Recombinant Aeromonas hydrophila subsp. hydrophila NADH-quinone oxidoreductase subunit A (nuoA)

What is the genomic organization of NADH-quinone oxidoreductase subunit A (nuoA) in Aeromonas hydrophila?

NADH-quinone oxidoreductase subunit A (nuoA) in Aeromonas hydrophila is part of the nuo operon, which encodes the multisubunit NADH:ubiquinone oxidoreductase (Complex I) of the respiratory chain. The nuoA gene is typically located at the beginning of the nuo operon, followed by other subunit genes (nuoB through nuoN). This respiratory complex plays a crucial role in energy metabolism as highlighted by studies showing that inhibition of respiratory enzymes including NADH:ubiquinone oxidoreductase (complex I) is a secondary mechanism of action for antibiotics like colistin . In A. hydrophila, the nuoA gene typically spans approximately 300-400 base pairs, encoding a hydrophobic protein that forms part of the membrane domain of Complex I.

How does NADH-quinone oxidoreductase function in Aeromonas hydrophila metabolism?

NADH-quinone oxidoreductase (Complex I) functions as the primary entry point for electrons into the respiratory chain of A. hydrophila. The complex catalyzes the transfer of electrons from NADH to ubiquinone coupled with proton translocation across the inner membrane, contributing to the proton motive force needed for ATP synthesis. Research indicates that respiratory enzymes, including NADH:ubiquinone oxidoreductase, are targets of antimicrobial agents like colistin . The nuoA subunit specifically contributes to the membrane domain structure that facilitates proton pumping. The complex's activity directly impacts cellular bioenergetics, with inhibition leading to reduced ATP production and potentially cell death. Impaired function of this complex has been associated with decreased virulence and growth rates in several bacterial pathogens.

What methods are used to purify recombinant NADH-quinone oxidoreductase subunit A from Aeromonas hydrophila?

Purification of recombinant nuoA from A. hydrophila typically follows this methodological approach:

• Cloning of the nuoA gene into an expression vector with an affinity tag (His-tag is commonly used)

• Expression in a suitable host system (often E. coli BL21(DE3) or similar strains)

• Cell disruption via sonication or French press in buffer containing detergents appropriate for membrane proteins

• Solubilization using mild detergents (n-dodecyl β-D-maltoside or CHAPS)

• Purification via affinity chromatography (Ni-NTA for His-tagged constructs)

• Size exclusion chromatography for further purification

• Verification of purity via SDS-PAGE and Western blotting

For functional studies, careful consideration of detergent choice is critical as the hydrophobic nature of nuoA makes it challenging to maintain in a properly folded state. Unlike detection methods developed for A. hydrophila virulence factors that employ nucleic acid amplification techniques like recombinase-assisted amplification (RAA) assays , protein purification requires maintaining structural integrity through specialized buffer conditions.

How do mutations in NADH-quinone oxidoreductase subunit A impact antibiotic resistance in Aeromonas hydrophila?

Mutations in nuoA can significantly alter antibiotic susceptibility in A. hydrophila through multiple mechanisms. Research has demonstrated that respiratory chain components, including NADH:ubiquinone oxidoreductase (Complex I), are secondary targets for antibiotics like colistin . Alterations in nuoA can affect:

• Membrane potential and proton gradient – influencing uptake of cationic antimicrobials

• Redox balance – affecting oxidative stress responses

• Energy production – impacting efflux pump activity

In A. hydrophila, studies have shown that inhibition of vital respiratory enzymes represents a secondary antibacterial mechanism of colistin . While the hypothetical protein gene1038 has been directly connected to colistin resistance by reducing antibiotic function in the inner membrane , alterations in nuoA could similarly affect the interaction of antibiotics with their membrane targets.

The experimental approach to studying such mutations typically involves:

• Site-directed mutagenesis of conserved regions in nuoA

• Expression of mutant proteins in A. hydrophila

• Assessment of minimum inhibitory concentrations (MICs) for various antibiotics

• Measurement of membrane potential and respiratory rates

• Proteomic analysis to identify compensatory changes

What expression systems yield optimal production of functional recombinant NADH-quinone oxidoreductase subunit A?

The optimization of expression systems for recombinant nuoA production requires addressing several technical challenges inherent to membrane proteins:

For optimal expression, these methodological considerations are critical:

• Temperature reduction (16-20°C) during induction to slow protein synthesis

• Use of weak promoters to prevent overwhelming the membrane insertion machinery

• Co-expression with chaperones (GroEL/GroES)

• Addition of specific detergents to culture media

• Fusion with solubility-enhancing tags (MBP, thioredoxin)

Unlike nucleic acid-based detection methods for A. hydrophila that can achieve rapid results (approximately 45 minutes) , protein expression optimization is an iterative process requiring multiple experimental cycles to achieve acceptable yields of functional protein.

