Recombinant Lactobacillus reuteri Elongation factor Tu (tuf)

What is Elongation factor Tu (EF-Tu) in Lactobacillus reuteri?

Elongation factor Tu (EF-Tu) in Lactobacillus reuteri is a multifunctional protein primarily known for its canonical role in protein synthesis, where it facilitates the selection of aminoacyl-tRNA by bacterial ribosomes. Beyond this function, L. reuteri EF-Tu has been characterized as a cell surface-associated protein that exhibits sulfated carbohydrate-binding properties. Research has demonstrated that EF-Tu is present in cell surface fractions isolated from various Lactobacillus strains, suggesting its role extends beyond protein synthesis to mediating interactions with host tissues, particularly in the gastrointestinal tract .

To study this protein, researchers typically begin with genomic analysis of the tuf gene sequence in L. reuteri strains, followed by recombinant expression in suitable bacterial systems (commonly E. coli) and purification using affinity chromatography methods. Functional assays frequently involve adhesion studies with intestinal cell lines or purified mucin to evaluate binding characteristics.

How does the structure of L. reuteri EF-Tu differ from EF-Tu in other bacterial species?

While the primary structure of EF-Tu is highly conserved across bacterial species as it performs essential translational functions, L. reuteri EF-Tu exhibits specific structural features that contribute to its moonlighting functions. The protein consists of three domains: domain I contains the GTP-binding site, while domains II and III are involved in binding aminoacyl-tRNA during protein synthesis .

Research methodologies to investigate structural differences typically include:

• Comparative sequence analysis between L. reuteri EF-Tu and other bacterial species

• X-ray crystallography or cryo-electron microscopy to determine tertiary structure

• Surface plasmon resonance to evaluate binding characteristics

• Molecular dynamics simulations to identify functional motifs and binding sites

The carbohydrate-binding regions of L. reuteri EF-Tu, particularly those that interact with sulfated carbohydrates, represent unique structural adaptations that distinguish it from EF-Tu proteins in non-probiotic bacteria and contribute to its role in adhesion to gastric mucin .

What experimental systems are suitable for expressing recombinant L. reuteri EF-Tu?

For successful expression of recombinant L. reuteri EF-Tu, researchers should consider:

• Expression vector selection: pET series vectors with histidine tags are commonly used, as demonstrated in studies producing His-EF-Tu for functional analysis .

• Expression host: While E. coli BL21(DE3) is the most common system due to its high yield and ease of use, expression in Lactobacillus-based systems may preserve native folding and post-translational modifications.

• Induction conditions: Optimization of temperature (typically 16-25°C), inducer concentration, and expression duration is critical to maximize soluble protein yield.

• Purification strategy: Immobilized metal affinity chromatography (IMAC) using nickel or cobalt resins is effective for His-tagged proteins, followed by size exclusion chromatography to achieve higher purity.

• Protein solubility: Addition of solubility enhancers like SUMO tags may improve yield of functional protein.

The experimental approach should be tailored to the intended application of the recombinant protein, with particular attention to preserving the carbohydrate-binding functions if adhesion studies are planned .

How can the inhibitory mechanism of recombinant L. reuteri EF-Tu against H. pylori adhesion be characterized at the molecular level?

Characterizing the inhibitory mechanism of L. reuteri EF-Tu against H. pylori adhesion requires a multifaceted approach:

• Binding site mapping: Use site-directed mutagenesis of recombinant EF-Tu to identify specific amino acid residues involved in sulfated carbohydrate binding. Critical residues can be mutated and the resulting variants can be tested for their ability to inhibit H. pylori adhesion.

• Competition assays: Develop quantitative assays using labeled recombinant His-EF-Tu to measure direct competition with H. pylori ligands for binding to sulfated carbohydrates on gastric mucin. Research has shown that His-EF-Tu exhibits concentration-dependent inhibitory effects on H. pylori adhesion to porcine gastric mucin (PGM) .

• Receptor depletion studies: As demonstrated in previous research, treating PGM with sulfatase significantly reduces H. pylori adhesion, and His-EF-Tu shows minimal effect on H. pylori adhesion to sulfatase-treated PGM. This confirms the importance of sulfated carbohydrates in the inhibitory mechanism .

