Recombinant Bifidobacterium adolescentis Methionyl-tRNA formyltransferase (fmt)

What is Methionyl-tRNA formyltransferase (fmt) and what is its role in bacterial translation?

Methionyl-tRNA formyltransferase (fmt) is an essential enzyme that catalyzes the formylation of methionyl-tRNA (Met-tRNAfMet) to produce formyl-methionyl-tRNA (fMet-tRNAfMet). This formylation is critical for efficient translation initiation in bacteria, mitochondria, and chloroplasts . The reaction involves transferring a formyl group from 10-formyl-tetrahydrofolate (10-CHO-THF) to the alpha-amino group of methionine attached to tRNAfMet.

Recent studies have demonstrated that fmt can also utilize 10-formyl-dihydrofolate (10-CHO-DHF) as an alternative formyl group donor . This flexibility in substrate usage may have implications for bacterial survival under various metabolic conditions.

The formylation process is particularly important for translation initiation fidelity, as it helps distinguish initiator tRNA from elongator tRNAs. Studies have shown that mutations in human mitochondrial MTF can significantly reduce mitochondrial translation efficiency, leading to combined oxidative phosphorylation deficiency and conditions such as Leigh syndrome .

To assess fmt activity experimentally, researchers can use in vitro formylation assays where:

• Deacylated tRNA preparations are charged with methionine using methionyl-tRNA synthetase (MetRS)

• The Met-tRNAfMet is then incubated with fmt and formyl donors

• Formylated Met-tRNAfMet can be detected using acid urea PAGE and Northern blotting with tRNAfMet-specific probes

Which vectors are most effective for gene expression in Bifidobacterium adolescentis?

For heterologous gene expression in Bifidobacterium adolescentis, several Escherichia coli/Bifidobacterium shuttle vectors have demonstrated efficacy. The table below summarizes key vectors and their properties:

The pMB1 replicon has been specifically demonstrated to work in B. adolescentis, using the theta replication mechanism which generally offers greater stability than rolling circle plasmids . When selecting an appropriate vector for fmt expression, consider:

• Replicon stability in the target strain

• Selection marker compatibility with your experimental system

• Promoter strength and inducibility requirements

• Insert size limitations

For optimal results, preliminary transformation efficiency tests with different vectors should be conducted with your specific B. adolescentis strain before proceeding with fmt cloning.

How do cultivation conditions affect transformation efficiency in B. adolescentis?

Transformation efficiency in B. adolescentis is significantly influenced by cultivation conditions prior to and during the transformation process. The following methodological considerations are critical:

Growth Medium Composition:

• MRS medium supplemented with 0.05% L-cysteine hydrochloride provides optimal anaerobic conditions for B. adolescentis growth

• Addition of 0.5% glucose can enhance cell density and competence development

• Presence of glycine (1-2%) in pre-transformation culture weakens the cell wall, improving DNA uptake

Growth Phase and Cell Density:

• Early to mid-logarithmic phase cells (OD600 0.4-0.6) typically yield higher transformation frequencies

• Older cultures demonstrate reduced competence due to changes in cell wall structure and composition

Buffer Composition for Electroporation:

• Sucrose-based buffers (typically 0.5M sucrose) maintain osmotic balance during electroporation

• Addition of glycerol (10%) can improve cell viability post-electroporation

• Ensuring the buffer is ice-cold throughout the procedure preserves cell integrity

Optimization of these parameters can increase transformation efficiency by 1-2 orders of magnitude, which is particularly important when working with recombinant B. adolescentis strains expressing fmt.

How can successful expression of fmt in recombinant B. adolescentis be verified?

Verification of successful fmt expression in recombinant B. adolescentis requires a multi-faceted approach:

Genetic Verification:

• PCR amplification of the fmt gene from plasmid DNA extracted from transformants

• DNA sequencing to confirm the absence of mutations in the fmt coding region

• RT-PCR and qRT-PCR to verify transcription of the fmt gene

Protein Expression Analysis:

• Western blotting using anti-fmt antibodies or antibodies against an epitope tag fused to fmt

• SDS-PAGE coupled with mass spectrometry to detect and identify the fmt protein

• Enzyme-linked immunosorbent assay (ELISA) for quantitative measurement of fmt expression levels

Functional Assays:

• In vitro formylation assay using cell lysates and Met-tRNAfMet substrate

• Monitoring the formation of fMet-tRNAfMet using acid urea PAGE and Northern blotting

• Comparative analysis of translation initiation efficiency between wild-type and recombinant strains

Successful expression should be confirmed by at least one method from each category to ensure both the presence and functionality of the recombinant fmt protein.

