Recombinant Edwardsiella ictaluri Peptide chain release factor 1 (prfA)

What is Edwardsiella ictaluri and its significance in research?

Edwardsiella ictaluri is a Gram-negative intracellular bacterial pathogen responsible for Enteric Septicemia of Catfish (ESC), also known as "Hole in the Head disease." This bacterium primarily affects farm-raised channel catfish and represents the most prevalent disease in U.S. catfish aquaculture . The pathology of E. ictaluri infection manifests as either chronic encephalitis or acute septicemia, with weight loss due to anorexia typically being the first observable symptom . Beyond its economic importance in aquaculture, E. ictaluri serves as a valuable model organism for studying host-pathogen interactions, bacterial virulence mechanisms, and intracellular survival strategies. The bacterium possesses sophisticated secretion systems, including Type III and Type VI secretion systems (T3SS and T6SS), which contribute to its pathogenicity .

What is Peptide Chain Release Factor 1 (prfA) and its function in bacteria?

Peptide Chain Release Factor 1 (prfA) is a critical protein involved in translation termination in bacterial protein synthesis. During protein synthesis, prfA recognizes the stop codons UAA and UAG in the mRNA, triggering the hydrolysis of the ester bond between the completed polypeptide chain and the tRNA in the ribosome's P-site. This process results in the release of the newly synthesized protein from the ribosome. In E. ictaluri, prfA likely plays a similar essential role in translation termination, and its function may be integrated with the bacterium's virulence mechanisms and survival strategies, particularly under stress conditions encountered during host infection.

How does E. ictaluri adapt to iron-limited conditions during infection?

E. ictaluri, like other bacterial pathogens, has developed sophisticated iron acquisition systems to overcome the iron limitation imposed by host defense mechanisms. Research has shown that E. ictaluri contains a heme-hemoglobin uptake system regulated by the Ferric Uptake Regulator (Fur) protein . Unlike many other pathogens, E. ictaluri does not secrete detectable siderophores for iron acquisition . The Fur protein in E. ictaluri is evolutionarily distinct, being smaller than other fur family members, suggesting possible genome degradation during its evolution . This iron uptake system is crucial for the bacteria's survival and multiplication within the host, as iron is an essential nutrient for bacterial growth and metabolic processes.

How does the expression pattern of recombinant E. ictaluri prfA compare under different environmental conditions?

The expression of recombinant E. ictaluri prfA, like many bacterial proteins, is significantly influenced by environmental conditions. Research indicates that E. ictaluri virulence factors show differential expression under conditions mimicking the phagosomal environment, particularly under low pH and phosphate limitation . Quantitative PCR and Western blotting analyses have confirmed this environmental regulation for various E. ictaluri systems .

For recombinant prfA expression, researchers should consider the following conditions and their impact:

Understanding these expression patterns is crucial for optimizing recombinant production and for gaining insights into the protein's physiological role during infection.

What are the structural and functional differences between E. ictaluri prfA and prfA proteins from other bacterial species?

While the search results don't provide specific information about E. ictaluri prfA structure, comparative analysis with prfA from other bacterial species would likely reveal both conserved domains essential for translation termination and species-specific variations that may reflect adaptation to E. ictaluri's ecological niche and pathogenic lifestyle.

The functional domains typically found in bacterial prfA proteins include:

• Stop codon recognition domain

• Peptidyl-transferase center interaction domain

• GGQ motif (essential for peptidyl-tRNA hydrolysis)

Researchers investigating E. ictaluri prfA should conduct phylogenetic analyses comparing the protein sequence with those from related Edwardsiella species and more distant bacterial taxa to identify conserved and divergent regions. Such comparisons could provide insights into the evolution of this essential protein and potentially identify features unique to E. ictaluri that might be related to its pathogenicity or host adaptation.

How does mutation of prfA affect E. ictaluri virulence and intracellular survival?