• Structural biology approaches: Employ X-ray crystallography or cryo-EM to visualize the complex of EF-Tu with sulfated carbohydrates, providing insights into the binding interface.

• Molecular dynamics simulations: Model the interaction between EF-Tu, H. pylori adhesins, and sulfated carbohydrates to predict binding energies and identify key interaction points.

The research methodology should incorporate controls using PGM34 antibody (which recognizes sulfated carbohydrates) to compare inhibition rates with recombinant EF-Tu .

What are the methodological challenges in studying EF-Tu's dual functionality in protein synthesis and bacterial adhesion?

Investigating the dual functionality of EF-Tu presents several methodological challenges that researchers must address:

• Functional isolation: Separating EF-Tu's translational role from its adhesin function requires careful experimental design. Site-directed mutagenesis can generate variants that maintain one function while disrupting the other, but this requires precise knowledge of the structural determinants for each function .

• Subcellular localization: Determining how EF-Tu translocates to the cell surface despite lacking typical secretion signals requires specialized techniques:

  • Immunoelectron microscopy to visualize surface-associated EF-Tu

  • Cell fractionation followed by Western blotting to quantify EF-Tu in different cellular compartments

  • Reporter fusion constructs to track protein localization

• Post-translational modifications: Identifying modifications that may differentiate cytoplasmic from surface-associated EF-Tu requires mass spectrometry techniques and proteomic analysis.

• Dynamic analysis: Understanding how EF-Tu's functions are regulated under different conditions requires techniques such as real-time PCR to measure expression levels in response to environmental stimuli .

• In vivo relevance: Translating in vitro findings to physiological contexts requires animal models with appropriate controls and markers to track EF-Tu localization and function in the gastrointestinal environment.

Research has shown that EF-Tu expression can be upregulated when lactobacilli are exposed to gastrointestinal conditions, suggesting adaptive regulation of this dual-function protein .

How can genetic engineering approaches be used to enhance the adhesion-inhibitory properties of L. reuteri EF-Tu?

Genetic engineering strategies to enhance the adhesion-inhibitory properties of L. reuteri EF-Tu include:

• Directed evolution: Create libraries of EF-Tu variants through error-prone PCR and screen for enhanced binding to sulfated carbohydrates and improved inhibition of H. pylori adhesion.

• Domain swapping: Exchange domains between EF-Tu proteins from different Lactobacillus species to identify regions contributing to superior binding characteristics.

• Surface display systems: Develop L. reuteri strains that overexpress EF-Tu on their cell surface to increase the local concentration of this adhesin inhibitor. This approach would require appropriate surface anchoring motifs and expression control systems .

• Fusion proteins: Create chimeric proteins combining EF-Tu with other adhesion inhibitors or antimicrobial peptides to develop multifunctional inhibitory molecules.

• Conditional expression systems: Design genetic circuits that upregulate EF-Tu expression specifically in response to H. pylori presence or in the gastric environment.

Implementation of these approaches requires:

• Efficient transformation protocols for L. reuteri

• Stable integration of modified genes into the chromosome

• Careful assessment of effects on bacterial fitness and survival during gastrointestinal transit

• Functional validation using in vitro adhesion assays and animal models

Recent advances in recombineering techniques have enabled efficient genetic manipulation of L. reuteri strains, facilitating the development of modified EF-Tu variants with enhanced therapeutic potential .

What are the optimal conditions for purifying recombinant L. reuteri EF-Tu while maintaining its carbohydrate-binding activity?

Purification of recombinant L. reuteri EF-Tu with preserved carbohydrate-binding activity requires careful attention to several parameters:

• Expression conditions:

  • Induction at lower temperatures (16-20°C)

  • Extended expression periods (overnight)

  • Reduced inducer concentration to promote proper folding

• Lysis buffer composition:

  • pH 7.5-8.0 (physiological range)

  • Inclusion of stabilizing agents such as 5-10% glycerol

  • Addition of protease inhibitors to prevent degradation

  • Mild detergents (0.1% Triton X-100) may help solubilize membrane-associated forms

• Purification steps:

  • IMAC using Ni-NTA for His-tagged EF-Tu

  • Ion exchange chromatography to remove contaminants

  • Size exclusion chromatography as a final polishing step

  • Avoid harsh elution conditions that might denature the protein

• Activity preservation:

  • Include sulfated carbohydrates or their analogs in storage buffers

  • Store at -80°C in small aliquots to avoid freeze-thaw cycles

  • Consider lyophilization with appropriate cryoprotectants

• Validation of activity:

  • Develop a binding assay using porcine gastric mucin (PGM)-coated plates

  • Measure concentration-dependent inhibition of H. pylori adhesion to confirm functionality

  • Use circular dichroism to confirm proper folding

The quality of the purified recombinant EF-Tu should be assessed using multiple criteria, including SDS-PAGE for purity, Western blotting for identity, and functional assays for binding activity .

