Recombinant Bartonella henselae NADH-quinone oxidoreductase subunit C (nuoC)

What is Bartonella henselae NADH-quinone oxidoreductase subunit C (nuoC)?

Bartonella henselae NADH-quinone oxidoreductase subunit C (nuoC) is a protein component of the bacterial respiratory chain complex I. This enzyme (EC 1.6.99.5) plays a critical role in the electron transport chain of B. henselae, contributing to energy metabolism through NADH oxidation and electron transfer to quinones. As part of the NADH dehydrogenase I complex, nuoC is essential for bacterial survival and may represent a potential target for antimicrobial development . The protein is structurally related to similar subunits found in other bacterial species, including the closely related Bartonella bacilliformis .

What is the biological significance of studying B. henselae nuoC?

Studying B. henselae nuoC provides important insights into bacterial metabolism and potential virulence mechanisms. As B. henselae is the causative agent of cat scratch disease and serious conditions like endocarditis and bacillary angiomatosis, understanding its core metabolic machinery is crucial for developing targeted therapies . The NADH-quinone oxidoreductase complex represents a fundamental component of bacterial energy production, making it relevant to research on bacterial survival under varying host conditions. Recent research indicates that B. henselae undergoes host-specific adaptations during its life cycle, suggesting that metabolic enzymes like nuoC may play roles in this adaptive process .

How is recombinant B. henselae nuoC typically produced for research applications?

Recombinant B. henselae nuoC is typically produced using expression systems in laboratory hosts such as E. coli, yeast, baculovirus, or mammalian cells . The production process generally follows these methodological steps:

• Gene isolation from B. henselae genomic DNA using PCR amplification

• Cloning of the nuoC gene into an appropriate expression vector

• Transformation of the construct into the chosen expression system

• Induction of protein expression under controlled conditions

• Protein purification using affinity chromatography (often His-tag based)

• Quality control assessment including purity verification (>90% purity standard) and functional testing

The resulting purified protein is typically stored in a liquid buffer containing glycerol at -20°C or -80°C for long-term storage, with working aliquots maintained at 4°C for up to one week .

What are the recommended protocols for optimizing expression of recombinant B. henselae nuoC?

Optimizing expression of recombinant B. henselae nuoC requires systematic adjustment of several parameters:

For researchers working with B. henselae proteins, establishing protein functionality post-purification is essential, as purification methods may impact the native conformation and activity of respiratory chain components . Expression system selection should consider that membrane proteins like nuoC may require specialized approaches compared to soluble proteins.

How can researchers effectively validate the structural integrity and functionality of purified recombinant B. henselae nuoC?

A comprehensive validation approach for recombinant B. henselae nuoC should include:

• Structural Integrity Assessment:

  • SDS-PAGE and Western blotting to confirm molecular weight and immunoreactivity

  • Circular dichroism spectroscopy to evaluate secondary structure elements

  • Limited proteolysis to assess proper protein folding

  • Size exclusion chromatography to evaluate oligomeric state

• Functional Validation:

  • NADH oxidation assay measuring reaction kinetics (Km and Vmax values)

  • Electron transfer capacity using artificial electron acceptors

  • Reconstitution experiments with other complex I subunits to assess proper assembly

  • Membrane incorporation studies to evaluate integration into lipid bilayers

• Comparative Analysis:

  • Activity comparison with native enzyme complexes isolated from B. henselae

  • Cross-species comparison with related Bartonella species such as B. bacilliformis

When interpreting results, researchers should consider that nuoC functions as part of a multi-subunit complex, and isolated subunit activity may differ from the activity observed in the intact complex.

What experimental controls are critical when working with recombinant B. henselae nuoC in enzyme activity assays?

When designing enzyme activity assays for recombinant B. henselae nuoC, the following controls are essential:

• Negative Controls:

  • Heat-inactivated enzyme preparation to establish baseline activity

  • Buffer-only samples to account for spontaneous substrate oxidation

  • Preparations from expression systems transformed with empty vectors

• Positive Controls:

  • Commercial NADH dehydrogenase preparations when available

  • Well-characterized homologous proteins from related species

• Specificity Controls:

  • Substrate specificity testing with NADH analogs

  • Inhibitor panels including rotenone and piericidin A

  • pH and temperature gradient tests to establish optimal conditions

• System Validation Controls:

  • Reconstitution with membrane fractions to assess integration

  • Electron acceptor variation to confirm proper electron transport chain function

Researchers should establish standard curves for each assay and ensure measurements fall within the linear range of detection to accurately quantify enzymatic activity.

