Recombinant Aeromonas salmonicida Probable ubiquinone biosynthesis protein UbiB (ubiB)

What is Aeromonas salmonicida UbiB protein and what is its function in bacterial metabolism?

Aeromonas salmonicida UbiB (UniProt accession: A4STK9) is a probable ubiquinone biosynthesis protein involved in electron transport chain functionality. As its name suggests, it plays a role in the biosynthetic pathway of ubiquinone (coenzyme Q), which is essential for aerobic cellular respiration. In A. salmonicida, UbiB functions within the ubiquinone biosynthetic pathway, potentially catalyzing reactions necessary for the production of this critical electron carrier. This protein is part of the broader metabolic network that allows A. salmonicida to generate energy through respiratory processes .

How does A. salmonicida UbiB relate to bacterial pathogenicity?

While UbiB is primarily involved in ubiquinone biosynthesis rather than direct virulence, its role in energy metabolism indirectly contributes to pathogenicity. A. salmonicida is a significant fish pathogen causing furunculosis, a disease characterized by sepsis, hemorrhages, muscle lesions, intestinal inflammation, and spleen enlargement in freshwater fish, particularly salmonids .

Metabolic proteins like UbiB enable bacterial survival and persistence in host environments by ensuring efficient energy production. Disruption of energy metabolism through targeting proteins like UbiB could potentially attenuate bacterial virulence by limiting the pathogen's ability to proliferate during infection. Recent genomic studies of re-emergent A. salmonicida outbreaks have highlighted the importance of metabolic factors in adaptation to different environments and hosts .

Recombinant Protein Production and Characterization

Standard recombinant UbiB protein purification typically achieves >85% purity as determined by SDS-PAGE . A methodological approach to high-purity UbiB isolation involves:

• Initial Capture: Affinity chromatography using tag-specific matrices (depending on the tag used during expression)

• Intermediate Purification: Ion exchange chromatography to separate based on charge differences

• Polishing: Size exclusion chromatography to achieve final purity

The tag type varies between different commercial preparations and is determined during the production process. For research requiring exceptionally high purity (>95%), additional steps may be necessary:

• Hydrophobic interaction chromatography

• Multimodal chromatography

• Optimized buffer conditions with stabilizing agents

Quality control via SDS-PAGE, Western blotting, and mass spectrometry confirms purity and identity of the final product .

What is the optimal storage protocol for maintaining UbiB stability and activity?

Based on manufacturer recommendations, the optimal storage protocol for recombinant UbiB involves:

For lyophilized protein:

• Store at -20°C to -80°C

• Shelf life is approximately 12 months under these conditions

For reconstituted protein:

• Briefly centrifuge vial prior to opening

• Reconstitute to 0.1-1.0 mg/mL using deionized sterile water

• Add glycerol to a final concentration of 5-50% (50% is recommended)

• Aliquot to minimize freeze-thaw cycles

• Store at -20°C to -80°C for long-term storage (6 months)

• Working aliquots can be stored at 4°C for up to one week

Critical considerations:

• Avoid repeated freeze-thaw cycles as this significantly reduces protein activity

• Buffer composition (typically Tris/PBS-based) should be optimized for specific application requirements

• For specific functional assays, the addition of stabilizing agents like DTT or specific cofactors may be necessary .

What experimental techniques are most effective for studying UbiB function?

Several experimental approaches have proven effective for investigating UbiB function:

• Enzymatic Activity Assays:

  • Monitoring ubiquinone biosynthesis intermediates via HPLC or LC-MS

  • Coupled enzyme assays measuring electron transfer activities

  • Oxygen consumption measurements in reconstituted systems

• Structural Analysis:

  • X-ray crystallography for high-resolution structure determination

  • Cryo-EM for visualization of protein complexes

  • NMR for dynamics and ligand binding studies in solution

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation with other components of the ubiquinone biosynthesis pathway

  • Bacterial two-hybrid systems

  • Surface plasmon resonance for binding kinetics

• Genetic Approaches:

  • Gene knockout and complementation studies

  • Site-directed mutagenesis of conserved residues

  • Suppressor analysis to identify functional partners

For initial characterization in research settings, Western blotting and ELISA techniques are commonly employed to confirm protein expression and analyze interactions with other components of metabolic pathways .

