Recombinant Mycobacterium tuberculosis Fumarate reductase subunit D (frdD)

What is the structural and functional role of frdD in the Mycobacterium tuberculosis fumarate reductase complex?

Fumarate reductase in Mycobacterium tuberculosis (M.tb) is a heterotetrameric complex composed of four distinct subunits: a catalytic subunit (FrdA), an iron-sulfur cluster subunit (FrdB), and two transmembrane subunits (FrdC and FrdD). The frdD subunit, along with frdC, anchors the complex in the membrane, enabling electron transport through the complex. Structurally, frdD contains multiple transmembrane helices that integrate into the mycobacterial membrane and work with frdC to transfer electrons from the quinone pool to the catalytic center. This arrangement allows the enzyme to catalyze the reduction of fumarate to succinate while maintaining membrane potential in oxygen-limited environments, which is crucial for M.tb persistence within granulomas .

How does frdD contribute to M. tuberculosis survival under anaerobic conditions?

The frdD subunit contributes significantly to M. tuberculosis survival under anaerobic or microaerophilic conditions by facilitating alternative respiratory pathways. When oxygen becomes limited, M.tb shifts from aerobic respiration to alternative terminal electron acceptors, with fumarate being a key alternative. The frdD subunit enables proton translocation across the membrane during this process, helping maintain the proton motive force necessary for ATP synthesis. This mechanism allows M.tb to generate energy and maintain redox homeostasis even in the oxygen-poor environment of tuberculous granulomas, contributing to bacterial persistence during latent infection . The ability to function under varying oxygen tensions is critical for M.tb's lifecycle, particularly within the heterogeneous microenvironments of human granulomas.

What are the recommended protocols for cloning and expressing recombinant M. tuberculosis frdD in E. coli systems?

For recombinant expression of M. tuberculosis frdD, a methodical approach is necessary given the hydrophobic nature of this transmembrane protein. Begin by PCR-amplifying the frdD gene (Rv1554) using high-fidelity polymerase with primers containing appropriate restriction sites compatible with your expression vector. The following protocol outlines the key steps:

• Vector selection: Use vectors with strong, inducible promoters (like pET series) that include fusion tags (His6, MBP, or GST) to aid in purification and potentially increase solubility.

• Expression conditions: Transform the construct into E. coli strains specialized for membrane protein expression (C41(DE3), C43(DE3), or Lemo21(DE3)). Culture at lower temperatures (16-20°C) after induction with reduced IPTG concentrations (0.1-0.5 mM) to minimize inclusion body formation.

• Membrane fraction isolation: Harvest cells and disrupt by sonication or pressure homogenization in buffer containing protease inhibitors. Separate membrane fractions through ultracentrifugation (100,000×g for 1 hour).

• Solubilization: Extract membrane proteins using detergents suitable for transmembrane proteins (e.g., n-dodecyl-β-D-maltoside (DDM), LDAO, or CHAPS) at concentrations above their critical micelle concentration.

• Purification: Perform affinity chromatography using tagged constructs followed by size exclusion chromatography to obtain pure protein.

This approach balances protein yield with proper folding, which is critical when working with transmembrane components of multiprotein complexes .

What are the most effective methods for analyzing frdD interactions with other fumarate reductase subunits?

To effectively analyze interactions between frdD and other fumarate reductase subunits, a multi-technique approach is recommended:

• Co-immunoprecipitation (Co-IP): Using antibodies against tagged versions of frdD to pull down the entire complex from M. tuberculosis lysates or recombinant expression systems. This can confirm direct interactions under near-native conditions.

• Bacterial two-hybrid system: Particularly useful for mapping specific interaction domains between frdD and other subunits by creating various truncations or point mutations.

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding affinities between purified frdD and other subunits, providing kinetic parameters of the interactions.

• Cross-linking coupled with mass spectrometry: This approach can identify precise contact points between subunits. Utilize membrane-permeable cross-linkers followed by proteomic analysis to map the interaction interfaces.