What technical challenges exist in expressing heterologous fmt in B. adolescentis?

Expression of heterologous fmt in Bifidobacterium adolescentis presents several technical challenges that researchers should anticipate and address:

Codon Usage Bias:
B. adolescentis has a high GC content (approximately 59-60%) which creates a distinct codon usage bias. Heterologous fmt genes may contain rare codons that limit translation efficiency in B. adolescentis. Codon optimization of the fmt gene sequence for B. adolescentis is recommended.

Protein Folding and Stability:
The cytoplasmic environment of B. adolescentis may differ from the source organism of the fmt gene, potentially affecting protein folding and stability. Consider:

• Adding chaperone co-expression systems to assist proper folding

• Including stabilizing domains or fusion partners

• Testing multiple N- and C-terminal fusion configurations

Anaerobic Expression Conditions:
B. adolescentis is a strict anaerobe, and protein expression must occur under oxygen-free conditions. This complicates:

• Experimental setup requiring specialized anaerobic chambers

• Potential oxygen exposure during sampling affecting enzyme activity

• Challenges in scaling up production while maintaining anaerobic conditions

Enzymatic Activity Requirements:
Fmt requires specific cofactors like 10-CHO-THF or 10-CHO-DHF . Ensuring sufficient availability of these cofactors in B. adolescentis may require:

• Metabolic engineering of folate synthesis pathways

• Supplementation of precursors in growth media

• Co-expression of enzymes involved in formyl donor generation

Toxicity Concerns:
Overexpression of fmt may disrupt the normal translational balance in B. adolescentis, potentially leading to:

• Growth inhibition due to excessive initiation events

• Competition with endogenous fmt for substrates

• Metabolic burden from high-level heterologous protein production

To address these challenges, an iterative optimization approach is recommended, beginning with inducible expression systems that allow tight control over fmt expression levels.

How does the interaction between fmt and folate metabolism affect recombinant B. adolescentis?

The interaction between fmt and folate metabolism is critical for successful recombinant expression in B. adolescentis, as fmt activity directly depends on folate-derived formyl donors.

Folate Pathway Integration:
Fmt utilizes 10-formyl-THF (10-CHO-THF) as its primary formyl donor, which is generated by the folate dehydrogenase-cyclohydrolase (FolD) enzyme from 5,10-methylene tetrahydrofolate (5,10-CH2-THF) . Recent research has also demonstrated that fmt can use 10-formyl-dihydrofolate (10-CHO-DHF) as an alternative substrate . This metabolic flexibility has important implications for expression systems:

Metabolic Engineering Considerations:
To optimize fmt activity in recombinant B. adolescentis, researchers should consider:

• Analysis of the folate synthesis pathway in B. adolescentis to identify potential bottlenecks

• Co-expression of FolD to increase 10-CHO-THF availability

• Supplementation of growth media with folate precursors

• Engineering of fmt variants with enhanced affinity for available formyl donors

Metabolic Impact Assessment:
Overexpression of fmt may create a metabolic drain on the folate pool, affecting other cellular processes. This can be assessed by:

• Metabolomic analysis of folate-related metabolites in wild-type versus recombinant strains

• Transcriptomic analysis to detect compensatory changes in folate metabolism genes

• Growth rate comparison under folate-limited versus folate-abundant conditions

Antibiotic Sensitivity Implications:
Research shows FolD-deficient mutants and fmt-overexpressing strains demonstrate increased sensitivity to trimethoprim (TMP) . This suggests recombinant B. adolescentis expressing fmt may have altered antibiotic sensitivity profiles, which should be evaluated during strain development.

What methods can be used to quantify the enzymatic activity of recombinant fmt in B. adolescentis?