How can researchers design effective experiments to investigate UbiB's role in A. salmonicida pathogenesis?

Research into UbiB's role in A. salmonicida pathogenesis requires a multifaceted approach:

Genetic Manipulation Strategy:

• Generate UbiB deletion mutants in A. salmonicida using CRISPR-Cas9 or homologous recombination

• Create complemented strains expressing wild-type and mutant versions of UbiB

• Construct reporter strains with tagged UbiB to track expression during infection

Functional Analysis Pipeline:

• Compare growth kinetics of wild-type, mutant, and complemented strains under various conditions

• Assess biofilm formation capacity and stress resistance

• Measure ubiquinone levels by analytical chemistry methods

• Evaluate membrane potential and respiratory capacity

Virulence Assessment:

• In vitro infection models using fish cell lines

• Ex vivo tissue explant challenge models

• Small-scale in vivo models with appropriate ethical approvals

• Transcriptomic and proteomic analysis of host-pathogen interactions

This comprehensive experimental design allows correlation between UbiB function, bacterial metabolism, and virulence phenotypes. The research should be contextualized within the broader understanding of A. salmonicida pathogenesis, including its known virulence factors and the specific role of metabolic adaptations during infection .

What are the key considerations for developing inhibitors targeting UbiB protein?

Developing inhibitors against UbiB requires systematic consideration of several factors:

Target Validation and Characterization:

• Confirm essentiality of UbiB for A. salmonicida viability through genetic knockdown experiments

• Determine biochemical mechanism and kinetic parameters

• Resolve crystal structure for structure-based drug design

Inhibitor Design Strategy:

• Identify active site or allosteric binding pockets through computational modeling

• Design competitive inhibitors that mimic natural substrates

• Consider mechanism-based inactivators that form covalent bonds with catalytic residues

Selectivity Considerations:

• Compare sequence and structural homology with host (fish) proteins

• Design inhibitors that exploit differences between bacterial and eukaryotic homologs

• Consider potential off-target effects on commensal microbiota

Practical Development Pipeline:

• In silico screening and molecular docking

• Biochemical assays with purified recombinant protein

• Whole-cell activity testing against A. salmonicida

• Cytotoxicity assessment in fish cell lines

• Pharmacokinetic and stability studies in relevant aquatic conditions

The ultimate goal would be developing compounds that specifically disrupt bacterial ubiquinone biosynthesis without affecting host metabolism, potentially providing alternatives to conventional antibiotics for controlling A. salmonicida infections in aquaculture settings .

How does A. salmonicida UbiB compare to UbiB homologs in other bacterial species?

A comparative analysis of UbiB across bacterial species reveals important insights:

These comparisons provide valuable perspectives on evolutionary conservation of ubiquinone biosynthesis pathways. The high conservation suggests essential metabolic functions, while species-specific variations may reflect adaptations to different ecological niches. Researchers can leverage better-characterized homologs (like E. coli UbiB) as models when studying the less-characterized A. salmonicida protein .

What role might UbiB play in antibiotic resistance mechanisms in A. salmonicida?

UbiB's potential involvement in antibiotic resistance represents an important research consideration:

Direct Resistance Mechanisms:

• Alterations in respiratory metabolism can affect uptake of certain antibiotics

• Changes in membrane potential due to altered ubiquinone levels may reduce accumulation of cationic antimicrobials

• Metabolic adaptations involving ubiquinone pathway may enable survival during antibiotic stress

Indirect Contributions to Resistance:

• Energetic support for efflux pump activity

• Involvement in oxidative stress management during antibiotic exposure

• Potential role in formation of persister cells with reduced metabolic activity

Research Approaches to Investigate This Connection:

• Compare UbiB expression levels in antibiotic-resistant vs. sensitive strains

• Assess antibiotic susceptibility in UbiB mutants or overexpression strains

• Evaluate synergistic effects between UbiB inhibitors and conventional antibiotics

• Analyze metabolomic profiles during antibiotic challenge

Given the increasing concern about antibiotic resistance in aquaculture pathogens, understanding the intersection between metabolic functions like UbiB and resistance mechanisms could provide new approaches for combating A. salmonicida infections .