How can recombinant B. henselae nuoC be utilized in studies of host-pathogen interactions?

Recombinant B. henselae nuoC offers several methodological approaches for investigating host-pathogen interactions:

• Immunological Studies:

  • Development of specific antibodies for immunolocalization studies

  • Screening of patient sera to evaluate host immune responses during infection

  • T-cell epitope mapping to understand cellular immunity

• Metabolic Adaptation Research:

  • Comparison of nuoC expression and activity under different host conditions

  • Investigation of metabolic shifts during transition between hosts (feline to human)

  • Analysis of nuoC mutations in laboratory-adapted versus clinical isolates

• Interaction Studies:

  • Identification of potential host cell proteins that interact with bacterial respiratory complexes

  • Investigation of how host metabolic states affect bacterial energy production

  • Evaluation of nuoC role in bacterial survival within professional phagocytes

This research is particularly relevant given that B. henselae demonstrates genetic adaptation during its life cycle with alternating host conditions, which may involve modifications to core metabolic machinery including respiratory chain components .

What are the current challenges and limitations in structural studies of B. henselae nuoC?

Researchers face several methodological challenges when conducting structural studies of B. henselae nuoC:

• Membrane Protein Crystallization Barriers:

  • Inherent difficulty in crystallizing membrane-associated proteins

  • Challenges in maintaining native conformation during purification

  • Need for appropriate detergents and lipid environments

• Complex Formation Considerations:

  • nuoC functions as part of a multi-subunit complex, complicating structural analysis

  • Potential for artificial conformations when studied in isolation

  • Challenges in reconstituting functional complexes for structural studies

• Technical Limitations:

  • Limited availability of high-resolution structures of homologous proteins

  • Challenges in producing sufficient quantities of properly folded protein

  • Difficulty in obtaining homogeneous protein preparations

Recent advances in cryo-electron microscopy and long-read sequencing techniques offer promising approaches to overcome some of these limitations. For instance, long-read sequencing has already revealed genetic adaptations in B. henselae that might extend to respiratory complex components .

How can researchers leverage comparative genomics to enhance understanding of B. henselae nuoC function?

Comparative genomics approaches provide powerful tools for elucidating nuoC function:

• Cross-Species Comparison Protocol:

  • Sequence alignment of nuoC across Bartonella species (e.g., B. henselae vs. B. bacilliformis)

  • Identification of conserved domains suggesting functional importance

  • Analysis of selection pressure on different protein regions

  • Structural modeling based on homologous proteins with known structures

• Evolutionary Analysis Methods:

  • Phylogenetic tree construction of nuoC across alpha-proteobacteria

  • Identification of horizontal gene transfer events

  • Examination of co-evolution patterns with other respiratory complex components

• Host Adaptation Analysis:

  • Comparison of nuoC sequences from human, feline, and laboratory-adapted isolates

  • Investigation of single nucleotide polymorphisms in clinical isolates

  • Assessment of expression patterns across different host conditions

This comparative approach has already yielded insights into B. henselae adaptation mechanisms, as demonstrated by recent long-read sequencing studies revealing genomic differences between human, feline, and laboratory-adapted B. henselae isolates .

What molecular detection methods are most effective for studying native and recombinant B. henselae nuoC expression?

Several molecular detection methods can be effectively employed to study nuoC expression:

• PCR-Based Methods:

  • Quantitative real-time PCR for gene expression analysis

  • Bispecific targeted PCR assays similar to those used for Bartonella species detection

  • Reverse transcription PCR to evaluate mRNA levels under different conditions

• Sequencing Approaches:

  • Next-generation sequencing for transcriptome analysis

  • Long-read sequencing for identifying genomic context and potential mutations

  • 16S rRNA amplicon sequencing for species confirmation in mixed samples

• Protein Detection Methods:

  • Western blotting with specific antibodies

  • Mass spectrometry for proteomic analysis and post-translational modifications

  • ELISA-based quantification methods

When interpreting results, researchers should consider that different detection methods may yield varying results based on sensitivity and specificity. For instance, studies of Bartonella species have shown that PCR targeting specific genes can provide more definitive species-level identification than 16S rRNA sequencing alone .

How should researchers interpret discrepancies in experimental results when working with recombinant versus native B. henselae nuoC?

