Recombinant Campylobacter fetus subsp. fetus Elongation factor Tu (tuf)

What is Campylobacter fetus and why is it significant in research?

Campylobacter fetus comprises two closely related mammal-associated subspecies: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv). Cff is primarily found in the gastrointestinal tract of healthy ruminants but can cause sporadic abortions in cattle and more frequently in sheep. It's also recognized as an opportunistic pathogen in humans, particularly in immunocompromised patients .

The significance of C. fetus in research stems from:

• Its role as an emerging pathogen with veterinary and public health importance

• The economic impact of bovine genital campylobacteriosis on the cattle industry

• Its potential as a model organism for studying bacterial host adaptation

• The distinct ecological niches occupied by its subspecies despite their close genetic relatedness

How do recombinant technologies advance C. fetus research?

Recombinant technologies have significantly improved our ability to study C. fetus, which was previously hampered by a lack of genetic tools. Key advances include:

• Development of Escherichia coli-Campylobacter shuttle vectors specifically designed for C. fetus

• Introduction of C. fetus-specific promoters for efficient gene expression

• Creation of compatible plasmid systems for simultaneous expression of multiple genes

• Application of reporter gene systems for monitoring gene expression in vivo

These technologies enable researchers to:

• Complement mutant phenotypes

• Express reporter genes to track bacterial localization and gene expression

• Investigate virulence mechanisms through genetic manipulation

• Study host-pathogen interactions at the molecular level

What are the optimal conditions for expressing recombinant C. fetus Elongation factor Tu?

Based on protocols for related recombinant proteins, optimal expression of C. fetus Elongation factor Tu can be achieved using the following methodology:

Expression System Selection:

• E. coli BL21(DE3) or equivalent strain for high-level expression

• Mammalian cell expression systems for proper folding and post-translational modifications

Expression Vector Considerations:

• Incorporate a strong C. fetus promoter (essential for expression in C. fetus hosts)

• Include a 6xHis or other affinity tag for purification

• Consider fusion partners (like MBP or GST) to enhance solubility

Culture Conditions:

• Temperature: 30°C (rather than 37°C) often improves protein solubility

• Induction: 0.5-1.0 mM IPTG for E. coli systems

• Growth medium: Rich media (like LB with appropriate antibiotics)

• Incubation: Microaerobic conditions are essential when expressing in Campylobacter hosts (3-5 days for C. jejuni, 8-14 days for C. fetus)

What purification strategies are most effective for recombinant C. fetus Elongation factor Tu?

A multi-step purification approach is recommended for obtaining high-purity recombinant Elongation factor Tu:

• Initial Capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged proteins

  • Ensure lysis buffer contains 20-50 mM imidazole to reduce non-specific binding

• Intermediate Purification:

  • Ion exchange chromatography (typically Q-Sepharose) to separate based on charge differences

  • Buffer conditions: pH 7.5-8.0 (slightly above the predicted pI of the protein)

• Polishing Step:

  • Size exclusion chromatography to remove aggregates and achieve >95% purity

  • Buffer recommendation: 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 1 mM DTT

• Storage Recommendations:

  • Add 5-50% glycerol (final concentration) to purified protein

  • Aliquot and store at -20°C/-80°C to avoid repeated freeze-thaw cycles

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

How can I verify the functionality of recombinant C. fetus Elongation factor Tu?

Functional verification of recombinant Elongation factor Tu can be accomplished through several complementary approaches:

Biochemical Activity Assays:

• GTP Binding and Hydrolysis:

  • Measure GTP binding using fluorescent GTP analogs

  • Quantify GTPase activity through malachite green phosphate assay

• Aminoacyl-tRNA Binding:

  • Assess binding to aminoacyl-tRNA using filter binding assays

  • Fluorescence anisotropy with labeled tRNAs

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy:

  • Confirm proper secondary structure composition

  • Compare with native protein if available

• Thermal Shift Assays:

  • Measure protein stability under various conditions

  • Optimize buffer conditions for maximum stability

Functional Complementation:

• Transform the recombinant tuf gene into a conditional E. coli tuf mutant

• Assess the ability to restore growth under non-permissive conditions

How can recombinant C. fetus Elongation factor Tu be used in studying host-pathogen interactions?

Recombinant C. fetus Elongation factor Tu offers several approaches for investigating host-pathogen interactions:

Immunological Studies:

• Elongation factor Tu can be released extracellularly during infection and may interact with host cells

• The recombinant protein can be used to:

  • Study binding to host cell receptors

  • Investigate immune recognition by host Pattern Recognition Receptors (PRRs)

  • Examine its potential role in modulating host immune responses

Structural Biology Applications:

• Crystal structures of recombinant Elongation factor Tu can reveal:

  • Potential binding sites for host molecules

  • Structural differences from host translation factors

  • Epitopes recognized by host antibodies

In vivo Studies:

• Tagged recombinant Elongation factor Tu expressed from compatible shuttle vectors can:

  • Track protein localization during infection

  • Identify potential moonlighting functions beyond translation

  • Serve as a reporter for C. fetus growth and metabolism in host tissues

What genetic tools are available for manipulating the tuf gene in C. fetus?