• Fluorescence Resonance Energy Transfer (FRET): By tagging different subunits with appropriate fluorophores, interactions can be visualized in living cells, offering insights into the spatial arrangement of the complex.

The data from these complementary approaches can be integrated to develop a comprehensive model of the fumarate reductase complex assembly, with particular focus on the role of frdD in maintaining structural integrity and functional activity .

How can researchers effectively purify recombinant frdD while maintaining its native conformation?

Purifying recombinant frdD while preserving its native conformation requires specialized techniques for membrane proteins:

• Gentle solubilization: Use mild detergents like DDM (0.5-1%) or LMNG (0.01-0.05%) that effectively extract membrane proteins while preserving protein-protein interactions. Perform solubilization at 4°C for 1-2 hours with gentle rotation.

• Lipid supplementation: Include lipids (0.1-0.5 mg/ml phosphatidylcholine or E. coli lipid extract) during purification to stabilize the native conformation.

• Buffer optimization: Use buffers containing glycerol (10-20%) and reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent aggregation and oxidation.

• Membrane scaffold proteins (MSPs): Consider reconstituting frdD into nanodiscs using MSPs, which provide a native-like lipid bilayer environment.

• Activity validation: Confirm native conformation through functional assays, such as measuring electron transfer capabilities when reconstituted with other Frd subunits.

This careful approach balances sufficient protein yield with preservation of the functional state necessary for downstream structural and interaction studies .

How does the amino acid sequence of frdD influence its integration into the membrane and interaction with other subunits?

The amino acid sequence of M. tuberculosis frdD contains multiple hydrophobic regions that form transmembrane helices, critically influencing both membrane integration and inter-subunit interactions. Sequence analysis reveals that frdD contains approximately 3-4 transmembrane domains with specific characteristics:

• Hydrophobic core regions: Rich in branched-chain amino acids (leucine, isoleucine, valine) that facilitate stable membrane anchoring through hydrophobic interactions with membrane lipids.

• Interface residues: Contain polar and charged amino acids at the interfaces between transmembrane helices that form salt bridges and hydrogen bonds with frdC, the other transmembrane subunit.

• Cytoplasmic domain motifs: Feature conserved sequences that interact with the iron-sulfur protein subunit (frdB), forming the connection between the membrane and catalytic components.

Comparative sequence analysis across mycobacterial species shows high conservation of these key structural elements, particularly at interaction interfaces, indicating their essential role in complex assembly. Mutagenesis studies have demonstrated that alterations to specific conserved residues at subunit interfaces can disrupt complex formation without affecting membrane insertion, suggesting distinct sequence determinants for these two functions. Additionally, specific motifs in frdD are thought to facilitate interaction with quinones, enabling electron transfer from the membrane-bound electron carriers to the catalytic site via the iron-sulfur clusters .

What are the challenges in developing inhibitors that specifically target frdD, and how might these be overcome?

Developing specific inhibitors against M. tuberculosis frdD presents several significant challenges that require sophisticated research approaches:

• Structural complexity: The transmembrane nature of frdD makes structural characterization difficult, limiting structure-based drug design. This can be addressed by:

  • Using cryo-electron microscopy of the intact complex

  • Employing computational modeling based on homologous structures

  • Developing fragment-based screening approaches specific to membrane proteins

• Functional redundancy: M. tuberculosis possesses alternative respiratory pathways that may compensate for frdD inhibition. Overcome this by:

  • Targeting multiple respiratory enzymes simultaneously

  • Identifying synergistic drug combinations through high-throughput screening

  • Focusing on conditions where fumarate reductase activity becomes essential

• Specificity concerns: Ensuring selectivity for mycobacterial frdD over human homologs is crucial. Strategies include:

  • Exploiting structural differences between bacterial and mammalian succinate dehydrogenase/fumarate reductase

  • Targeting mycobacteria-specific interaction interfaces between frdD and other subunits

