Recombinant Campylobacter jejuni subsp. jejuni serotype O:6 Elongation factor Ts (tsf)

What methodologies are effective for cloning and expressing recombinant tsf from C. jejuni?

Cloning and expression strategies for recombinant tsf typically involve His tag affinity chromatography for purification, as demonstrated in studies on Pseudomonas aeruginosa EF-Ts . Key steps include:

• Gene cloning: Amplification of the tsf gene via PCR, followed by insertion into expression vectors (e.g., pET or pUC19 derivatives) with appropriate restriction sites .

• Sequence validation: Confirmation of sequence homology with E. coli EF-Ts (55% identity in P. aeruginosa) to predict functional domains and ensure proper folding .

• Expression optimization: Use of E. coli systems (e.g., BL21(DE3)) for high-yield production, followed by purification to >95% homogeneity .

Functional validation is critical, including GDP exchange assays to assess nucleotide binding kinetics (e.g., K_D for GDP: 30–75 nM; GTP: 125–200 nM) .

How does the interaction between tsf and EF-Tu affect protein synthesis in C. jejuni?

tsf facilitates GDP/GTP exchange on EF-Tu, enabling the latter to recycle into the active GTP-bound state for ternary complex formation with aminoacylated tRNA. Key findings include:

• Kinetic enhancement: EF-Ts (tsf) increases GDP exchange rates by 10-fold compared to EF-Tu alone, reducing K_D from 33 μM to 2 μM .

• Ternary complex formation: Recombinant tsf supports EF-Tu-GTP-tRNA interaction, critical for ribosomal elongation in P. aeruginosa systems, suggesting conserved mechanisms in C. jejuni .

• Stress adaptation: EF-Ts may stabilize EF-Tu under stress, as implied by C. jejuni’s broader stress tolerance compared to other enterobacteria .

What are the key considerations when designing functional assays to study tsf’s role in vivo?

Functional assays for tsf require addressing host-pathogen dynamics and mucosal interactions:

• Mucosal delivery models: Chitosan-based nanoparticles (CS-TPP) for intra-gastric immunization, as shown for C. jejuni Hcp protein, enhance intestinal sIgA and systemic IgY responses .

• Competitive colonization: Use of isogenic tagged strains (e.g., antibiotic resistance cassettes) to track C. jejuni colonization efficiency in chicken models .

• Stress response integration: Incorporation of stressors (e.g., oxidative agents) to mimic in vivo conditions, given C. jejuni’s unique stress adaptation mechanisms .

How do sequence homologies between C. jejuni tsf and other bacteria’s EF-Ts influence experimental design?

Sequence homology data inform domain-specific mutagenesis and cross-species functional studies:

• Conserved regions: High homology in GDP-binding domains (e.g., 55% identity with E. coli) allows extrapolation of kinetic parameters (e.g., K_D values) .

• Divergent regions: Unique motifs in C. jejuni tsf may require species-specific assays (e.g., poly(U)-dependent ribosomal binding) to validate activity .

What challenges exist in translating in vitro findings on tsf to in vivo models, and how to address them?

Translational challenges include host-specific factors and stress heterogeneity:

How to analyze data contradictions when comparing tsf’s role in different Campylobacter strains or hosts?

Data discrepancies often stem from experimental variability or strain-specific traits:

• Strain heterogeneity: Sialylated LOS (e.g., serotype O:6) enhances invasion in certain strains, requiring ganglioside mimicry analysis to contextualize tsf findings .

• Host model differences: Chicken vs. murine models may show divergent immune responses; species-specific qPCR primers for tag tracking mitigate this in mixed infections .

What are the best practices for purifying recombinant tsf to ensure functional activity?

Purification protocols must prioritize activity retention:

• His tag chromatography: Elute at low imidazole concentrations (<50 mM) to avoid denaturation .

• Gel filtration: Validate oligomeric state (e.g., monomeric vs. dimeric) and exclude aggregates .

• Functional testing: Conduct GDP binding assays (e.g., filter-based assays) post-purification to confirm activity .

How can tsf be targeted in vaccine development, and what delivery methods are effective?

tsf’s role in translation makes it a vaccine candidate, though efficacy depends on immune targeting:

• Mucosal immunization: Chitosan nanoparticles enhance intestinal sIgA responses, reducing cecal colonization in chickens .

• Systemic vs. local immunity: Alum-based adjuvants (e.g., IFA) may boost IgY titers but are less effective than mucosal routes for gut clearance .

What are the implications of tsf’s stress-related functions for antibiotic resistance studies?

tsf’s interaction with EF-Tu may influence stress-driven persistence:

• Stress adaptation: EF-Tu-Ts recycling under oxidative stress could enhance survival, potentially modulating antibiotic efficacy .

• Target validation: Inhibitors disrupting GDP exchange (e.g., small molecules) could be tested for synergy with antibiotics .

How to design experiments to study tsf’s interaction with other virulence factors in C. jejuni?

Synergy studies require combinatorial approaches:

What are the limitations of current tsf research, and how to address them?

Knowledge gaps:

• Structural data: Limited X-ray crystallography for C. jejuni tsf; prioritize cryo-EM for conformational studies.

• Host-pathogen dynamics: Need for multi-omics (e.g., proteomics/metabolomics) to map tsf’s regulatory network .