How can structural analysis of NADH-quinone oxidoreductase subunit A inform antimicrobial development?

Structural analysis of nuoA provides valuable insights for developing targeted antimicrobials through the following methodological approaches:

• Cryo-EM Analysis: Determination of the complete complex structure in native lipid environments at resolutions of 2.5-3.5 Å, revealing:

  • Interaction interfaces between nuoA and other subunits

  • Conformational changes during catalytic cycle

  • Potential inhibitor binding pockets

• Molecular Dynamics Simulations: Computational analysis of:

  • Proton translocation pathways through the membrane domain

  • Lipid-protein interactions specific to A. hydrophila

  • Conformational flexibility under different conditions

• Structure-Based Drug Design: Using structural data to:

  • Identify unique structural features in A. hydrophila nuoA versus human homologs

  • Design small molecules that selectively bind to A. hydrophila complex I

  • Develop peptide inhibitors targeting the assembly interface

This approach differs from methods targeting pathogenicity factors like aerolysin (aerA) and hemolysin (hlyA) , as it focuses on essential metabolic machinery rather than virulence factors. While virulence-based detection methods can identify pathogenic A. hydrophila strains with high sensitivity and specificity , structural approaches to respiratory chain components provide opportunities for broad-spectrum antimicrobial development targeting energy metabolism.

What protocols are most effective for measuring NADH-quinone oxidoreductase activity in Aeromonas hydrophila?

Effective measurement of NADH-quinone oxidoreductase activity in A. hydrophila can be accomplished through several complementary approaches:

• Spectrophotometric Assays:

  • NADH oxidation measured at 340 nm (ε = 6.22 mM⁻¹cm⁻¹)

  • DCPIP reduction monitored at 600 nm in the presence of NADH

  • Inhibition profiles using rotenone or piericidin A

• Oxygen Consumption Measurements:

  • Clark-type electrode systems measuring oxygen uptake rates

  • High-resolution respirometry for detailed kinetic analysis

  • Substrate-dependent respiration with NADH, malate, or succinate

• Membrane Potential Monitoring:

  • Fluorescent probes (DiSC3(5), TMRM) to quantify Δψ

  • Calibration with ionophores (valinomycin, CCCP)

  • Real-time monitoring during antibiotic exposure

The methodological approach should include careful preparation of inverted membrane vesicles or proteoliposomes to maintain native-like activity of the enzyme complex. Unlike virulence gene detection methods that can be performed in simple reaction tubes at 37°C , enzyme activity assays require strict control of temperature, pH, and ionic conditions, with appropriate negative controls using specific inhibitors.

How can CRISPR-Cas systems be optimized for studying NADH-quinone oxidoreductase function in Aeromonas hydrophila?

CRISPR-Cas systems provide powerful tools for studying nuoA function through precise genetic manipulation. The optimization process involves:

• sgRNA Design and Validation:

  • In silico prediction of optimal target sites within nuoA

  • Testing multiple sgRNAs to identify highest efficiency

  • Minimizing off-target effects through careful sequence selection

• Delivery System Selection:

  • Plasmid-based expression for stable editing

  • Ribonucleoprotein (RNP) delivery for transient modifications

  • Electroporation protocols optimized for A. hydrophila

• Modification Strategies:

  • Gene knockout through NHEJ repair

  • Point mutations via homology-directed repair

  • CRISPRi for tunable gene repression

  • Base editing for specific nucleotide changes without double-strand breaks

While CRISPR-Cas12a has been successfully employed for the detection of pathogenic A. hydrophila through nucleic acid amplification and trans-cleavage activity , its application for genetic engineering requires different optimization parameters. The dRAA-CRISPR/Cas12a detection method targeting virulence genes has demonstrated high sensitivity (as low as 2 copies of genomic DNA per reaction) , but genetic modification applications require careful consideration of transformation efficiency and editing precision.

How do modifications in NADH-quinone oxidoreductase subunit A correlate with Aeromonas hydrophila virulence in animal models?