When encountering discrepancies between recombinant and native nuoC results, researchers should systematically evaluate:

• Structural Differences:

  • Presence of fusion tags in recombinant proteins

  • Post-translational modifications present in native but absent in recombinant protein

  • Conformational variations due to isolation from natural complex

• Functional Context:

  • Native nuoC functions within a multi-subunit complex in bacterial membranes

  • Recombinant proteins may lack proper interaction partners

  • Different lipid environments between recombinant and native systems

• Experimental Variables:

  • Different buffer conditions between assays

  • Variations in protein concentration and purity

  • Potential contamination with host proteins in recombinant preparations

• Methodological Approach to Resolve Discrepancies:

  • Reconstitution experiments with whole complex components

  • Site-directed mutagenesis to identify critical residues

  • Comparison across multiple expression systems

Researchers should document all experimental conditions comprehensively to facilitate accurate interpretation of discrepancies and consider that the true biological function might be best understood through complementary approaches using both recombinant and native protein systems.

What bioinformatic approaches are recommended for analyzing the structure-function relationship of B. henselae nuoC?

For comprehensive structure-function analysis of B. henselae nuoC, researchers should employ these bioinformatic approaches:

• Sequence Analysis Workflow:

  • Multiple sequence alignment with homologous proteins

  • Identification of conserved domains and motifs

  • Analysis of potentially critical residues for electron transport

• Structural Prediction Methods:

  • Homology modeling based on related proteins with known structures

  • Ab initio modeling for regions lacking homologous templates

  • Molecular dynamics simulations to evaluate conformational flexibility

  • Protein-protein docking with other respiratory complex components

• Functional Prediction Approaches:

  • Ligand binding site prediction

  • Electrostatic surface analysis

  • Prediction of protein-protein interaction interfaces

  • Conservation analysis across bacterial species

• Integration with Experimental Data:

  • Mapping of biochemical data onto structural models

  • Correlation of genetic variants with functional outcomes

  • Prediction of the impact of site-directed mutations

These approaches should be implemented with appropriate validation strategies, including experimental verification of key predictions whenever possible.

How might B. henselae nuoC research contribute to novel antimicrobial development strategies?

The study of B. henselae nuoC offers several promising avenues for antimicrobial development:

• Target-Based Drug Design:

  • Identification of nuoC-specific inhibitors that don't affect human mitochondrial complexes

  • Structure-based virtual screening against predicted binding pockets

  • Fragment-based drug discovery targeting critical functional domains

• Metabolic Vulnerability Exploitation:

  • Development of compounds that disrupt bacterial energy production

  • Identification of synergistic drug combinations targeting different aspects of bacterial metabolism

  • Design of prodrugs activated by bacterial respiratory chain components

• Innovative Therapeutic Approaches:

  • Creation of immunogenic conjugates for vaccine development

  • Design of nanoparticle-delivered inhibitors with specificity for bacterial respiratory complexes

  • CRISPR-based antimicrobials targeting nuoC genes

Recent funding initiatives, such as the $4.8 million grant for developing treatments for Bartonella-related diseases, demonstrate the clinical importance of this research direction . The emerging understanding of B. henselae's metabolic adaptations during host infection suggests that targeting respiratory chain components could be particularly effective against this pathogen .

What are the implications of host adaptation studies for understanding B. henselae nuoC function?

Research on host adaptation provides critical insights into nuoC function:

• Metabolic Flexibility Analysis:

  • Investigation of how nuoC activity changes during transition between hosts

  • Examination of expression level variations between feline and human isolates

  • Analysis of potential post-translational modifications in different host environments

• Evolutionary Considerations:

  • Assessment of selection pressure on nuoC during host specialization

  • Identification of host-specific mutations that alter enzyme efficiency

  • Comparison of regulatory mechanisms across different host-adapted strains

• Functional Consequences:

  • Evaluation of how host adaptation affects electron transport chain efficiency

  • Investigation of potential alternate electron acceptors in different host niches

  • Examination of metabolic network adaptations involving respiratory complexes

Recent long-read sequencing studies have revealed that human, feline, and laboratory-adapted B. henselae isolates display genomic and phenotypic differences . These findings suggest that core metabolic machinery, including respiratory chain components like nuoC, may undergo adaptive changes to optimize bacterial survival in different host environments.

How can systems biology approaches enhance our understanding of B. henselae nuoC in the context of whole-cell metabolism?

