Recombinant Aeromonas salmonicida Elongation factor Tu (tuf1)

What is Elongation Factor Tu (tuf1) in Aeromonas salmonicida?

Elongation Factor Tu (EF-Tu), encoded by the tuf1 gene in A. salmonicida, is a highly conserved cytoplasmic protein primarily involved in protein synthesis. It functions during the elongation phase of translation by delivering aminoacyl-tRNAs to the ribosome. Interestingly, despite its classical cytoplasmic role, EF-Tu has been consistently detected in the extracellular proteome (secretome) of A. salmonicida . Proteomic analyses have revealed that EF-Tu is among the top seven most abundant proteins in A. salmonicida supernatants, suggesting additional functions beyond translation . This phenomenon, where proteins perform functions unrelated to their primary role, is known as "moonlighting."

What evidence suggests EF-Tu has extracellular localization in A. salmonicida?

The extracellular presence of EF-Tu in A. salmonicida is supported by several lines of evidence from proteomic studies:

• EF-Tu was consistently detected in the supernatant fractions of A. salmonicida cultures during both exponential and stationary growth phases .

• The presence of EF-Tu in supernatants does not appear to result from cell lysis, as approximately 83% (in exponential phase) and 90% (in stationary phase) of detected cytoplasmic proteins in pellets were never present in wild-type supernatants .

• Semi-quantitative proteomic analyses identified EF-Tu among the most abundant proteins in the extracellular fraction .

This non-classical secretion phenomenon has been observed for several highly conserved cytoplasmic proteins, including EF-Tu, suggesting specific export mechanisms or membrane associations that facilitate their extracellular localization.

What methods are optimal for cloning and expressing recombinant A. salmonicida EF-Tu?

Based on approaches used for similar proteins in A. salmonicida, the following methodological workflow is recommended:

Gene Cloning Strategy:

• Extract genomic DNA from A. salmonicida using standard protocols.

• Amplify the tuf1 gene using PCR with specific primers designed based on the published A. salmonicida genome sequence.

• Clone the amplified gene into an appropriate expression vector (e.g., pET systems for E. coli expression) .

Expression Optimization:

• Transform the recombinant plasmid into a suitable E. coli expression strain (BL21(DE3) or derivatives).

• Test various induction conditions (IPTG concentration, temperature, duration) to optimize protein yield and solubility.

• Consider using fusion tags (His-tag, GST, etc.) to facilitate purification and potentially enhance solubility.

Purification Protocol:

• Extract the recombinant protein using either native or denaturing conditions depending on solubility.

• Employ affinity chromatography (Ni-NTA for His-tagged proteins) as the primary purification step.

• Further purify using ion-exchange or size-exclusion chromatography if higher purity is required.

• Verify protein identity and purity using SDS-PAGE, Western blotting, and mass spectrometry.

The success of heterologous expression may be influenced by codon usage differences between A. salmonicida and E. coli, potentially requiring codon optimization for efficient expression.

How can researchers investigate the potential moonlighting functions of A. salmonicida EF-Tu?

Investigating the moonlighting functions of EF-Tu requires multifaceted approaches:

Protein-Protein Interaction Studies:

• Use pull-down assays with purified recombinant EF-Tu to identify potential host or bacterial binding partners.

• Employ yeast two-hybrid or bacterial two-hybrid systems to screen for interacting proteins.

• Perform co-immunoprecipitation experiments using anti-EF-Tu antibodies to capture protein complexes from bacterial cultures or infection models.

Functional Assays:

• Test purified EF-Tu for binding to host components (extracellular matrix proteins, cell surface receptors).

• Assess the ability of EF-Tu to induce immune responses in fish cell cultures (measure cytokine production, immune cell activation).

• Evaluate potential enzymatic activities beyond GTP binding/hydrolysis.

Localization Studies:

• Use immunofluorescence microscopy with anti-EF-Tu antibodies to visualize its distribution in bacterial cells and during infection.

• Employ cell fractionation techniques to quantify EF-Tu distribution across different cellular compartments.

Mutational Analysis:

• Create point mutations in functional domains to differentiate between translational and moonlighting functions.

• Develop tuf1 gene deletion or conditional mutants (if viable) to assess phenotypic changes.

These approaches should be complementary and may reveal functions related to adhesion, immune modulation, or other virulence-associated activities .

What is the relationship between EF-Tu secretion and the Type Three Secretion System (T3SS) in A. salmonicida?