How can researchers quantitatively measure the interaction between L. reuteri EF-Tu and sulfated carbohydrates?

Quantitative measurement of L. reuteri EF-Tu interactions with sulfated carbohydrates can be performed using several complementary techniques:

• Surface Plasmon Resonance (SPR):

  • Immobilize sulfated carbohydrates or gastric mucin on sensor chips

  • Flow recombinant EF-Tu at various concentrations to determine association/dissociation kinetics

  • Calculate binding constants (Ka, Kd) and thermodynamic parameters

• Isothermal Titration Calorimetry (ITC):

  • Directly measure heat changes during binding events

  • Determine stoichiometry, binding constants, and thermodynamic parameters (ΔH, ΔS, ΔG)

• Microscale Thermophoresis (MST):

  • Measure changes in movement of fluorescently labeled EF-Tu in temperature gradients upon binding

  • Requires minimal sample amounts and works in solution

• Fluorescence-based assays:

  • Develop competitive binding assays using fluorescently labeled sulfated carbohydrates

  • Measure displacement by unlabeled compounds to determine relative affinities

• Enzyme-Linked Immunosorbent Assay (ELISA):

  • Coat plates with gastric mucin or specific sulfated carbohydrates

  • Detect bound His-EF-Tu using anti-His antibodies

  • Compare binding before and after sulfatase treatment of substrates

Control experiments should include:

• PGM34 antibody as a positive control for sulfated carbohydrate recognition

• Sulfatase-treated substrates to confirm specificity

• Heat-denatured EF-Tu as a negative control

Researchers have demonstrated that sulfatase treatment of PGM reduces H. pylori adhesion and diminishes the inhibitory effect of His-EF-Tu, confirming the specificity of the interaction with sulfated carbohydrates .

What analytical methods are most effective for studying the conformational changes in EF-Tu during its interaction with ribosomes versus its adhesin function?

To investigate the distinct conformational states of EF-Tu in its ribosomal versus adhesin functions, researchers should employ these analytical methods:

• Cryo-electron microscopy (Cryo-EM):

  • Captures EF-Tu in different functional states

  • Can resolve structures at near-atomic resolution

  • Enables visualization of EF-Tu bound to ribosomes versus in its adhesin conformation

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps regions with different solvent accessibility in various functional states

  • Identifies conformational changes without requiring crystallization

  • Can be performed in near-physiological conditions

• Single-molecule Fluorescence Resonance Energy Transfer (smFRET):

  • Labels specific domains of EF-Tu with fluorophore pairs

  • Monitors real-time conformational changes during different functional interactions

  • Has been successfully used to study EF-Tu dynamics on ribosomes

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Provides atomic-level information on protein dynamics

  • Can detect local conformational changes and binding interfaces

  • Most effective for isolated domains or smaller fragments of EF-Tu

• Molecular Dynamics simulations:

  • Models conformational transitions between different functional states

  • Identifies key residues involved in conformational switching

  • Generates hypotheses for experimental validation

Research has demonstrated that EF-Tu undergoes significant conformational rearrangements during its interaction with the ribosome, particularly after GTP hydrolysis . Comparative analysis of these states with the conformation adopted during sulfated carbohydrate binding would provide valuable insights into the structural basis of EF-Tu's functional duality.

How can recombinant L. reuteri EF-Tu be developed as a therapeutic agent for H. pylori infection?