Based on our understanding of bacterial pathogenesis, mutation of an essential gene like prfA would likely have profound effects on E. ictaluri's virulence and survival capabilities. To investigate this question, researchers should consider:

• Construction of a defined prfA deletion mutant following established protocols for E. ictaluri gene deletion, similar to the approaches used for other E. ictaluri genes . This would involve:

  • Amplifying upstream and downstream regions of prfA

  • Using splicing overlap extension PCR to generate a deletion fragment

  • Cloning into a suicide vector like pMEG-375

  • Conjugational transfer and selection for allelic exchange

• Comparative analysis of the ΔprfA mutant with wild-type E. ictaluri for:

  • Growth kinetics in standard and stress conditions

  • Replication within channel catfish head-kidney-derived macrophages (HKDM)

  • Virulence in channel catfish models

  • Expression of T3SS and T6SS components

  • Response to iron limitation and other environmental stressors

Previous research on E. ictaluri regulatory genes (like esrA and esrB) has shown that mutation of key regulatory proteins can lead to complete attenuation in the catfish host and loss of ability to replicate in macrophages . Whether prfA mutation would have similarly dramatic effects or more subtle phenotypes would depend on the degree to which its function can be compensated by other cellular mechanisms.

What interactions exist between prfA and the E. ictaluri Fur-regulated iron uptake system?

The Fur protein in E. ictaluri regulates not only iron uptake-related genes but also genes important for virulence . Investigating potential interactions between prfA and the Fur-regulated iron uptake system would require:

• Comparative proteomic analysis of wild-type E. ictaluri and Fur mutant strains under iron-replete and iron-limited conditions to determine if prfA expression is Fur-regulated

• Chromatin immunoprecipitation (ChIP) assays to identify potential Fur binding sites in the prfA promoter region

• RNA-seq analysis to examine global transcriptional changes in prfA mutants, with particular attention to iron metabolism genes

• Co-immunoprecipitation experiments to detect potential physical interactions between prfA and components of the iron uptake system

Understanding these interactions could reveal how E. ictaluri coordinates translation termination with iron metabolism during infection, potentially identifying new targets for therapeutic intervention.

What are the optimal conditions for expressing recombinant E. ictaluri prfA in heterologous systems?

For expressing recombinant E. ictaluri prfA in heterologous systems, researchers should consider the following methodological approaches:

• Expression System Selection:

  • E. coli BL21(DE3) or similar strains are typically suitable for initial expression attempts

  • Consider using E. ictaluri's own promoter for expression in related Edwardsiella species

  • For difficult-to-express constructs, consider specialized expression strains like Rosetta (for rare codons) or SHuffle (for disulfide bond formation)

• Vector Design:

  • Include appropriate affinity tags (His6, GST, etc.) for purification

  • Consider using vectors with tightly controlled promoters (T7 lac, arabinose-inducible, etc.)

• Expression Conditions:

  • Test multiple induction temperatures (16°C, 25°C, 37°C)

  • Optimize inducer concentration (IPTG: 0.1-1.0 mM range)

  • Consider extended expression times (4-24 hours) at lower temperatures

  • Test expression in media mimicking conditions encountered during infection (low pH, low phosphate)

• Purification Strategy:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Size exclusion chromatography for final polishing

  • Ion exchange chromatography if electrostatic properties are favorable

The optimal conditions will need to be determined empirically, as protein-specific factors often dictate the success of recombinant expression.

How can functional assays be designed to assess recombinant E. ictaluri prfA activity?

To evaluate the functionality of recombinant E. ictaluri prfA, researchers can employ several complementary approaches:

• In vitro Translation Termination Assay:

  • Utilize a reconstituted translation system with purified ribosomes, mRNAs containing stop codons, and other necessary translation components

  • Measure peptide release efficiency in the presence of recombinant prfA

  • Compare activity with and without potential inhibitors or cofactors

• Complementation Assays:

  • Introduce recombinant prfA into a temperature-sensitive E. coli prfA mutant

  • Assess restoration of growth at non-permissive temperatures

  • Similar complementation approaches in E. ictaluri prfA mutants

• Stop Codon Readthrough Reporter System:

  • Construct dual reporter systems with a stop codon between two reporter genes

  • Measure readthrough efficiency in the presence of wild-type vs. mutant prfA

  • Test under various environmental conditions mimicking host environments

• Structural Integrity Assessment:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to identify flexible or exposed regions

These functional assays would provide comprehensive information about the activity, specificity, and regulation of E. ictaluri prfA, enabling comparisons with prfA proteins from other bacterial species.