The relationship between EF-Tu secretion and the T3SS in A. salmonicida can be explored by comparing proteomic data from wild-type and T3SS-deficient strains:

Comparative Secretome Analysis:
Research has compared the secretomes of wild-type A. salmonicida (hypervirulent) and a T3SS-deficient mutant (ΔascV, extremely low-virulent) . While the T3SS is a major virulence determinant , EF-Tu secretion appears to occur independently of this system, as it was detected in the supernatants of both strains .

Experimental Observations:

• EF-Tu was detected in culture supernatants of both wild-type and ΔascV mutant strains during both exponential and stationary growth phases.

• This suggests that EF-Tu utilizes a secretion mechanism distinct from the T3SS.

Secretion Mechanisms:
While the T3SS translocates specific effector proteins directly into host cells through a needle-like structure, EF-Tu likely employs a non-classical secretion pathway. This could involve:

• Membrane vesicles or outer membrane vesicles (OMVs)

• Specific transporters or secretion channels

• Transient membrane association and release

Understanding the mechanism of EF-Tu secretion is crucial for characterizing its potential role in virulence and could reveal novel secretion pathways in A. salmonicida.

How does growth phase affect the expression and secretion of EF-Tu in A. salmonicida?

Growth phase significantly impacts the protein expression profile of A. salmonicida, including EF-Tu levels:

Proteomic Observations:

• In supernatants, more proteins were detected in stationary phase (326 for wild-type vs. 329 for mutant) than in exponential phase (275 for wild-type vs. 263 for mutant) .

• EF-Tu was consistently among the most abundant proteins detected in bacterial supernatants across different growth phases .

Growth Phase-Dependent Changes:
The transition from exponential to stationary phase involves significant physiological changes that may affect EF-Tu expression and secretion:

Environmental Factors:
Studies on A. salmonicida growth under different conditions reveal that factors like temperature and pH significantly impact bacterial physiology . For instance, growth at 30°C resulted in the loss of A-layer expression due to genetic rearrangement . Similar environmental responses might influence EF-Tu expression and secretion patterns.

Researchers can monitor these changes using:

• Quantitative proteomics to measure EF-Tu levels across growth phases

• qRT-PCR to analyze tuf1 gene expression

• Reporter gene constructs fused to the tuf1 promoter

What is the potential of A. salmonicida EF-Tu as a vaccine candidate against furunculosis?

EF-Tu shows promising characteristics as a vaccine candidate against furunculosis:

Favorable Properties:

• Abundance: EF-Tu is among the most abundant proteins in A. salmonicida supernatants .

• Conservation: High conservation across A. salmonicida strains may provide broad protection.

• Extracellular Presence: Its consistent detection in the secretome suggests accessibility to the host immune system .

• Immunogenicity: As a bacterial protein with potential moonlighting functions, it likely possesses immunogenic epitopes.

Vaccine Development Strategies:

• Whole Protein Approach: Using purified recombinant EF-Tu as an antigen.

• Epitope-Based Approach: Identifying and using protective epitopes, as demonstrated with YopE (homologous to AexT) in Yersinia, where the N-terminal domain was protective while the whole protein was not .

• Delivery Systems: Evaluating different adjuvants and delivery methods optimized for fish vaccination.

Evaluation Methods:

• Fish immunization trials with subsequent challenge using virulent A. salmonicida

• Measurement of specific antibody production and cellular immune responses

• Comparative protection studies against different A. salmonicida strains

Research on T3SS components as vaccine candidates provides a methodological framework that could be applied to EF-Tu . The potential of EF-Tu as a vaccine component should be evaluated alone and in combination with other A. salmonicida antigens for optimal protection.

What are the optimal conditions for studying A. salmonicida EF-Tu expression and secretion in vitro?

Based on the research protocols described in the literature, the following conditions are recommended for studying A. salmonicida EF-Tu:

Culture Conditions:

• Media: Tryptic Soy Broth (TSB) is commonly used for A. salmonicida culture .

• Temperature: 18°C is optimal for virulence expression, as higher temperatures (e.g., 30°C) can cause loss of virulence factors .

• Agitation: Moderate shaking (160 rpm) to ensure aeration .

• Growth Monitoring: Track growth by measuring optical density (OD600).

Sampling Points:

• Exponential Phase: Harvest at OD600 ≈ 1.5 .

• Stationary Phase: Harvest at OD600 > 2.0 .