Development of recombinant L. reuteri EF-Tu as a therapeutic agent against H. pylori infection requires a systematic approach:

• Formulation optimization:

  • Stability studies to determine optimal pH, temperature, and excipients

  • Protection strategies for gastrointestinal transit (encapsulation, pH-responsive delivery)

  • Dosage determination through dose-response studies in animal models

• Preclinical efficacy testing:

  • In vitro inhibition assays against diverse clinical H. pylori isolates

  • Ex vivo adhesion studies using human gastric tissue explants

  • Animal models of H. pylori infection to assess colonization reduction

• Combination therapy evaluation:

  • Synergy testing with conventional antibiotics

  • Co-administration with other probiotic strains or components

  • Integration with existing H. pylori eradication protocols

• Genetic engineering approaches:

  • Development of L. reuteri strains with enhanced EF-Tu expression

  • Creation of genetically modified L. reuteri with reduced colonization potential but maintained EF-Tu functionality

  • Design of chimeric proteins combining EF-Tu with other anti-H. pylori effectors

• Bioavailability and pharmacokinetic studies:

  • Transit time and degradation rates in gastrointestinal conditions

  • Mucoadhesion studies to determine residence time at gastric epithelium

  • Tissue distribution and clearance profiles

Research has demonstrated that recombinant His-EF-Tu exhibits concentration-dependent inhibition of H. pylori adhesion to gastric mucin, suggesting potential therapeutic applications . Additionally, engineered L. reuteri strains with modified adhesin profiles retain their ability to survive gastrointestinal transit, making them promising candidates for therapeutic delivery .

What experimental approaches can evaluate the immunomodulatory effects of L. reuteri EF-Tu in inflammatory disease models?

To evaluate the immunomodulatory effects of L. reuteri EF-Tu in inflammatory disease models, researchers should implement these approaches:

• In vitro immune cell assays:

  • Dendritic cell maturation and cytokine production profiles

  • T-cell polarization (Th1, Th2, Th17, Treg) in response to EF-Tu exposure

  • Macrophage activation states and inflammatory mediator production

  • Intestinal epithelial cell inflammatory responses and barrier function

• Ex vivo tissue explant cultures:

  • Cytokine production from intestinal tissue biopsies exposed to EF-Tu

  • Histological assessment of tissue inflammatory markers

  • Gene expression analysis of inflammatory pathway components

• Animal models of inflammation:

  • DSS-induced or TNBS-induced colitis models

  • Radiation-induced intestinal inflammation

  • H. pylori-induced gastritis models

  • Systemic inflammation models (endotoxemia)

• Mechanistic studies:

  • Pattern recognition receptor binding assays (TLRs, NODs)

  • Signaling pathway analysis (NF-κB, MAPK, STAT)

  • Epigenetic modifications in immune cells after EF-Tu exposure

  • Microbiome analysis to assess indirect effects via microbiota modulation

• Translational relevance:

  • Correlation studies between EF-Tu exposure and inflammatory biomarkers

  • Comparative analysis with other L. reuteri components known to have immunomodulatory effects

L. reuteri strains have demonstrated the ability to reduce pro-inflammatory cytokine production while promoting regulatory T cell development and function . Research into whether EF-Tu specifically contributes to these effects would provide valuable insights for therapeutic applications in inflammatory diseases.

How does the function and structure of EF-Tu differ across Lactobacillus species, and what are the implications for host interactions?

To investigate functional and structural differences in EF-Tu across Lactobacillus species and their implications for host interactions, researchers should employ these methodologies:

• Comparative genomics:

  • Phylogenetic analysis of tuf genes across Lactobacillus species

  • Identification of conserved domains versus variable regions

  • Analysis of selection pressure on different protein regions

• Structural comparison:

  • Homology modeling of EF-Tu proteins from different Lactobacillus species

  • Superimposition analysis to identify structural deviations

  • Surface charge and hydrophobicity mapping to predict interaction potential

• Functional comparative assays:

  • Side-by-side binding assays to sulfated carbohydrates

  • Comparison of inhibitory effects against pathogen adhesion

  • Species-specific differences in subcellular localization

• Host-specificity testing:

  • Binding assays using mucins from different host species

  • Comparison of adhesion to cell lines derived from different hosts

  • Analysis of host-specific adaptations in EF-Tu sequence

• Data correlation and analysis:

  • Statistical methods to correlate sequence/structural features with functional properties

  • Machine learning approaches to identify predictive features for host interaction

  • Database development of EF-Tu variants and their functional characteristics

Research has shown that EF-Tu is present in cell surface fractions of several Lactobacillus strains , suggesting conservation of this moonlighting function across species. Comparative studies would reveal whether species-specific adaptations in EF-Tu structure contribute to host-specific colonization patterns observed in various Lactobacillus species .