How might structural variations in UbiB between psychrophilic and mesophilic A. salmonicida strains affect function?

Analysis of UbiB sequences from different A. salmonicida strains reveals potential adaptations to different temperature preferences:

Structural Adaptations in Psychrophilic vs. Mesophilic UbiB:

• Psychrophilic strains may exhibit increased flexibility in key catalytic regions

• Higher proportion of hydrophobic residues exposed to solvent in psychrophilic variants

• Reduced electrostatic interactions that might be destabilizing at low temperatures

• Potential differences in cofactor binding regions affecting catalytic efficiency at different temperatures

Functional Implications:

• Different kinetic parameters (Km, kcat) at various temperatures

• Altered thermal stability profiles

• Differential interaction with other components of the respiratory chain

• Varying susceptibility to potential inhibitors

Research Methodologies to Explore These Differences:

• Comparative expression and purification of UbiB from both psychrophilic and mesophilic strains

• Enzyme kinetics studies across temperature ranges

• Thermal stability assays (e.g., differential scanning fluorimetry)

• Site-directed mutagenesis to create chimeric proteins with domains from each variant

Understanding these adaptations could provide insights into A. salmonicida's ability to cause disease in different aquatic environments and host species with varying temperature requirements .

What are the most promising approaches for developing vaccines targeting metabolic proteins like UbiB?

While traditional vaccines typically target surface antigens, metabolic proteins like UbiB present interesting alternative candidates:

Vaccine Development Strategies:

• Recombinant Subunit Vaccines:

  • Purified UbiB protein or immunogenic peptides derived from it

  • Potentially combined with other metabolic antigens to create multi-component vaccines

  • Requires appropriate adjuvants to enhance immunogenicity

• DNA Vaccine Approaches:

  • Plasmid DNA encoding UbiB for in vivo expression

  • Potential for co-expression with immunostimulatory molecules

  • Advantages in stability and manufacturing

• Attenuated Live Vaccines:

  • A. salmonicida strains with modified UbiB or other metabolic genes

  • Balance between attenuation and immunogenicity

  • Safety considerations for release in aquatic environments

Research Challenges to Address:

• Determining if antibodies against UbiB can access the protein in intact bacteria

• Assessing whether anti-UbiB responses provide protective immunity

• Developing delivery systems appropriate for fish vaccination

• Understanding correlates of protection in fish immune responses

Experimental Approaches:

• Immunization studies with recombinant UbiB in fish models

• Challenge experiments to assess protection

• Immunological assays to characterize responses (antibody titers, T-cell responses)

• Comparative trials against conventional vaccine formulations

This represents a novel direction in vaccine development against A. salmonicida, exploring whether targeting metabolic machinery can provide effective protection against furunculosis .

How can systems biology approaches integrate UbiB function into broader metabolic networks of A. salmonicida?

Systems biology offers powerful frameworks for understanding UbiB within the complex metabolic landscape of A. salmonicida:

Integrative Approaches:

• Genome-Scale Metabolic Modeling:

  • Incorporation of UbiB function into stoichiometric models of A. salmonicida metabolism

  • Flux balance analysis to predict effects of UbiB perturbation

  • Identification of synthetic lethal interactions with other metabolic genes

• Multi-Omics Integration:

  • Correlation of transcriptomic, proteomic, and metabolomic data

  • Temporal modeling of metabolic shifts during infection

  • Network analysis to identify regulatory hubs connected to UbiB

• Host-Pathogen Systems Analysis:

  • Modeling metabolic interactions between A. salmonicida and fish hosts

  • Identifying critical nodes in host-pathogen metabolic interfaces

  • Predicting emergent properties of the infection system

Methodological Framework:

• Generate comprehensive datasets across conditions (temperature, oxygen levels, growth phase)

• Develop computational models incorporating UbiB and related pathways

• Experimentally validate model predictions through targeted perturbations

• Refine models iteratively based on new data

This systems-level understanding would place UbiB research in its proper biological context, potentially revealing unexpected connections to virulence, stress response, and host adaptation that might not be apparent from reductionist approaches .