The correlation between nuoA modifications and A. hydrophila virulence has been investigated through various animal models with the following methodological approaches:

• Fish Challenge Models:

  • Wild-type versus nuoA mutant strains administered via injection or immersion

  • Monitoring survival rates, bacterial load, and tissue damage

  • Comparative transcriptomics of host immune responses

• Mouse Infection Models:

  • Intraperitoneal or oral administration of defined bacterial doses

  • Measurement of LD50 values for different strains

  • Histopathological examination of affected tissues

  • Cytokine profiling to assess inflammatory responses

Research findings indicate that disruption of respiratory function through nuoA modification often results in attenuated virulence due to:

• Reduced growth rates in vivo

• Decreased expression of type III secretion systems

• Altered biofilm formation capabilities

• Increased susceptibility to host defense mechanisms

Unlike virulence factors such as aerolysin and hemolysin that directly damage host cells , nuoA affects pathogenicity indirectly by influencing bacterial fitness and energy production. Studies investigating A. hydrophila virulence have identified multiple virulence factors playing key roles through cooperation or independently , suggesting that energy metabolism through respiratory complexes provides the necessary support for virulence factor expression and function.

What biomarkers can identify changes in NADH-quinone oxidoreductase activity during Aeromonas hydrophila infection?

Monitoring changes in NADH-quinone oxidoreductase activity during infection requires sensitive biomarkers that can be detected in complex biological samples:

• Metabolomic Markers:

  • NADH/NAD+ ratio in tissue samples

  • Lactate accumulation indicating shifted metabolism

  • TCA cycle intermediates reflecting respiratory chain dysfunction

• Proteomic Signatures:

  • Altered expression of compensatory energy production pathways

  • Post-translational modifications of respiratory complex proteins

  • Stress response proteins upregulated during respiratory inhibition

• Transcriptomic Indicators:

  • Differential expression of nuo operon genes

  • Changes in alternative respiratory pathways

  • Stress response gene networks activation

• Direct Enzyme Activity Measurements:

  • Tissue biopsies analyzed for complex I activity

  • Circulating bacterial membrane vesicles containing respiratory complexes

  • Isotope tracing to monitor respiratory flux

While detection methods for pathogenic A. hydrophila typically focus on virulence genes like aerA and hlyA , monitoring respiratory function provides insight into metabolic adaptation during infection. This approach complements virulence-based detection by assessing the functional state of the pathogen rather than merely its identity or virulence potential.

How might targeting NADH-quinone oxidoreductase subunit A contribute to novel antimicrobial strategies against multidrug-resistant Aeromonas hydrophila?

Targeting nuoA represents a promising avenue for antimicrobial development against multidrug-resistant A. hydrophila through several innovative approaches:

• Structure-Based Inhibitor Design:

  • Development of small molecules specifically binding to A. hydrophila nuoA

  • Peptide inhibitors disrupting complex assembly

  • Allosteric modulators affecting conformational changes during catalytic cycle

• Combination Therapies:

  • Respiratory chain inhibitors combined with existing antibiotics

  • Synergistic effects with membrane-targeting antimicrobials

  • Metabolic sensitization to enhance antibiotic efficacy

• Antimicrobial Peptides Targeting Complex I:

  • Designed peptides that interact with membrane-embedded regions

  • Cell-penetrating peptides carrying complex I inhibitors

  • Disruption of protein-protein interactions within the complex

Research on colistin resistance in A. hydrophila has shown that vital respiratory enzymes, including NADH:ubiquinone oxidoreductase, are secondary targets of this antibiotic . This suggests that direct targeting of respiratory complexes could circumvent resistance mechanisms that protect primary antibiotic targets. While the hypothetical protein gene1038 contributes to colistin resistance by reducing antibiotic function in the inner membrane , nuoA inhibitors could directly compromise energy production regardless of membrane modifications.

What computational approaches can predict interactions between NADH-quinone oxidoreductase subunit A and potential inhibitors?

Advanced computational approaches for predicting nuoA-inhibitor interactions include:

• Molecular Docking:

  • Virtual screening of compound libraries against nuoA binding sites

  • Flexible docking to account for protein dynamics

  • Consensus scoring across multiple algorithms to improve prediction accuracy

• Molecular Dynamics Simulations:

  • Binding free energy calculations using methods such as MM/PBSA

  • Analysis of residence time and dissociation pathways

  • Investigation of water networks and their role in ligand binding

• Machine Learning Models:

  • Development of predictive models trained on known respiratory chain inhibitors

  • Feature extraction from successful inhibitors of complex I

  • Deep learning approaches incorporating protein sequence and structural information

• Quantum Mechanical Calculations:

  • QM/MM methods to investigate electronic properties of binding interactions

  • Understanding proton transfer mechanisms affected by inhibitors

  • Reaction coordinate analysis for enzyme-inhibitor complexes

These computational approaches can significantly accelerate the identification of potential nuoA inhibitors before experimental validation. Unlike detection methods that utilize optimized RAA conditions and specific crRNA design for identifying A. hydrophila , computational drug discovery requires extensive validation through in vitro and in vivo testing to confirm predicted binding modes and antimicrobial efficacy.