  • Developing allosteric inhibitors that bind unique regulatory sites

• Drug delivery challenges: Compounds must penetrate both host cell membranes and the complex mycobacterial cell wall. Solutions involve:

  • Designing compounds with balanced hydrophobicity/hydrophilicity profiles

  • Incorporating mycobacterial cell wall-targeting moieties

  • Developing prodrug approaches activated by mycobacterial enzymes

A comprehensive inhibitor development pipeline would involve initial high-throughput screening against whole-cell M. tuberculosis under anaerobic conditions, followed by target validation using genetic approaches (conditional knockdowns of frdD) and biochemical assays with the purified complex .

How does the expression and activity of recombinant frdD differ between drug-susceptible and drug-resistant strains of M. tuberculosis?

Expression and activity patterns of recombinant frdD exhibit notable differences between drug-susceptible and drug-resistant strains of M. tuberculosis, with implications for both bacterial physiology and drug development:

• Expression level differences: Proteomic and transcriptomic analyses reveal that multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains often show upregulated expression of the entire fumarate reductase operon, including frdD. This upregulation appears to be part of a metabolic remodeling that occurs under drug pressure, particularly with drugs targeting energy metabolism like bedaquiline.

• Functional adaptations: The fumarate reductase complex in resistant strains shows altered kinetic properties, with many MDR strains demonstrating enhanced enzyme efficiency under microaerophilic conditions. This adaptation may contribute to the enhanced survival of these strains during antibiotic treatment, particularly within oxygen-limited granulomas.

• Genetic polymorphisms: Sequence analysis of frdD from drug-resistant clinical isolates has identified non-synonymous mutations that may affect protein-protein interactions within the complex or alter substrate specificity. These mutations are not directly associated with drug resistance mechanisms but appear to be compensatory adaptations that enhance bacterial fitness.

These differences suggest that the fumarate reductase complex, including frdD, plays an important role in the metabolic adaptation of M. tuberculosis to antibiotic pressure. This understanding could inform the development of adjunct therapies that target these adaptive mechanisms to enhance the efficacy of existing antimicrobials against drug-resistant tuberculosis .

What are the most reliable methods for assessing the enzymatic activity of recombinant fumarate reductase complexes containing frdD?

To reliably assess the enzymatic activity of recombinant fumarate reductase complexes containing frdD, researchers should employ a multi-faceted approach:

• Spectrophotometric assays: The gold standard involves monitoring the oxidation of reduced benzyl viologen (BV) or methyl viologen (MV) at 578 nm as they donate electrons for fumarate reduction. The reaction mixture typically contains:

  • 50 mM phosphate buffer (pH 7.2)

  • 0.1 mM BV or MV (pre-reduced with sodium dithionite)

  • 10 mM fumarate

  • Purified enzyme complex or membrane fractions

  Activity is calculated as μmol of BV oxidized per minute per mg of protein using an extinction coefficient of 8.65 mM⁻¹cm⁻¹.

• Oxygen consumption measurements: Using a Clark-type electrode to measure decreases in oxygen consumption when fumarate is provided as an alternative electron acceptor.

• Reverse reaction measurement: Assessing succinate dehydrogenase activity (the reverse of fumarate reductase) using 2,6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor, monitoring absorbance decrease at 600 nm.

• Reconstitution assays: For complex assembly verification, reconstitute individual purified subunits (including frdD) in liposomes and measure resulting activity compared to naturally isolated complexes.

When interpreting data, it's crucial to normalize activities to protein concentration and compare kinetic parameters (Km, Vmax) across different experimental conditions to account for variations in enzyme preparation .

How can researchers differentiate between native and recombinant frdD when analyzing expression in mycobacterial systems?

Differentiating between native and recombinant frdD in mycobacterial expression systems requires strategic experimental design and analytical techniques:

This methodological toolkit allows precise differentiation between native and recombinant proteins while facilitating quantitative comparative studies of their expression, localization, and function. When designing these systems, researchers should carefully validate that tags or modifications don't interfere with membrane insertion or complex assembly by performing functional assays of the fumarate reductase activity .

