomes from non-incorporated protein by sucrose gradient centrifugation

• Verify reconstitution by freeze-fracture electron microscopy or protease protection assays

This approach enables reliable transport measurements with signal-to-noise ratios suitable for kinetic analyses.

What methods can be used to study CorA interactions with other bacterial proteins and systems?

Investigating CorA's interactions with other bacterial proteins and cellular systems requires specialized approaches:

Protein-Protein Interaction Methods:

• Bacterial two-hybrid systems adapted for membrane proteins

• Pull-down assays using tagged CorA as bait

• Cross-linking followed by mass spectrometry (XL-MS)

• Förster resonance energy transfer (FRET) between fluorescently labeled proteins

Genetic Interaction Approaches:

• Synthetic genetic arrays to identify genes with functional relationships to corA

• Suppressor screens to identify proteins that can compensate for CorA dysfunction

• CRISPR interference (CRISPRi) screens in the presence/absence of functional CorA

Transcriptional Profiling:
Analysis of gene expression changes in response to CorA manipulation can reveal functional connections. For example, qRT-PCR analysis of key genes involved in various cellular processes revealed connections between CorA function and:

• Respiratory chain components

• ATP generation pathways

• Protein folding mechanisms

• Anti-ROS systems

• Secretory pathway components

• Central carbon metabolism

Systems Biology Approaches:
The Control Ratio (CoRa) approach can be applied to quantify the contribution of feedback control mechanisms related to magnesium homeostasis. This mathematical framework evaluates a biological system with feedback control compared to a locally analogous system without feedback, isolating the contribution of the feedback control . Such approaches help understand how CorA functions within the larger context of bacterial physiology and adaptation.

How does H. influenzae CorA differ functionally from homologs in other bacterial species?

H. influenzae CorA shows both similarities and important differences compared to homologs in other bacterial species:

Cation Selectivity Profiles:

• H. influenzae CorA belongs to subgroup B of CorA proteins, which are primarily Mg²⁺-selective

• This contrasts with subgroup A (including Thermotoga maritima CorA), which shows preference for Co²⁺

• The key structural determinant appears to be threonine residues in subgroup A versus serine residues in subgroup B at specific positions in TM1

Transport Energetics:

• Most CorA proteins, including H. influenzae CorA, are driven by membrane potential

• In contrast, some related transporters (like ZntB) are stimulated by proton gradients

• This indicates evolutionary divergence in transport mechanisms within the same protein scaffold

Metal Ion Specificity:
Comparative studies with various CorA homologs show that while all can transport Mg²⁺, Co²⁺, Ni²⁺, and Zn²⁺, they differ in their preferences:

What genetic and structural factors influence CorA expression and function in clinical H. influenzae isolates?

Several genetic and structural factors impact CorA expression and function in clinical H. influenzae isolates:

Genetic Diversity:

• H. influenzae shows extensive genetic diversity, necessitating discriminatory analytical approaches to evaluate its population structure

• Core genome multilocus sequence typing (cgMLST) analyses have been developed to characterize H. influenzae variants

• These genetic variations can potentially affect CorA expression levels and functionality

Strain-Specific Expression Patterns:

• Expression of transporters like CorA can vary significantly between clinical isolates

• Some clinical isolates (particularly nontypeable H. influenzae) present challenges for genetic manipulation and protein expression

• Specialized conjugal expression systems have been developed to overcome transformation limitations in these strains

Structural Adaptations:

• Variations in the CorA sequence, particularly in the transmembrane regions, can alter substrate specificity and transport rates

• These adaptations may contribute to strain-specific phenotypes such as antibiotic resistance or survival in magnesium-limited environments

Host Environment Influences:

• H. influenzae colonizes the human respiratory tract, where it faces varying magnesium availability

• In the Navajo and White Mountain Apache populations, specific H. influenzae variants show unique carriage patterns that may reflect adaptations to host-specific environments

• These adaptations could include modifications to CorA function or regulation

Understanding these factors is essential for interpreting the role of CorA in H. influenzae pathogenesis and for developing targeted interventions against specific clinical isolates.

How might CorA function contribute to H. influenzae pathogenesis and host interactions?