Recent developments in C. fetus genetic tools have expanded options for tuf gene manipulation:

Vector Systems:

• pIP1455-based Vectors:

  • Wide host range across Campylobacter species

  • Require C. fetus-specific promoters for gene expression

  • Compatible with C. fetus subsp. fetus and C. fetus subsp. venerealis

• pCFV108-based Vectors:

  • C. fetus-specific with smaller size

  • Compatible with pIP1455-based vectors for co-expression

  • Stable maintenance in C. fetus subsp. venerealis for extended periods

Genetic Manipulation Techniques:

• Homologous recombination for chromosomal integration

• Allelic exchange for gene replacement

• Complementation of mutant phenotypes (demonstrated with gyrA)

Expression Control Systems:

• Strong C. fetus promoters for constitutive expression

• Inducible expression systems adapted for C. fetus

Transfer Methods:

How does sequence variation in the tuf gene contribute to C. fetus subspecies differentiation?

The tuf gene shows potential as a molecular marker for C. fetus subspecies differentiation, with important research implications:

Sequence Conservation and Variation:

• The tuf gene is generally highly conserved due to its essential function

• Subtle sequence variations may exist between C. fetus subspecies

• These variations can be exploited for molecular diagnostics and evolutionary studies

Pangenomic Context:

• Comparative genomic analysis of C. fetus strains reveals:

  • Core genome conservation across subspecies

  • Accessory genome variations that correlate with host adaptation

  • Potential horizontal gene transfer events affecting translation machinery genes

  • Subspecies-specific gene content (such as the pqqL gene identified as Cfv-specific)

Diagnostic Applications:

• PCR targeting tuf gene polymorphisms could complement existing molecular methods

• Multi-locus sequence typing (MLST) schemes including tuf may improve discrimination

• Whole genome sequencing provides the most accurate subspecies identification

What challenges exist in working with recombinant C. fetus proteins and how can they be addressed?

Researchers face several challenges when working with recombinant C. fetus proteins, including Elongation factor Tu:

Challenge 1: Microaerophilic Growth Requirements

• C. fetus requires microaerobic conditions (reduced oxygen) for optimal growth

• Solution: Use specialized incubation systems (gas packs, anaerobic jars, or tri-gas incubators)

• Alternative: Express recombinant proteins in heterologous hosts (E. coli)

Challenge 2: Slow Growth Rate

• C. fetus grows slower than many other bacteria (8-14 days versus 3-5 days for C. jejuni)

• Solution: Plan experiments with extended timeframes and optimize media formulations

• Alternative: Use shuttle vectors with dual expression capability in faster-growing hosts

Challenge 3: Genetic Instability

• The sap locus in C. fetus undergoes high-frequency DNA rearrangements

• Solution: Verify genetic stability through frequent sequencing and phenotypic checks

• Strategy: Use stable genomic integration rather than plasmid-based expression when possible

Challenge 4: Post-translational Modifications

• Bacterial post-translational modifications may differ between expression systems

• Solution: Consider native purification from C. fetus when modifications are critical

• Alternative: Use eukaryotic expression systems (insect or mammalian cells) for specific modifications

How can recombinant C. fetus Elongation factor Tu contribute to improved diagnostic methods?

Recombinant C. fetus Elongation factor Tu offers promising applications for enhancing diagnostic capabilities:

Serological Diagnostics:

• Recombinant Elongation factor Tu can serve as an antigen in ELISA-based assays

• Potential advantages over whole-cell antigens:

  • Improved standardization of diagnostic reagents

  • Enhanced specificity through use of a defined antigen

  • Potential for differentiating immune responses to specific C. fetus antigens

Development of Novel Molecular Tests:

• Anti-Elongation factor Tu antibodies can be used in:

  • Immunofluorescence assays with improved sensitivity

  • Capture ELISA systems for direct detection from clinical samples

  • Lateral flow immunochromatographic tests for field diagnostics

Comparison with Current Diagnostic Methods:

These methods could significantly improve the diagnosis of bovine genital campylobacteriosis and human C. fetus infections .

What role might C. fetus Elongation factor Tu play in vaccine development?

Elongation factor Tu shows potential as a vaccine candidate against C. fetus infections:

Advantages as a Vaccine Antigen:

• Highly conserved across strains, potentially providing broad protection

• Surface-exposed in some conditions, making it accessible to immune recognition

• Essential for bacterial survival, limiting escape mutants

• Demonstrated immunogenicity in other bacterial species

Vaccine Development Approaches:

• Subunit Vaccines:

  • Recombinant Elongation factor Tu alone or combined with other antigens

  • Formulated with appropriate adjuvants for veterinary use

• DNA Vaccines:

  • Utilizing the tuf gene in expression vectors

  • Potentially co-expressing immunostimulatory molecules

• Attenuated Live Vaccines:

  • C. fetus strains engineered to overexpress Elongation factor Tu

  • Utilizing the genetic tools developed for C. fetus manipulation

Research Considerations:

• Evaluate cross-protection against both subspecies

• Assess duration of immunity

• Determine correlates of protection

• Consider route of administration for optimal mucosal immunity

How does C. fetus Elongation factor Tu compare structurally and functionally with homologs from other Campylobacter species?

