Recombinant Aeromonas salmonicida Elongation factor Ts (tsf)

What is Elongation Factor Ts (tsf) in Aeromonas salmonicida?

Elongation Factor Ts (EF-Ts) is a protein encoded by the tsf gene in Aeromonas salmonicida, a Gram-negative bacterium that causes furunculosis in fish. It functions primarily as a guanine nucleotide exchange factor for Elongation Factor Tu (EF-Tu). Proteomic studies have identified EF-Ts in the exoproteome of A. salmonicida, suggesting it may have additional functions beyond its traditional role in translation . EF-Ts has been detected in both exponential and stationary growth phases of A. salmonicida cultures, with its presence in supernatants indicating potential active secretion rather than mere cell lysis .

What is the canonical function of Elongation Factor Ts in bacterial translation?

In bacterial protein synthesis, Elongation Factor Ts (EF-Ts) serves as a guanine nucleotide exchange factor that regenerates active EF-Tu by catalyzing the exchange of GDP for GTP. The process follows several distinct steps:

• EF-Tu delivers aminoacyl-tRNA to the ribosome while bound to GTP

• GTP hydrolysis occurs upon codon recognition

• EF-Tu·GDP is released from the ribosome

• EF-Ts binds to EF-Tu·GDP, causing a conformational change

• GDP is released, forming an EF-Tu·EF-Ts intermediate

• GTP binds to EF-Tu, displacing EF-Ts

• The regenerated EF-Tu·GTP is ready for another round of elongation

This nucleotide exchange function is essential for maintaining translation efficiency in A. salmonicida and contributes to bacterial growth and adaptation .

How conserved is Elongation Factor Ts across Aeromonas species?

Elongation Factor Ts shows high conservation across Aeromonas species, reflecting its essential role in bacterial protein synthesis. According to proteomic analysis, approximately 81% of identified proteins in A. salmonicida were common to the Aeromonas genus, with EF-Ts being among these highly conserved proteins . The conservation pattern can be summarized as follows:

The high level of conservation suggests strong evolutionary pressure to maintain EF-Ts structure and function, though species-specific variations may exist that could affect protein-protein interactions or potential moonlighting functions .

What are the potential moonlighting functions of Elongation Factor Ts in A. salmonicida?

Elongation Factor Ts in A. salmonicida may possess moonlighting functions beyond its primary role in translation, particularly given its detection in the bacterial exoproteome. Evidence from proteomic studies shows that numerous highly conserved cytoplasmic proteins, including translation factors like EF-Ts, were found in A. salmonicida supernatants, with several indicators suggesting that "their extracellular localization was not the result of cell lysis" .

Potential moonlighting functions may include:

• Immunomodulation: Interaction with host immune components to influence immune responses

• Adhesion: Facilitating bacterial attachment to host tissues

• Stress response: Contributing to bacterial adaptation to environmental stressors

• Virulence: Participating in pathogenesis pathways independent of translation

• Biofilm formation: Supporting bacterial persistence through biofilm development

These potential functions would require experimental validation through techniques such as protein-protein interaction studies, knockout mutants, and in vivo infection models to establish their biological relevance in A. salmonicida pathogenicity .

How does the secretion of EF-Ts in A. salmonicida relate to virulence mechanisms?

The secretion of EF-Ts in A. salmonicida has intriguing implications for understanding virulence mechanisms. Proteomic analysis revealed that EF-Ts was detected in supernatants of both wild-type and Type III Secretion System (T3SS)-deficient mutant strains . This finding suggests that its secretion occurs independently of the T3SS, which is a major virulence determinant in this bacterium.

The relationship between secreted EF-Ts and virulence might involve:

• Immune Evasion: Secreted EF-Ts might interfere with host immune recognition or effector functions

• Host Cell Manipulation: The protein could modulate host cell processes to facilitate bacterial survival

• Nutritional Immunity: EF-Ts might help counteract host strategies to sequester essential nutrients

• Biofilm Development: Contributing to community structures that enhance bacterial persistence

Importantly, the search results indicate that many cytoplasmic proteins found in supernatants "were demonstrated to be immunogenic and recognized by sera from diseased hosts, confirming that they should be extracellularly presented to the immune system by bacteria during the pathogenesis" . While EF-Ts is not specifically named in this context, its consistent presence in the exoproteome suggests it may contribute to the antigenic profile of A. salmonicida during infection.