Systems biology offers powerful frameworks for contextualizing nuoC function:

• Metabolic Network Modeling:

  • Integration of nuoC into genome-scale metabolic models of B. henselae

  • Flux balance analysis to predict metabolic changes when nuoC function is altered

  • Identification of synthetic lethal interactions with other metabolic genes

• Multi-omics Integration Strategies:

  • Correlation of nuoC expression with global transcriptomic changes

  • Proteomic analysis of respiratory complex assembly and regulation

  • Metabolomic profiling to identify shifts in energy metabolism

• Host-Pathogen Interaction Modeling:

  • Simulation of metabolic interactions between host and pathogen

  • Prediction of critical nodes in host-pathogen metabolic networks

  • Identification of emergent properties in complex host-pathogen systems

This systems-level understanding is particularly relevant given the complex lifecycle of B. henselae, which involves adaptation to different host environments and potentially different metabolic states within each host . The approach may reveal unexpected connections between respiratory chain function and virulence mechanisms.

What are the best practices for designing experiments to study potential post-translational modifications of B. henselae nuoC?

Comprehensive investigation of post-translational modifications (PTMs) in B. henselae nuoC requires:

• Analytical Workflow:

  • Enrichment strategies for different PTM types (phosphorylation, acetylation, etc.)

  • High-resolution mass spectrometry with multiple fragmentation techniques

  • Targeted analysis of modification sites based on predictive algorithms

  • Comparison of PTM patterns across different growth conditions

• Validation Methods:

  • Generation of modification-specific antibodies

  • Site-directed mutagenesis of predicted modification sites

  • In vitro modification assays with purified enzymes

  • Functional assessment of modified versus unmodified protein

• Physiological Relevance Assessment:

  • Correlation of modifications with bacterial growth phase

  • Analysis of modification changes during host cell infection

  • Comparison of modification patterns between different Bartonella species

Researchers should consider that recombinant expression systems may not reproduce native bacterial PTM patterns, necessitating comparative studies between recombinant and native proteins.

How can researchers effectively incorporate B. henselae nuoC into in vitro reconstitution studies of the complete NADH dehydrogenase complex?

Successful reconstitution of NADH dehydrogenase complexes incorporating B. henselae nuoC requires:

• Component Preparation Strategy:

  • Expression and purification of all essential subunits with compatible tags

  • Careful selection of detergents and lipids for membrane protein stabilization

  • Stepwise assembly protocol based on known complex architecture

  • Quality control at each assembly stage

• Reconstitution Protocol:

  • Controlled removal of detergents using dialysis or adsorption methods

  • Incorporation into liposomes or nanodiscs for membrane environment mimicry

  • Optimization of protein-to-lipid ratios for optimal complex formation

  • Verification of proper assembly using analytical ultracentrifugation or negative-stain electron microscopy

• Functional Verification Methods:

  • NADH oxidation activity measurements with various electron acceptors

  • Proton pumping assays using pH-sensitive fluorescent dyes

  • Electron paramagnetic resonance spectroscopy to monitor iron-sulfur clusters

  • Inhibitor sensitivity profiling compared to native complexes

This approach enables detailed structure-function studies that cannot be performed with isolated subunits and provides a platform for investigating the impact of mutations or modifications on complex assembly and function.

What considerations are important when designing antibodies against B. henselae nuoC for research applications?

Effective antibody development for B. henselae nuoC research requires:

• Epitope Selection Strategy:

  • Bioinformatic analysis to identify surface-exposed, unique regions

  • Consideration of sequence conservation across Bartonella species

  • Avoidance of regions involved in complex formation

  • Selection of multiple epitopes for polyclonal development

• Production Considerations:

  • Choice between monoclonal and polyclonal approaches based on application

  • Selection of appropriate host species for immunization

  • Purification methods that preserve antibody functionality

  • Validation against both recombinant and native protein sources

• Validation Requirements:

  • Specificity testing against related proteins from Bartonella and other bacteria

  • Cross-reactivity assessment with host tissues when applicable

  • Functionality verification in multiple applications (Western blot, immunoprecipitation, immunofluorescence)

  • Lot-to-lot consistency evaluation for reproducible results

• Application-Specific Optimization:

  • Determination of optimal working dilutions for each application

  • Buffer compatibility assessment

  • Storage conditions for maximum stability

  • Conjugation protocols for specialized applications

Properly validated antibodies against B. henselae nuoC can serve as valuable tools for studying protein expression, localization, and interactions in both in vitro and ex vivo systems.