Protein Isolation Protocol:

• Supernatant Preparation:

  • Separate bacterial cells by centrifugation (6,000 × g, 10 min, 4°C)

  • Filter through 0.22 μm filter to remove remaining cells

  • Add protease inhibitors to prevent protein degradation

  • Precipitate proteins using trichloroacetic acid (20% final concentration)

• Pellet Preparation:

  • Resuspend bacterial pellet in PBS

  • Process for protein extraction using appropriate buffers

• Proteomic Analysis:

  • SDS-PAGE separation followed by LC-MS/MS for comprehensive protein identification

  • Western blotting with specific antibodies for targeted EF-Tu detection

Including protease inhibitors during sample preparation is critical to preserve EF-Tu integrity, as proteolytic degradation can significantly impact results.

How can researchers distinguish between active secretion and passive release of EF-Tu?

Distinguishing between active secretion and passive release due to cell lysis is crucial for understanding EF-Tu's extracellular function:

Methodological Approaches:

• Cell Viability Assessment:

  • Measure bacterial viability throughout the experiment using colony counts or live/dead staining

  • Monitor membrane integrity using dyes like propidium iodide

• Lysis Marker Analysis:

  • Quantify strictly intracellular markers (e.g., DNA-binding proteins) in supernatants

  • Research has shown that 83-90% of detected cytoplasmic proteins in A. salmonicida pellets were never present in supernatants, suggesting specific secretion rather than general lysis

• Selective Inhibition:

  • Use specific inhibitors of secretion pathways to identify the mechanism responsible for EF-Tu export

  • Compare secretion patterns in mutants with defects in specific secretion systems

• Pulse-Chase Experiments:

  • Label newly synthesized proteins and track their localization over time

  • Determine if EF-Tu is preferentially secreted compared to other cytoplasmic proteins

• Secretion Kinetics:

  • Compare the kinetics of EF-Tu appearance in supernatants with known secreted proteins and lysis markers

  • Rapid appearance before significant cell death would suggest active secretion

Evidence from proteomic studies supports the theory that EF-Tu's extracellular localization is not the result of cell lysis but rather represents a specific export mechanism .

What approaches can be used to study the interaction between EF-Tu and host cells?

Understanding EF-Tu's interaction with host cells requires multiple complementary approaches:

In Vitro Binding Studies:

• Direct Binding Assays:

  • Incubate purified recombinant EF-Tu with fish cell lines

  • Use fluorescently labeled EF-Tu to track binding and potential internalization

  • Identify cellular receptors through crosslinking followed by mass spectrometry

• Competition Assays:

  • Determine if EF-Tu competes with other A. salmonicida adhesins for host receptors

  • Use peptide fragments to map binding domains

Functional Impact Assessment:

• Immune Response Measurement:

  • Quantify cytokine production in fish cell lines exposed to EF-Tu

  • Assess changes in gene expression using RNA-seq or qPCR arrays

  • Measure immune cell activation (respiratory burst, phagocytosis)

• Signal Transduction Analysis:

  • Monitor host cell signaling pathways activated upon EF-Tu exposure

  • Use specific inhibitors to block signaling pathways and assess functional outcomes

In Vivo Studies:

• Localization During Infection:

  • Use immunohistochemistry to detect EF-Tu in infected fish tissues

  • Track labeled EF-Tu after injection into fish

• Vaccination Studies:

  • Immunize fish with recombinant EF-Tu and assess protection against challenge

  • Evaluate both humoral and cell-mediated immune responses

The model of pathogenesis where A. salmonicida induces temporary immunosuppression in fish should be considered when studying EF-Tu's potential role in host-pathogen interactions.

How should researchers interpret contradictory findings regarding EF-Tu function?

Contradictory findings regarding EF-Tu function are common given its dual roles in translation and potential moonlighting activities:

Sources of Contradictions:

• Strain Variation: Different A. salmonicida strains may exhibit variations in EF-Tu expression, secretion, or function.

• Experimental Conditions: Growth conditions, sample preparation methods, and analytical techniques can significantly impact results.

• Dual Functionality: The same protein can exhibit different functions depending on cellular location and environmental context.

Analytical Framework:

Resolution Strategies:

• Critical Evaluation: Assess methodological differences between studies.

• Replication: Repeat key experiments under multiple conditions.

• Integrative Analysis: Consider how seemingly contradictory functions might be reconciled in the broader context of bacterial physiology and host-pathogen interactions.

• Domain-Specific Analysis: Different domains of EF-Tu may be responsible for different functions.

When interpreting results, researchers should consider that moonlighting proteins like EF-Tu can genuinely possess multiple, seemingly unrelated functions depending on context.