What proteomics approaches are most effective for studying E. ictaluri prfA interactions?

Based on the proteomics methodologies previously applied to E. ictaluri , the following approaches would be most effective for studying prfA interactions:

• Affinity Purification-Mass Spectrometry (AP-MS):

  • Express tagged prfA (His, FLAG, etc.) in E. ictaluri

  • Perform pull-downs under various conditions (different growth phases, stress conditions)

  • Identify co-purifying proteins by LC-MS/MS

  • Validate interactions by reciprocal pull-downs or orthogonal methods

• Crosslinking Mass Spectrometry (XL-MS):

  • Use chemical crosslinkers to stabilize transient interactions

  • Digest and analyze crosslinked peptides by specialized MS methods

  • Map interaction interfaces at amino acid resolution

• 2D-LC ESI MS/MS and 2-DE MALDI TOF/TOF MS:

  • These methods have been successfully applied to E. ictaluri proteome analysis

  • Compare protein expression profiles between wild-type and prfA mutant strains

  • Identify proteins whose expression is affected by prfA mutation

• Protein-Protein Interaction Network Analysis:

  • Integrate experimental data with computational predictions

  • Analyze using clusters of orthologous groups (COG) and pathway analysis as previously applied to E. ictaluri proteome data

  • This approach previously identified 788 unique E. ictaluri proteins and 12 significantly represented pathways

The combined use of these proteomics approaches would provide a comprehensive view of prfA's interactome and its functional context within E. ictaluri's cellular processes.

How can genetic manipulation techniques be optimized for studying prfA in E. ictaluri?

Based on the genetic manipulation techniques described in the search results for E. ictaluri, researchers should consider the following approaches for studying prfA:

• Construction of Defined Deletion Mutants:

  • Follow established protocols for creating in-frame deletions in E. ictaluri

  • Use splicing overlap extension PCR to join these fragments

  • Clone into suicide vectors like pMEG-375 or pRE107

  • Select for double-crossover events using appropriate antibiotics and counter-selection (e.g., sacB)

• Complementation Strategies:

  • Clone prfA with its native promoter into vectors like pAYCY184 or pEZ151

  • Introduce the complementation construct into the prfA mutant

  • Verify restoration of phenotype

• Site-Directed Mutagenesis:

  • Target conserved residues in prfA functional domains

  • Create a library of point mutants affecting specific functions

  • Analyze phenotypic effects in vivo and biochemical properties in vitro

• Conditional Expression Systems:

  • Develop inducible or repressible promoter systems for E. ictaluri

  • Place prfA under controlled expression to study dosage effects

  • Use for studying essential genes where complete deletion may be lethal

How should researchers address inclusion body formation when expressing recombinant E. ictaluri prfA?

Inclusion body formation is a common challenge when expressing recombinant bacterial proteins. For E. ictaluri prfA, researchers should consider the following strategies:

• Prevention Approaches:

  • Lower expression temperature (16-20°C)

  • Reduce inducer concentration

  • Co-express molecular chaperones (GroEL/ES, DnaK/J/GrpE)

  • Use fusion partners known to enhance solubility (SUMO, MBP, TrxA)

  • Express in specialized E. coli strains (Arctic Express, SHuffle)

• Refolding Strategies:

  • If prevention fails, optimize inclusion body isolation:

    • Multiple washing steps to remove contaminants

    • Use mild detergents to preserve secondary structure

  • Develop refolding protocol:

    • Gradual dilution or dialysis to remove denaturants

    • Test various additives (L-arginine, glycerol, sucrose)

    • Monitor refolding by functional assays and structural analyses

• Comparative Troubleshooting:

  Common Issue	Possible Solution	Success Indicator
  Complete insolubility	Express as fusion with SUMO or MBP	Appearance of soluble fraction on SDS-PAGE
  Partial solubility	Lower temperature to 16°C	Increased ratio of soluble to insoluble protein
  Inactive protein	Optimize refolding conditions	Recovery of functional activity in in vitro assays
  Degradation	Add protease inhibitors, use protease-deficient strains	Intact protein band on Western blot

• Alternative Expression Strategies:

  • Cell-free protein synthesis systems

  • Periplasmic expression to facilitate disulfide bond formation if relevant

  • Expression in Edwardsiella species if E. coli systems fail

By systematically addressing inclusion body formation, researchers can maximize the yield of correctly folded, functional recombinant E. ictaluri prfA for subsequent studies.

What controls are essential when analyzing the effects of prfA mutation on E. ictaluri virulence?

When analyzing the effects of prfA mutation on E. ictaluri virulence, researchers must include several controls to ensure valid interpretations:

• Genetic Controls:

  • Wild-type E. ictaluri (positive control for virulence)

  • Complemented prfA mutant (to confirm phenotype restoration)

  • Known attenuated mutant (e.g., esrA or esrB mutant ) as reference

  • prfA point mutants affecting specific functions (to dissect functional domains)

• Experimental Controls for In Vitro Studies:

  • Growth curve comparison in standard media

  • Stress response assays (acid stress, oxidative stress, iron limitation)

  • Expression analysis of key virulence genes (T3SS, T6SS components)

  • Protein synthesis rate measurement using radiolabeled amino acids

• Experimental Controls for Ex Vivo Studies:

  • Macrophage viability assessment (uninfected cells)

  • Known attenuated strains in parallel infections

  • Blocking phagocytosis to distinguish attachment from internalization

  • Complemented mutant to confirm phenotype is due to prfA deletion

• Experimental Controls for In Vivo Studies:

  • Mock-infected animals

  • Wild-type infection at various doses

  • Complemented mutant to confirm genetic basis of attenuation

  • Sequential time points to track infection progression

These comprehensive controls would allow researchers to confidently attribute observed phenotypes to prfA mutation and distinguish direct effects from indirect consequences of altered translation termination.

How should researchers interpret contradictory data between in vitro and in vivo studies of recombinant E. ictaluri prfA?

Contradictions between in vitro and in vivo findings are common in bacterial pathogenesis research. When studying E. ictaluri prfA, researchers should apply the following analytical framework:

By systematically addressing contradictions, researchers can develop more nuanced models of prfA function that account for the complexity of host-pathogen interactions in real infection scenarios.

What bioinformatics tools are most appropriate for analyzing E. ictaluri prfA sequence and structure?

For comprehensive analysis of E. ictaluri prfA sequence and structure, researchers should utilize the following bioinformatics tools and approaches:

• Sequence Analysis:

  • BLAST and HMMER for homology searches and domain identification

  • MUSCLE or CLUSTAL for multiple sequence alignment with prfA from related species

  • MEGA or MrBayes for phylogenetic analysis

  • ConSurf for evolutionary conservation mapping

  • PROVEAN or SIFT for predicting the impact of amino acid substitutions

• Structural Analysis:

  • AlphaFold or RoseTTAFold for protein structure prediction

  • SWISS-MODEL for homology modeling if crystal structures of related prfA proteins exist

  • UCSF Chimera or PyMOL for structure visualization and analysis

  • MDAnalysis or GROMACS for molecular dynamics simulations

  • CASTp or POCASA for binding pocket prediction

• Functional Prediction:

  • InterProScan for functional domain prediction

  • STRING for protein-protein interaction network analysis

  • Predict Protein for secondary structure and functional site prediction

  • DiANNA for disulfide bond prediction if relevant

  • NetSurfP for surface accessibility prediction

• Genomic Context Analysis:

  • RAST or Prokka for genome annotation

  • BPROM or BDGP for promoter prediction

  • RBS finder for ribosome binding site identification

  • OperonDB for operon structure prediction

  • DOOR for determining operons and regulons

These bioinformatics approaches would provide a comprehensive understanding of E. ictaluri prfA's sequence features, structural properties, and genomic context, facilitating experimental design and interpretation of functional studies.