Comparative analysis of Elongation factor Tu across Campylobacter species reveals important structural and functional insights:

Sequence Conservation:

• High sequence similarity expected between C. fetus and C. jejuni Elongation factor Tu

• Conserved functional domains including:

  • GTP-binding domain

  • tRNA-binding domain

  • Ribosome-binding regions

Functional Comparisons:

• Core translational functions are highly conserved

• Species-specific adaptations may relate to:

  • Optimal temperature for activity (37°C for C. jejuni vs. variable for C. fetus)

  • pH optima reflecting niche adaptation

  • Interactions with species-specific translation factors

Structural Features:

• Predicted three-dimensional structure based on C. jejuni homolog:

  • Three-domain architecture typical of bacterial Elongation factor Tu

  • Species-specific surface residues that may interact with host molecules

  • Conservation of GTP-binding pocket across species

Evolutionary Implications:

• Phylogenetic analysis using tuf sequences can provide insights into:

  • Evolutionary relationships within the Campylobacter genus

  • Host adaptation processes

  • Horizontal gene transfer events

What emerging technologies might enhance our understanding of C. fetus Elongation factor Tu functions?

Several cutting-edge technologies show promise for advancing our understanding of C. fetus Elongation factor Tu:

Cryo-Electron Microscopy:

• Enables visualization of Elongation factor Tu interactions with the ribosome

• Can capture dynamic states during translation

• Potential to reveal species-specific structural features

CRISPR-Cas9 Gene Editing:

• Precise modification of the tuf gene in its native context

• Creation of conditional mutations to study essential functions

• Analysis of the effects of specific mutations on C. fetus virulence

Transcriptomics and Proteomics:

• RNA-Seq to examine tuf expression under different conditions

• Proteomics to identify interaction partners beyond the translation machinery

• Analysis of post-translational modifications specific to C. fetus

Single-Cell Technologies:

• Tracking Elongation factor Tu dynamics in individual bacterial cells

• Examining heterogeneity in expression and localization

• Correlating with virulence phenotypes at the single-cell level

How might comparative studies of C. fetus subspecies advance our understanding of bacterial adaptation?

Comparative studies of C. fetus subspecies offer valuable insights into bacterial adaptation:

Genomic Comparisons:

• Recent pangenomic analysis of C. fetus isolates revealed:

  • Distinct clustering patterns correlating with subspecies designation

  • Novel sequence types (STs) specific to certain geographic regions

  • Subspecies-specific genes potentially involved in niche adaptation

  • Variations in accessory genome content reflecting different ecological niches

Functional Genomics Approaches:

• Transcriptomic comparisons between subspecies can reveal:

  • Differential gene expression patterns in response to host environments

  • Regulation of core genes (including tuf) across subspecies

  • RNA-based regulatory mechanisms

Experimental Evolution:

• Laboratory-based evolution experiments can track:

  • Adaptations to different host environments

  • Changes in translation machinery components

  • Selection pressures on highly conserved genes like tuf

Host-Pathogen Interaction Models:

• Comparative analysis of subspecies behavior in:

  • Cell culture infection models

  • Organ-on-chip systems

  • Animal models reflecting natural hosts

What are the implications of C. fetus research for understanding bacterial pathogenesis more broadly?

Research on C. fetus and its Elongation factor Tu has broader implications for understanding bacterial pathogenesis:

Moonlighting Functions of Conserved Proteins:

• Elongation factor Tu in various bacteria has been shown to:

  • Act as an adhesin for host cell attachment

  • Interact with components of the host immune system

  • Contribute to biofilm formation

  • Respond to environmental stresses beyond its role in translation

Host Adaptation Mechanisms:

• C. fetus subspecies provide a model for studying:

  • The molecular basis of host specificity

  • Evolution of pathogenicity in related bacterial lineages

  • Minimal genetic changes leading to distinct ecological niches

Bacterial Surface Architecture:

• C. fetus surface layer proteins (SLPs) represent a sophisticated virulence mechanism:

  • High-frequency DNA rearrangements generate antigenic variation

  • Multiple sapA homologues can be expressed

  • Surface proteins interact with host molecules

  • Potential interaction between Elongation factor Tu and surface structures

Implications for Other Pathogens:

• Insights gained from C. fetus research may inform studies of:

  • Other Campylobacter species pathogenesis

  • Mechanisms of persistent infection in other bacteria

  • Evolution of host-restricted bacterial pathogens

  • Roles of highly conserved proteins in virulence