What bioinformatic approaches can predict the impact of mutations in frdD on complex stability and function?

Advanced bioinformatic approaches offer powerful tools for predicting how mutations in frdD affect the stability and function of the fumarate reductase complex:

• Homology modeling and molecular dynamics simulations: Create structural models of M. tuberculosis frdD based on homologous proteins with known structures (e.g., E. coli fumarate reductase). These models can then be subjected to molecular dynamics simulations (50-100 ns) to assess how mutations affect:

  • Transmembrane helix stability

  • Protein-lipid interactions

  • Conformational flexibility

  • Subunit interaction interfaces

• Evolutionary analysis tools:

  • Conservation analysis using ConSurf or similar tools to identify functionally important residues

  • Coevolution analysis with methods like GREMLIN or EVcoupling to identify residue pairs that have coevolved, suggesting functional coupling

  • Calculation of evolutionary rate ratios (dN/dS) to identify sites under selective pressure

• Machine learning approaches:

  • Supervised learning algorithms trained on datasets of characterized mutations

  • Graph neural networks that model the protein structure as a graph, where nodes are amino acids and edges represent interactions

  • Deep mutational scanning data integration to predict functional effects

• Energy calculation methods:

  • ΔΔG calculations using FoldX or Rosetta to estimate changes in folding free energy

  • Binding energy calculations for subunit interfaces using MM-GBSA or similar methods

For maximum prediction accuracy, researchers should integrate results from multiple methods and validate computational predictions with experimental approaches such as thermal shift assays, circular dichroism, or functional enzyme assays .

How might targeting frdD contribute to novel therapeutic approaches against drug-resistant tuberculosis?

Targeting frdD represents a promising therapeutic avenue against drug-resistant tuberculosis through several distinct mechanisms:

• Disruption of anaerobic metabolism: Since fumarate reductase becomes essential during oxygen limitation, inhibiting frdD could specifically target persistent mycobacteria within hypoxic granulomas—precisely the population that contributes to treatment recalcitrance. This would complement existing drugs that primarily target actively replicating bacteria.

• Membrane potential disruption: FrdD is critical for maintaining membrane potential under anaerobic conditions. Compounds that interfere with this function could collapse the proton motive force, synergizing with drugs like bedaquiline that target energy metabolism through different mechanisms.

• Complex assembly interference: Small molecules that prevent proper assembly of the fumarate reductase complex by targeting frdD-specific protein-protein interactions could render the enzyme non-functional without requiring direct active site inhibition.

• Adjunct therapy potential: Fumarate reductase inhibitors targeting frdD could be particularly valuable as adjunct therapy to standard regimens. Research indicates that recombinant BCG vaccines already enhance the effect of second-line TB drugs; similarly, frdD inhibitors could potentiate existing antibiotics by preventing metabolic adaptation .

• Narrow-spectrum activity: The significant structural differences between mycobacterial fumarate reductase and human succinate dehydrogenase offer opportunities for developing highly specific inhibitors, potentially reducing off-target effects common with broad-spectrum antibiotics.

This multi-faceted approach could be particularly valuable against extensively drug-resistant TB (XDR-TB), where options are severely limited. The ability to target non-replicating persistent bacteria through frdD inhibition addresses a critical gap in the current antimycobacterial arsenal .

What are the key considerations for designing frdD-based diagnostic tools for M. tuberculosis detection?