What are the prospects for developing prfA-targeted antimicrobials against E. ictaluri infections?

The essential nature of peptide chain release factors in bacterial protein synthesis makes prfA a potentially attractive target for antimicrobial development against E. ictaluri. Future research should explore:

• Target Validation Studies:

  • Confirm essentiality of prfA in E. ictaluri under various conditions

  • Identify minimum inhibitory concentrations of translation inhibitors

  • Develop conditional prfA mutants to study partial inhibition effects

• Structure-Based Drug Design:

  • Determine high-resolution structure of E. ictaluri prfA

  • Identify unique binding pockets distinct from host translation factors

  • Perform virtual screening of compound libraries targeting these pockets

  • Validate hits with in vitro binding and functional assays

• Natural Product Screening:

  • Test known translation inhibitors against E. ictaluri

  • Screen aquatic microbiota for compounds with selective activity

  • Investigate traditional remedies used in aquaculture for active components

• Delivery Systems for Aquaculture Applications:

  • Develop feed-incorporated formulations for oral administration

  • Explore nanoparticle-based delivery for improved stability

  • Investigate water treatment approaches for hatchery applications

The development of prfA-targeted antimicrobials could provide new tools for controlling ESC in aquaculture settings, potentially with reduced environmental impact compared to current broad-spectrum antibiotics.

How might research on E. ictaluri prfA inform our understanding of bacterial adaptation to host environments?

Research on E. ictaluri prfA could provide valuable insights into bacterial adaptation mechanisms:

• Translation Regulation Under Stress:

  • Investigate how translation termination efficiency changes under host-relevant stresses

  • Examine potential regulatory modifications of prfA during infection

  • Study interactions between prfA and stress response pathways

• Host-Specific Adaptations:

  • Compare prfA sequences and activities across E. ictaluri strains from different fish hosts

  • Identify potential signatures of selection in prfA sequences

  • Examine how prfA function may be optimized for growth at fish body temperatures

• Coordination with Virulence Mechanisms:

  • Investigate potential regulatory links between prfA and the EsrAB/EsrC regulatory systems

  • Examine how translation termination efficiency affects expression of virulence factors

  • Study potential preferential translation of virulence-associated mRNAs

• Evolutionary Considerations:

  • Analyze whether prfA in E. ictaluri shows evidence of genome degradation similar to the Fur protein

  • Compare with homologs from non-pathogenic relatives to identify pathogen-specific features

  • Investigate horizontal gene transfer events that may have shaped prfA evolution

These studies would contribute to our fundamental understanding of how basic cellular processes like translation termination are integrated with pathogenesis and host adaptation.

What potential applications exist for recombinant E. ictaluri prfA in vaccine development?

The development of effective vaccines against E. ictaluri remains an important goal for aquaculture. Recombinant prfA could contribute to this effort through several approaches:

• Subunit Vaccine Development:

  • Evaluate recombinant prfA as a potential antigen

  • Identify immunogenic epitopes through epitope mapping

  • Test various adjuvant formulations for enhanced immunogenicity

  • Evaluate protective efficacy in challenge studies

• Live Attenuated Vaccine Improvement:

  • Engineer conditional prfA mutants as potential vaccine strains

  • Combine with other attenuating mutations (e.g., in T3SS or T6SS components )

  • Evaluate safety, immunogenicity, and protective efficacy

  • Assess genetic stability of attenuation

• Diagnostic Applications:

  • Develop antibody-based detection systems using recombinant prfA

  • Create serological assays to monitor vaccine responses

  • Design rapid tests for E. ictaluri infection using anti-prfA antibodies

• Immunomodulatory Studies:

  • Investigate whether prfA has immunomodulatory effects on fish immune cells

  • Examine potential adjuvant properties when co-administered with other antigens

  • Study impact on innate and adaptive immune responses in fish

The search results mention the successful development of live attenuated E. ictaluri vaccines based on T6SS components , suggesting that similar approaches targeting prfA or combining prfA modification with other attenuating strategies could be promising avenues for improved vaccine development.