What experimental approaches are most effective for expressing and purifying recombinant A. salmonicida EF-Ts?

Expressing and purifying recombinant A. salmonicida EF-Ts requires a systematic approach that optimizes yield, solubility, and biological activity. Based on established protocols for similar bacterial proteins, the following methodology is recommended:

Expression System Design:

• Vector Selection:

  • pET-based vectors with T7 promoter for high-level expression

  • Inclusion of affinity tags (His6 or GST) at N- or C-terminus

  • Codon optimization for the expression host

• Host Strain Selection:

  • E. coli BL21(DE3) or Rosetta for rare codon supplementation

  • Arctic Express or C41/C43 strains if protein folding is problematic

Optimized Expression Protocol:

Purification Strategy:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT

• Affinity chromatography (IMAC for His-tagged protein)

• Ion exchange chromatography for intermediate purification

• Size exclusion chromatography as final polishing step

• Activity assessment through nucleotide exchange assays with EF-Tu

This approach should yield highly pure, functional protein suitable for structural, biochemical, and immunological studies .

How can recombinant A. salmonicida EF-Ts be utilized in vaccine development against furunculosis?

Recombinant A. salmonicida EF-Ts offers promising potential for furunculosis vaccine development based on several lines of evidence from proteomic studies. The protein's presence in bacterial supernatants and potential immunogenicity make it a candidate for inclusion in next-generation vaccines.

Strategic Approaches for Vaccine Development:

• Subunit Vaccine Formulation:

  • Purified recombinant EF-Ts can be combined with other immunogenic proteins

  • Adjuvants significantly enhance protection, as demonstrated with other A. salmonicida antigens

  • Immunostimulants added to A. salmonicida bacterins enhance defense mechanisms in rainbow trout

• Multi-Epitope Vaccine Design:

  • Identification of immunodominant epitopes within EF-Ts

  • Construction of chimeric proteins combining multiple protective epitopes

  • Rational design to enhance presentation to fish immune system

• Delivery Systems:

  • Injectable preparations with appropriate adjuvants

  • Oral delivery systems using bioencapsulation

  • Immersion vaccines for mass vaccination

• Combination with Existing Strategies:

  • Current furunculosis vaccines primarily use "bacterial pellets inactivated with formalin, thereby avoiding the extracellular protein (ECP) fraction"

  • Incorporation of secreted proteins like EF-Ts could address this limitation

  • Combining traditional bacterins with recombinant EF-Ts may provide broader protection

The efficacy of such approaches would require validation through controlled challenge studies, evaluating different vaccine formulations, routes of administration, and protective correlates in various fish species under different environmental conditions .

What analytical methods are most effective for studying the interactions between EF-Ts and other bacterial proteins?

Understanding the interactions between EF-Ts and other bacterial proteins requires a multi-faceted approach combining complementary analytical methods:

Biophysical Techniques:

• Surface Plasmon Resonance (SPR):

  • Provides real-time measurements of association/dissociation kinetics

  • Determines binding affinity constants (KD values)

  • Allows analysis of the effects of nucleotides or other small molecules on binding

• Isothermal Titration Calorimetry (ITC):

  • Measures thermodynamic parameters (ΔH, ΔS, ΔG)

  • Determines binding stoichiometry

  • Provides label-free detection of interactions

Structural Biology Approaches:

• X-ray Crystallography:

  • Reveals atomic-level details of protein-protein interfaces

  • Captures conformational changes upon complex formation

  • Provides insights into the mechanism of nucleotide exchange

• Cryo-Electron Microscopy:

  • Useful for larger complexes (e.g., EF-Ts with ribosomes)

  • Doesn't require crystallization

  • Can capture different conformational states

Biochemical Methods:

• Co-Immunoprecipitation:

  • Identifies protein interactions in near-native conditions

  • Can be coupled with mass spectrometry for unbiased identification

  • Particularly useful for detecting novel interaction partners in A. salmonicida lysates

• Cross-linking Mass Spectrometry:

  • Maps specific residues at interaction interfaces

  • Captures transient interactions

  • Provides distance constraints for molecular modeling

These methods, used in combination, would provide comprehensive characterization of EF-Ts interactions with its canonical partner EF-Tu as well as potential non-canonical interaction partners that might explain its presence in the A. salmonicida exoproteome .

How do environmental factors affect the expression and secretion of EF-Ts in A. salmonicida?

The expression and secretion of EF-Ts in A. salmonicida are influenced by multiple environmental factors, with important implications for experimental design and interpretation. Proteomic analyses have revealed clear differences in protein secretion patterns between growth phases:

This data demonstrates that protein secretion increases during stationary phase, likely reflecting stress responses and adaptation mechanisms .

Key environmental factors to consider when studying EF-Ts expression include:

• Temperature Conditions:

  • A. salmonicida is psychrophilic, with optimal growth around 18°C as used in experimental studies

  • Temperature shifts may trigger stress responses affecting secretion patterns

• Media Composition:

  • Rich media (TSB) was used in proteomic studies

  • Nutrient limitation might alter secretion profiles

• Growth Phase:

  • Stationary phase shows increased protein secretion

  • Time course sampling is essential to capture dynamic changes

• Host-Derived Factors:

  • Fish serum or tissue extracts may induce virulence-associated secretion

  • Co-culture with host cells might reveal interaction-dependent secretion patterns

For robust experimental design, researchers should standardize these parameters and consider using environmentally relevant conditions that mimic the natural habitat of A. salmonicida in infected fish .

How to design experiments to identify potential epitopes in EF-Ts for vaccine development?

Designing experiments to identify potential epitopes in EF-Ts for vaccine development requires a systematic approach combining computational prediction and experimental validation:

Computational Epitope Prediction:

• B-cell Epitope Prediction:

  • Analysis of surface accessibility, hydrophilicity, and flexibility

  • Identification of regions with high antigenic propensity

  • Comparative analysis with homologous proteins from other fish pathogens

• T-cell Epitope Prediction:

  • MHC binding prediction for relevant fish species

  • Conservation analysis across A. salmonicida strains

  • Exclusion of epitopes with potential cross-reactivity to host proteins

Experimental Validation Pipeline:

In vivo Validation:

• Immunize fish with selected epitopes (as synthetic peptides or recombinant constructs)

• Measure antibody titers and cellular responses

• Perform challenge studies with virulent A. salmonicida

• Assess protection levels and immunological correlates

This approach would build on observations that cytoplasmic proteins found in A. salmonicida supernatants "were demonstrated to be immunogenic and recognized by sera from diseased hosts" , potentially leading to effective epitope-based vaccines against furunculosis.

How do we reconcile the presence of cytoplasmic proteins like EF-Ts in bacterial supernatants?

The presence of traditionally cytoplasmic proteins like EF-Ts in bacterial supernatants presents an intriguing paradox that requires careful experimental analysis. According to the research findings, numerous highly conserved cytoplasmic proteins, including translation factors like EF-Ts, EF-Tu, and EF-G, were detected in A. salmonicida supernatants . Importantly, "several evidences support the theory that their extracellular localization was not the result of cell lysis" .