What experimental protocols can best determine the role of EF-Tu in mediating bacterial interactions within complex microbial communities?

To investigate EF-Tu's role in bacterial interactions within complex microbial communities, researchers should implement these experimental protocols:

• Labeled protein interaction studies:

  • Fluorescently labeled recombinant EF-Tu to track binding to different bacterial species

  • Co-immunoprecipitation assays to identify bacterial binding partners

  • Surface plasmon resonance to quantify interspecies binding affinities

• In vitro community modeling:

  • Continuous culture systems (chemostats) with defined microbial communities

  • Addition of recombinant EF-Tu or EF-Tu-overexpressing strains to observe community shifts

  • Multi-omics analysis (metagenomics, metatranscriptomics, metaproteomics) to track community responses

• Genetic approaches:

  • Generation of EF-Tu variants with altered surface expression

  • Complementation studies in EF-Tu-deficient backgrounds

  • CRISPR interference to modulate EF-Tu expression levels without complete knockout

• Biofilm studies:

  • Mixed-species biofilm formation assays with and without EF-Tu supplementation

  • Confocal microscopy with fluorescently labeled bacteria to observe spatial organization

  • Biofilm matrix analysis to identify EF-Tu-dependent changes in extracellular polymeric substances

• Advanced microscopy techniques:

  • Super-resolution microscopy to visualize EF-Tu localization at bacterial interfaces

  • Atomic force microscopy to measure cell-cell adhesion forces

  • Live-cell imaging to track dynamics of bacterial interactions

Research has demonstrated that L. reuteri can inhibit the colonization of pathogenic microbes and remodel commensal microbiota composition in the host . Investigating whether EF-Tu contributes to these interactions would provide insights into the molecular mechanisms of probiotic activity and microbiome modulation.

What are the key knowledge gaps in understanding the molecular mechanisms of L. reuteri EF-Tu's dual functionality?

Despite significant advances in our understanding of L. reuteri EF-Tu, several critical knowledge gaps remain that warrant further investigation:

Research addressing these knowledge gaps would require interdisciplinary approaches combining structural biology, biochemistry, microbiology, and computational methods .

How might synthetic biology approaches expand the applications of engineered L. reuteri EF-Tu variants?

Synthetic biology offers exciting possibilities for expanding the applications of engineered L. reuteri EF-Tu variants:

• Domain fusion engineering:

  • Creation of chimeric proteins combining EF-Tu with other functional domains

  • Development of bifunctional molecules targeting multiple pathogenic mechanisms

  • Design of EF-Tu variants with enhanced binding affinity through directed evolution

• Controlled expression systems:

  • Inducible promoters responsive to specific environmental signals (pH, bile, pathogen presence)

  • Oscillatory expression systems to provide pulsed delivery of EF-Tu

  • Cell density-dependent expression to ensure optimal concentrations at colonization sites

• Delivery vehicle optimization:

  • Engineering L. reuteri strains with modified adhesin profiles for targeted tissue delivery

  • Development of controlled-release systems for recombinant EF-Tu

  • Creation of EF-Tu-displaying bacterial ghosts or membrane vesicles

• Circuit design for therapeutic applications:

  • Sense-and-respond systems that detect pathogen presence and upregulate EF-Tu

  • Genetic toggle switches to maintain therapeutic EF-Tu production after initial stimulus

  • Memory circuits to record exposure to gastrointestinal pathogens

• Novel interaction partners:

  • Screening of synthetic peptide libraries to identify partners enhancing EF-Tu functions

  • Computer-aided design of novel binding interfaces on EF-Tu

  • Incorporation of non-natural amino acids to introduce new functionalities

Research has already demonstrated the feasibility of engineering L. reuteri strains with modified adhesin profiles that maintain their ability to survive gastrointestinal transit and deliver therapeutic molecules in vivo . Building on this foundation, synthetic biology approaches could significantly expand the range of applications for engineered EF-Tu variants in biomedical research and therapeutic development.