What strategies can address poor expression or solubility of recombinant UbiB protein?

Researchers frequently encounter expression and solubility challenges with membrane-associated proteins like UbiB. Effective troubleshooting approaches include:

Expression Optimization:

• Codon optimization for the expression host

• Promoter selection - test inducible vs. constitutive systems

• Expression temperature modulation - often lower temperatures (16-20°C) improve solubility

• Induction parameters - optimize inducer concentration and induction duration

Solubility Enhancement:

• Fusion partners - MBP, SUMO, or thioredoxin tags can increase solubility

• Domain truncation - express soluble domains separately

• Chaperone co-expression - GroEL/ES, DnaK/J systems

• Detergent screening - identify optimal detergents for membrane-associated regions

Purification Adaptations:

• Denaturing/renaturing protocols - recover protein from inclusion bodies

• Buffer optimization - screen various pH conditions, salt concentrations

• Additives - glycerol, amino acids, or specific cofactors that might stabilize the protein

When standard approaches fail, consider alternative expression systems like cell-free protein synthesis or specialized strains designed for difficult proteins .

How can researchers troubleshoot inconsistent results in UbiB functional assays?

Functional assays for enzymes involved in ubiquinone biosynthesis can be challenging. Systematic troubleshooting includes:

Assay Component Validation:

• Protein quality assessment - verify folding using circular dichroism

• Substrate purity verification - HPLC analysis of substrates and standards

• Buffer component analysis - test for interfering substances

Methodological Refinements:

• Reaction conditions optimization - systematic testing of pH, temperature, ionic strength

• Enzyme concentration titration - determine optimal enzyme:substrate ratios

• Time course studies - establish linear range of reaction

Technical Considerations:

• Detection method validation - confirm linearity and sensitivity

• Control experiments - positive and negative controls, including heat-inactivated enzyme

• Reference standard inclusion - use well-characterized homologous enzymes when available

Data Analysis Approaches:

• Statistical methods for identifying outliers

• Normalization strategies to account for batch-to-batch variation

• Detailed documentation of all variables to identify potential confounding factors

Implementing a quality control checklist for each component of the assay system can help identify sources of variability and improve reproducibility .

What are the best practices for validating antibodies for detecting UbiB in A. salmonicida samples?

Proper antibody validation is critical for reliable immunodetection of UbiB:

Validation Criteria and Methodology:

• Specificity Testing:

  • Western blot against recombinant UbiB protein

  • Comparison of wild-type A. salmonicida vs. UbiB knockout strains

  • Peptide competition assays to confirm epitope specificity

  • Cross-reactivity assessment with related bacterial species

• Sensitivity Determination:

  • Limit of detection using purified protein standards

  • Signal-to-noise ratio optimization

  • Comparison across different sample types (pure protein, lysates, fixed samples)

• Reproducibility Assessment:

  • Intra- and inter-assay coefficient of variation

  • Lot-to-lot consistency testing

  • Performance across different experimental conditions

Application-Specific Validation:

• For Western Blotting:

  • Optimization of extraction methods for membrane-associated proteins

  • Determination of optimal blocking conditions

  • Evaluation of detection systems (chemiluminescence, fluorescence)

• For Immunohistochemistry/Immunofluorescence:

  • Fixation method comparison

  • Antigen retrieval optimization

  • Background reduction strategies

• For ELISA/Immunoprecipitation:

  • Capture efficiency determination

  • Non-specific binding assessment

  • Standard curve validation

Thorough documentation of validation procedures ensures reliable interpretation of results and facilitates troubleshooting when unexpected findings arise .