This apparent contradiction can be addressed through multiple hypotheses:

• Non-classical Secretion Mechanisms:

  • Some cytoplasmic proteins may be secreted through alternative pathways not requiring signal peptides

  • Membrane vesicles (outer membrane vesicles) might transport cytoplasmic proteins outside the cell

• Moonlighting Functions:

  • Dual functionality of proteins depending on cellular location

  • Evolutionary selection for proteins that can serve multiple roles

• Controlled Release:

  • Regulated autolysis of a subpopulation of bacteria

  • Programmed release of specific proteins during certain growth phases

• Experimental Design Considerations:

  • The proteomic study used protease inhibitors during cultivation , which helps rule out degradation as an explanation

  • Supernatants were filtered (0.22 μm) to eliminate intact bacteria

  • TCA precipitation was used to concentrate proteins from identical volumes

Experimental approaches to resolve this contradiction include:

• Electron microscopy to visualize membrane vesicles

• Pulse-chase experiments to track protein movement

• Genetic manipulation of potential secretion pathways

• Reporter protein fusions to monitor secretion in real-time

Understanding this phenomenon is crucial for interpreting the biological significance of extracellular EF-Ts in A. salmonicida pathogenesis .

What are the contradictions in current understanding of EF-Ts immunogenicity in fish?

The immunogenicity of EF-Ts in fish presents several unresolved contradictions in current research. While the search results don't specifically address EF-Ts immunogenicity, they provide context about related proteins and immunogenic responses to A. salmonicida components:

Contradictory Evidence:

Resolving these contradictions requires:

• Direct assessment of anti-EF-Ts antibody titers in naturally infected fish

• Comparative immunization studies with various EF-Ts formulations

• Analysis of protection correlates in challenge studies

• Investigation of cellular versus humoral responses to EF-Ts

Such studies would illuminate whether EF-Ts contributes significantly to protective immunity against A. salmonicida infection .

What are the most promising research directions for A. salmonicida EF-Ts studies?

Based on current knowledge of A. salmonicida EF-Ts, several research directions show particular promise for advancing our understanding of fish pathogen biology and developing effective control strategies for furunculosis.

The most promising research avenues include:

• Structural and Functional Characterization:

  • Determine the three-dimensional structure of A. salmonicida EF-Ts

  • Elucidate the molecular mechanisms of its interactions with EF-Tu and other proteins

  • Investigate potential moonlighting functions beyond translation

• Secretion Mechanism Exploration:

  • Identify the pathway(s) responsible for EF-Ts secretion

  • Determine whether secretion is active or passive

  • Investigate regulatory factors controlling secretion during infection

• Host-Pathogen Interaction Studies:

  • Examine interactions between EF-Ts and fish immune components

  • Investigate potential roles in immune evasion or modulation

  • Determine if EF-Ts contributes to bacterial adhesion or invasion

• Vaccine Development:

  • Assess EF-Ts as a subunit vaccine antigen

  • Identify protective epitopes within the protein

  • Develop optimal formulations with appropriate adjuvants

  • Evaluate cross-protection against diverse A. salmonicida strains

• Comparative Analysis Across Pathogens:

  • Compare EF-Ts properties across different fish pathogens

  • Identify conserved features that could be targeted for broad-spectrum interventions

  • Investigate species-specific adaptations that might contribute to host specificity

By pursuing these research directions, scientists can leverage the unexpected presence of EF-Ts in the A. salmonicida exoproteome to develop novel approaches for understanding and controlling furunculosis in aquaculture settings .

How might integrating multiple data types enhance our understanding of EF-Ts in A. salmonicida pathogenesis?

Integrating multiple data types offers a powerful approach to comprehensively understand the role of EF-Ts in A. salmonicida pathogenesis. This systems biology perspective can reveal insights not apparent from individual studies:

Multi-omics Integration:

Computational Framework:

• Network analysis to position EF-Ts within protein interaction networks

• Machine learning approaches to identify patterns across diverse datasets

• Predictive modeling to generate testable hypotheses about EF-Ts function

Experimental Validation:

• CRISPR-Cas9 genome editing to create precise tsf mutations

• In vivo imaging to track protein localization during infection

• Single-cell approaches to capture heterogeneity in bacterial populations

This integrated approach could resolve current paradoxes regarding the presence of EF-Ts in bacterial supernatants and its potential moonlighting functions. By combining molecular-level understanding with system-wide perspectives, researchers can develop a comprehensive model of how EF-Ts contributes to A. salmonicida virulence and identify optimal points for therapeutic intervention.