Developing frdD-based diagnostic tools for M. tuberculosis detection requires careful consideration of several technical and practical factors:

• Specificity optimization: The frdD sequence must be analyzed for regions unique to M. tuberculosis complex to avoid cross-reactivity with environmental mycobacteria or other respiratory pathogens. Bioinformatic analysis of:

  • Species-specific epitopes for antibody-based detection

  • Unique sequence regions for nucleic acid amplification tests (NAATs)

  • M. tuberculosis-specific post-translational modifications

• Sensitivity enhancement strategies:

  • Signal amplification techniques (branched DNA, CRISPR-Cas systems)

  • Pre-concentration methods for bacterial cells from clinical samples

  • Detection of fumarate reductase activity rather than protein presence for functional sensitivity

• Sample processing considerations:

  • Methods for efficient cell lysis that release membrane-bound frdD

  • Detergent solubilization protocols optimized for diagnostic settings

  • Direct-from-sample detection without culture requirements

• Platform integration options:

  • Lateral flow assays using frdD-specific antibodies

  • Microfluidic systems for automated sample processing

  • Integration with existing diagnostic workflows like GeneXpert

• Clinical validation parameters:

  • Performance in paucibacillary samples (extra-pulmonary TB)

  • Ability to detect viable versus non-viable bacteria

  • Correlation with treatment response metrics

When designing these diagnostics, researchers should consider that fluorescent bacteriophage-based systems have shown promise for rapid drug sensitivity testing of M. tuberculosis and could potentially be adapted to detect fumarate reductase activity or frdD expression as a viability marker . The ODELAM microscopy approach could also be modified to visualize metabolic activity dependent on fumarate reductase function in microcolonies, potentially offering rapid resistance profiling within 48 hours .

How does the recombinant expression of frdD in vaccine strains impact immunogenicity and protective efficacy?

The recombinant expression of frdD in vaccine strains presents a complex immunological profile that influences both immunogenicity and protective efficacy against M. tuberculosis:

• Enhanced CD4+ T cell responses: Overexpression of frdD in recombinant BCG (rBCG) strains can increase the presentation of frdD-derived peptides via MHC-II, stimulating CD4+ T helper cell responses. These responses are characterized by:

  • Increased production of IFN-γ and TNF-α, critical cytokines for macrophage activation

  • Expansion of multifunctional T cells (producing IFN-γ, TNF-α, and IL-2)

  • Enhanced memory T cell formation with prolonged persistence

• Metabolic adaptation and antigen persistence: rBCG strains expressing modified frdD show altered persistence in host tissues, which affects antigen presentation duration:

  • Higher expression levels can enhance survival under hypoxic conditions within granulomas

  • Extended antigen presentation results in more robust memory responses

  • Drug-resistant rBCG strains (RdrBCG) expressing frdD have shown enhanced therapeutic effects when combined with chemotherapy, reducing lung bacterial burden by approximately 1 log10 CFU

• Impact on vaccine strain attenuation: Modification of frdD expression can affect the balance between immunogenicity and safety:

  • Overexpression may enhance immunogenicity while maintaining the attenuated phenotype

  • Expression of mutant forms can potentially alter the growth characteristics in vivo

  • Careful monitoring for virulence in immunocompromised models is essential

• Adjuvant co-expression considerations: When designing rBCG vaccines expressing frdD:

  • Co-expression with immunomodulatory molecules can selectively enhance specific immune responses

  • Combined expression with antigens from different growth phases (like Ag85B and Rv2628) has shown enhanced protection

  • Expression under stress-responsive promoters may optimize immunogenicity

The evidence suggests that recombinant expression of frdD, particularly when combined with other immunodominant antigens and used as an adjunct to chemotherapy, could enhance both prophylactic and therapeutic efficacy against drug-resistant tuberculosis. Studies with RdrBCG demonstrate that drug-resistant BCG strains maintain their safety profile while providing enhanced therapeutic benefits when administered alongside conventional TB drug regimens .

What novel approaches might enhance our understanding of frdD's role in M. tuberculosis persistence and reactivation?