CorA function may significantly impact H. influenzae pathogenesis through several mechanisms:

Magnesium Acquisition in Host Environments:

• Magnesium availability varies across host niches, making efficient acquisition critical for survival

• CorA facilitates magnesium uptake in magnesium-limited environments encountered during infection

• This capability may be particularly important during invasion of epithelial cells, where free magnesium concentrations can be restricted

Contribution to Antibiotic Resistance:

• By analogy with findings in M. smegmatis, H. influenzae CorA may contribute to antibiotic resistance

• This could occur through direct effects on antibiotic efflux or indirect effects on membrane permeability

• Such mechanisms would be particularly relevant during chronic infections where antibiotic exposure is prolonged

Biofilm Formation:

• Expression of corA enhances biofilm formation in bacterial systems

• H. influenzae biofilms are associated with persistent infections, particularly in otitis media

• CorA-mediated enhancement of biofilm formation could contribute to treatment recalcitrance and chronic disease

Intracellular Survival:

• H. influenzae can invade and persist within host cells

• Proper magnesium homeostasis, mediated by CorA, may be essential for adaptation to the intracellular environment

• This function could complement other invasion factors like the HMW1 adhesin, which has been identified as crucial for intracellular invasion

The importance of these mechanisms is suggested by epidemiological data showing that H. influenzae remains a significant pathogen despite vaccination, particularly in certain populations. For example, from 2004-2016, the annual rate of Haemophilus influenzae type a (Hia) disease among Navajo and White Mountain Apache children <5 years was nearly 20 times higher than the rate of all non-type b disease combined among general US children . Understanding the role of CorA in these persistent infections could lead to new therapeutic approaches.

What novel approaches could advance our understanding of H. influenzae CorA function and regulation?

Several innovative approaches could significantly advance our understanding of H. influenzae CorA:

Single-Molecule Transport Studies:

• Apply single-molecule fluorescence techniques to track individual ion transport events

• Use nanodiscs containing single CorA pentamers for controlled studies

• Employ high-speed atomic force microscopy to visualize conformational changes during transport

Cryo-Electron Microscopy:

• Obtain high-resolution structures of H. influenzae CorA in different conformational states

• Capture the protein in the presence of various substrates and inhibitors

• Develop time-resolved cryo-EM approaches to visualize the transport cycle

Genome-Wide Association Studies:

• Correlate natural variations in corA sequences from clinical isolates with phenotypic differences

• Apply transformed recombinant enrichment profiling (TREP), which uses natural transformation to generate complex pools of recombinants followed by phenotypic selection and deep sequencing

• Identify genetic modifiers that influence CorA function in different genetic backgrounds

Synthetic Biology Approaches:

• Create chimeric transporters between different CorA homologs to define functional domains

• Develop optogenetic control of CorA function for precise temporal studies

• Engineer artificial feedback circuits to study CorA regulation

In Vivo Imaging:

• Develop fluorescent sensors for real-time tracking of magnesium flux in living bacteria

• Apply correlative light and electron microscopy to localize CorA in bacterial cells during infection

• Use intravital microscopy to study CorA-dependent processes during host-pathogen interactions

These approaches would complement existing methodologies and provide unprecedented insights into the molecular mechanisms of CorA function and its role in bacterial physiology and pathogenesis.

How might research on H. influenzae CorA inform the development of novel antimicrobial strategies?

Research on H. influenzae CorA presents several promising avenues for antimicrobial development:

Direct Inhibition Strategies:

• Design small molecule inhibitors targeting the conserved GxN motif or hydroxyl-bearing residues essential for transport

• Develop peptide-based inhibitors that disrupt the pentameric assembly

• Create transition-state analogs that block the transport cycle

Anti-Virulence Approaches:

• Target CorA-dependent biofilm formation to enhance antibiotic efficacy

• Disrupt CorA's contribution to antibiotic efflux to restore sensitivity

• Interfere with magnesium-dependent signaling pathways regulated by CorA activity

Combination Therapies:

• Identify synergistic effects between CorA inhibitors and existing antibiotics

• Develop magnesium chelation strategies that work in concert with CorA inhibition

• Target multiple magnesium transporters simultaneously to overcome redundancy

Vaccine Development:

• Explore recombinant CorA as a potential vaccine component

• The successful expression of recombinant H. influenzae proteins in E. coli (yielding 20-40 mg/L of soluble and active protein) provides a platform for vaccine antigen production

• Combine with existing vaccination strategies against H. influenzae to broaden protection

Host-Directed Therapies:

• Modulate host magnesium availability in infection sites

• Target host pathways that interact with bacterial magnesium homeostasis

• Develop probiotics that compete with pathogens for magnesium