What bioinformatic tools are most valuable for analyzing A. salmonicida EF-Tu?

Several bioinformatic approaches can provide valuable insights into A. salmonicida EF-Tu structure, function, and evolution:

Sequence Analysis Tools:

• Multiple Sequence Alignment: Tools like Clustal Omega, MUSCLE, or T-Coffee for comparing EF-Tu sequences across bacterial species.

• Phylogenetic Analysis: MEGA, PhyML, or MrBayes for evolutionary relationship analysis.

• Motif Identification: MEME, PROSITE, or InterProScan for detecting functional domains and motifs.

Structural Analysis:

• Protein Structure Prediction: AlphaFold2, I-TASSER, or SWISS-MODEL for generating 3D structural models.

• Molecular Docking: AutoDock, HADDOCK, or ClusPro for predicting interactions with potential binding partners.

• Molecular Dynamics: GROMACS or AMBER for simulating protein behavior under different conditions.

Functional Prediction:

• Gene Ontology Analysis: Tools like PANTHER or GO enrichment analysis to predict functional roles.

• Protein-Protein Interaction Prediction: STRING, STITCH for identifying potential interaction networks.

• Epitope Prediction: BepiPred, DiscoTope for identifying potential antigenic regions relevant to vaccine development.

Specialized Analyses:

• Non-Classical Secretion Prediction: SecretomeP for predicting proteins secreted through non-classical pathways.

• Codon Usage Analysis: CodonW or GCUA for analyzing codon optimization potential for recombinant expression.

• Post-Translational Modification Sites: NetPhos, UbPred for predicting potential modification sites that might regulate function.

These computational approaches should complement experimental data to develop comprehensive models of EF-Tu function in A. salmonicida.

How can researchers quantitatively assess the immunogenicity of EF-Tu for vaccine development?

Quantitative assessment of EF-Tu immunogenicity requires systematic evaluation using both in vitro and in vivo methods:

In Vitro Immunogenicity Assays:

• B-cell Epitope Mapping:

  • Peptide array analysis to identify antibody-binding regions

  • ELISA with overlapping peptides spanning the EF-Tu sequence

  • Competition assays to determine relative epitope importance

• T-cell Response Assessment:

  • Lymphocyte proliferation assays using fish head kidney or spleen cells

  • Cytokine production measurement following EF-Tu stimulation

  • IFN-γ ELISpot assays to quantify responding T-cells

In Vivo Immunogenicity Evaluation:

• Antibody Titer Measurement:

  • ELISA to quantify specific anti-EF-Tu antibodies following immunization

  • Western blot to confirm antibody specificity

  • Functional antibody assays (e.g., opsonization, neutralization)

• Protection Studies:

  • Dose-response trials with different EF-Tu formulations

  • Challenge studies with virulent A. salmonicida strains

  • Relative percent survival (RPS) calculation:

    RPS = (1 - [% mortality in vaccinated group / % mortality in control group]) × 100

• Comparative Analysis:

  • Side-by-side comparison with current commercial vaccines

  • Combination trials with other A. salmonicida antigens

Data Analysis Framework:

• Statistical comparison of antibody titers using appropriate tests (t-test, ANOVA)

• Correlation analysis between antibody levels and protection

• Multivariate analysis to identify factors influencing vaccine efficacy

Insights from studies of T3SS components as vaccine candidates can inform experimental design for EF-Tu vaccine development, particularly regarding epitope selection and delivery methods.

What are the most promising areas for future research on A. salmonicida EF-Tu?

Several high-priority research directions could significantly advance our understanding of A. salmonicida EF-Tu:

Molecular Mechanism Elucidation:

• Determine the precise mechanism of EF-Tu secretion/export

• Identify the specific domains responsible for moonlighting functions

• Characterize potential post-translational modifications that regulate function

Host-Pathogen Interaction Studies:

• Identify host receptors that interact with EF-Tu

• Characterize the impact of EF-Tu on fish immune responses

• Determine if EF-Tu contributes to the temporary immunosuppression state induced by A. salmonicida

Comparative Analysis:

• Compare EF-Tu function across different Aeromonas species and strains

• Investigate evolutionary adaptations in EF-Tu that may relate to host specificity

• Examine conservation of moonlighting functions across bacterial pathogens

Applied Research:

• Evaluate EF-Tu as a component of multivalent vaccines against furunculosis

• Develop EF-Tu-based diagnostic tests for A. salmonicida infection

• Explore therapeutic applications targeting EF-Tu functions

Technological Innovations:

• Apply CRISPR-Cas9 for precise genetic manipulation of the tuf1 gene

• Develop advanced imaging techniques to track EF-Tu during infection

• Utilize systems biology approaches to position EF-Tu within the broader virulence network

These research directions would address critical knowledge gaps and potentially lead to practical applications for controlling furunculosis in aquaculture.