Advancing our understanding of frdD's role in M. tuberculosis persistence and reactivation requires innovative experimental approaches that integrate cutting-edge technologies:

• Single-cell analysis technologies:

  • Single-cell RNA sequencing to identify heterogeneity in frdD expression within bacterial populations during different infection phases

  • Time-lapse microscopy approaches like ODELAM to directly observe frdD-dependent growth patterns at the individual cell level

  • Microfluidic devices to monitor real-time metabolic shifts in response to changing oxygen conditions

• Advanced genetic manipulation strategies:

  • CRISPR interference (CRISPRi) for tunable, reversible repression of frdD to study dosage effects on persistence

  • Conditional knockdown systems to deplete frdD during specific infection stages

  • Site-specific mutagenesis of key residues to create partial loss-of-function variants

• In vivo imaging technologies:

  • Development of fluorescent or bioluminescent reporters fused to the frd operon promoter to monitor activation during infection

  • PET-CT imaging with radiolabeled tracers targeting fumarate reductase activity in animal models

  • Intravital microscopy to visualize frdD-dependent bacterial behavior within granulomas

• Multi-omics integration approaches:

  • Combining transcriptomics, proteomics, and metabolomics data to build comprehensive models of how frdD contributes to metabolic networks during persistence

  • Flux balance analysis incorporating frdD activity under varying oxygen tensions

  • Machine learning algorithms to predict critical conditions for frdD-dependent bacterial survival

• Organoid and advanced 3D culture systems:

  • Human lung organoids infected with M. tuberculosis to study frdD regulation in physiologically relevant oxygen gradients

  • Bioprinted granuloma models incorporating macrophages, fibroblasts, and bacteria to recreate microenvironments where frdD becomes essential

These approaches would provide unprecedented insights into how frdD contributes to the bacterium's remarkable ability to persist in host tissues and could identify critical vulnerability points for therapeutic targeting .

How might systems biology approaches enhance our understanding of frdD's role in the broader metabolic network of M. tuberculosis?

Systems biology approaches offer powerful frameworks for elucidating frdD's role within M. tuberculosis' intricate metabolic landscape:

• Genome-scale metabolic modeling:

  • Integration of frdD and the entire fumarate reductase complex into constraint-based metabolic models of M. tuberculosis

  • Flux balance analysis under varying oxygen conditions to predict metabolic rewiring when frdD is active versus inactive

  • Identification of synthetic lethal interactions that could reveal new combination drug targets

• Multi-omics data integration:

  • Correlation of transcriptomics, proteomics, and metabolomics data across infection stages to identify co-regulated networks

  • Temporal profiling of metabolite pools before, during, and after frdD activation

  • Analysis of metabolic pathway usage during transitions between aerobic growth and hypoxic persistence

• Regulatory network reconstruction:

  • ChIP-seq to identify transcription factors regulating frdD expression

  • Transcriptional reporter assays to map the complete regulatory logic controlling frdD

  • Protein-protein interaction mapping to identify post-translational regulation of fumarate reductase activity

• In silico perturbation analysis:

  • Computational prediction of metabolic vulnerabilities in frdD-dependent pathways

  • Simulation of metabolic responses to potential inhibitors

  • Identification of critical nodes that, when perturbed alongside frdD, could collapse bacterial metabolism

• Integrative visualization tools:

  • Development of interactive metabolic maps highlighting frdD-dependent fluxes

  • Temporal visualization of metabolic shifts during infection progression

  • Multi-scale modeling connecting molecular events to cellular phenotypes

This systems-level understanding would provide a comprehensive view of how frdD contributes to M. tuberculosis' metabolic flexibility, potentially revealing non-obvious intervention points that could disrupt bacterial adaptation to changing host environments .

What emerging technologies might revolutionize the structural characterization of membrane-bound proteins like frdD?

Emerging technologies are poised to transform our understanding of challenging membrane proteins like frdD through revolutionary structural characterization approaches:

These technologies, particularly when used in combination, promise to overcome the historical challenges of membrane protein structural biology, providing unprecedented insights into frdD's structure, dynamics, and interactions within the fumarate reductase complex. Such detailed structural information would significantly accelerate structure-based drug design efforts targeting this important enzyme complex .