How might emerging technologies advance A. salmonicida EF-Tu research?

Emerging technologies offer exciting opportunities to address complex questions about A. salmonicida EF-Tu:

Advanced Imaging Technologies:

• Super-Resolution Microscopy: Techniques like STORM or PALM can visualize EF-Tu localization with nanometer precision.

• Correlative Light and Electron Microscopy (CLEM): Combining fluorescence and electron microscopy to track EF-Tu at different scales.

• Live-Cell Imaging: Using fluorescent protein fusions to monitor EF-Tu dynamics in real-time.

Genetic Engineering Advances:

• CRISPR-Cas Systems: Precise genome editing to create point mutations or domain deletions in the tuf1 gene.

• Inducible Expression Systems: Tetracycline-responsive or temperature-sensitive systems for controlled expression studies.

• Single-Cell Analysis: Examining heterogeneity in EF-Tu expression and secretion at the individual cell level.

Proteomic Innovations:

• Proximity Labeling: BioID or APEX2 methods to identify proteins interacting with EF-Tu in living cells.

• Crosslinking Mass Spectrometry: To capture transient protein-protein interactions.

• Targeted Proteomics: SRM/MRM approaches for precise quantification of EF-Tu and its modified forms.

Systems Biology Approaches:

• Multi-omics Integration: Combining transcriptomics, proteomics, and metabolomics data.

• Network Analysis: Positioning EF-Tu within global virulence networks.

• Machine Learning Applications: Predicting functional interactions and important structural features.

Immunological Methods:

• Single-Cell RNA-Seq: Characterizing host cell responses to EF-Tu at the single-cell level.

• Cytometry by Time of Flight (CyTOF): High-dimensional analysis of immune responses.

• Immune Repertoire Sequencing: Analyzing B and T cell receptor diversity following immunization.

These technologies could overcome current limitations in understanding EF-Tu's complex roles in A. salmonicida biology and pathogenesis.

What is the current consensus on the importance of EF-Tu in A. salmonicida pathogenesis?

• EF-Tu is consistently present in the extracellular proteome of A. salmonicida, suggesting functions beyond its classical role in translation .

• The extracellular presence of EF-Tu does not appear to result from cell lysis but rather from specific secretion mechanisms .

• As one of the most abundant proteins in A. salmonicida supernatants, EF-Tu likely plays significant roles during infection .

• EF-Tu may contribute to A. salmonicida's ability to induce temporary immunosuppression in fish hosts, facilitating bacterial dissemination .

• The high conservation of EF-Tu across A. salmonicida strains from diverse geographic locations suggests its fundamental importance to bacterial physiology and potentially pathogenesis .

The emerging consensus views EF-Tu as a multifunctional protein with significant potential as a research target for understanding and controlling furunculosis. Its dual roles in essential cellular processes and potential virulence functions make it particularly interesting from both basic science and applied perspectives.

What methodological challenges must be addressed for effective EF-Tu research?

Several methodological challenges need to be addressed to advance research on A. salmonicida EF-Tu:

Expression and Purification Challenges:

• Maintaining proper protein folding and activity in recombinant EF-Tu

• Distinguishing between monomeric and potential multimeric forms

• Preserving potential post-translational modifications

Functional Assessment Difficulties:

• Separating translation-related functions from moonlighting activities

• Creating viable mutants when studying an essential gene

• Developing appropriate in vitro and in vivo models that recapitulate infection conditions

Technical Limitations:

• Distinguishing between active secretion and passive release during sample preparation

• Developing specific antibodies that recognize native EF-Tu without cross-reactivity

• Tracking EF-Tu during infection in complex host tissues

Experimental Design Considerations:

• Accounting for strain variation and growth condition effects

• Standardizing protocols across laboratories for comparable results

• Integrating multiple methodological approaches for comprehensive analysis

Translation to Applications:

• Identifying the most immunogenic and protective regions for vaccine development

• Optimizing delivery systems for fish vaccination

• Developing cost-effective diagnostic tools based on EF-Tu detection

Addressing these challenges requires interdisciplinary approaches combining molecular biology, biochemistry, immunology, and fish pathology expertise, along with standardized reporting of experimental conditions to facilitate cross-study comparisons.