Accurate quantification of recombinant fmt enzymatic activity in B. adolescentis requires specialized techniques that address the anaerobic nature of the organism and the complexity of the formylation reaction:

In Vitro Formylation Assays:
The gold standard for fmt activity quantification involves:

• Preparation of total tRNA from an fmt-deficient strain (e.g., via a knockout approach)

• Charging of tRNA with methionine using purified methionyl-tRNA synthetase (MetRS)

• Incubation of Met-tRNAfMet with cell extracts containing recombinant fmt and formyl donors (10-CHO-THF or 10-CHO-DHF)

• Analysis of formylated Met-tRNAfMet by acid urea PAGE and Northern blotting

• Quantification of band intensity to determine the percentage of formylated versus non-formylated Met-tRNAfMet

LC-MS/MS-Based Assays:
For higher throughput and sensitivity:

• Conduct the formylation reaction as above

• Enzymatically digest the reaction products

• Analyze by LC-MS/MS to detect and quantify formylated methionine

• Monitor the formation of dihydrofolate (DHF) as a reaction by-product

Coupled Enzyme Assays:
Exploiting the folate metabolism connection:

• Link fmt activity to a spectrophotometrically detectable reaction

• Monitor the consumption of 10-CHO-THF or the production of THF

• Utilize recombinant enzymes that can convert reaction products to detectable compounds

Whole-Cell Translation Efficiency Measurement:
To assess the impact of fmt activity on global translation:

• Pulse-labeling with radioactive amino acids to measure protein synthesis rates

• Ribosome profiling to detect changes in translation initiation efficiency

• Comparative proteomics between wild-type and recombinant strains

Data Analysis Considerations:

When comparing mutant or engineered fmt variants, these parameters should be systematically evaluated to determine the impact of modifications on enzyme function.

How does recombinant fmt expression influence the probiotic properties of B. adolescentis?

Recombinant fmt expression can significantly impact the probiotic properties of Bifidobacterium adolescentis through several mechanisms:

Impact on Growth and Colonization:
The probiotic efficacy of B. adolescentis depends partly on its ability to colonize the intestinal tract. Recombinant fmt expression may affect:

• Growth rate in nutrient-limited environments due to metabolic burden

• Competitive fitness against other gut microbes

• Adherence to intestinal epithelial cells through alterations in surface proteins

Researchers should quantify colonization potential using:

• In vitro adhesion assays with human intestinal cell lines

• Competition assays with other gut commensals

• Gnotobiotic mouse models to assess colonization persistence

Immunomodulatory Effects:
B. adolescentis strains naturally exert anti-inflammatory effects by:

• Reducing pro-inflammatory cytokines (IL-6, IL-1β, IL-17A, IFN-γ, TNF-α)

• Promoting anti-inflammatory cytokines (IL-4, IL-10, TGF-β1)

Fmt overexpression may affect these properties through:

• Alterations in surface protein formylation patterns

• Changes in metabolite production profiles

• Modified interaction with host immune receptors

Metabolic Profile Changes:
B. adolescentis contains numerous pathways for carbohydrate metabolism , which contribute to its probiotic effects. Recombinant fmt expression might:

• Alter carbon flux through central metabolic pathways

• Affect production of short-chain fatty acids and other beneficial metabolites

• Change utilization patterns of prebiotic substrates

Stress Resistance Profiling:
A comprehensive analysis should include:

These analyses are essential for evaluating whether recombinant fmt expression maintains or enhances the therapeutic potential of B. adolescentis for conditions like inflammatory bowel disease, where it has shown promising effects .

What is the effect of fmt mutation on the colonization ability of B. adolescentis in experimental models?

Mutations in the fmt gene can significantly impact the colonization ability of Bifidobacterium adolescentis in experimental models through multiple mechanisms:

Translation Initiation Efficiency:
Fmt mutations may alter the efficiency of translation initiation, affecting:

Comparative Colonization Analysis:
To systematically evaluate colonization abilities, the following experimental approach is recommended:

• Generate defined fmt mutants:

  • Complete deletion mutants (Δfmt)

  • Point mutations affecting catalytic activity (e.g., mutations equivalent to S125L or S209L in human MTF)

  • Mutations affecting substrate binding

• Assess intestinal colonization using:

  • Gnotobiotic mouse models with controlled microbiota

  • Competitive index experiments (wild-type vs. mutant co-administration)

  • Longitudinal sampling of fecal content for quantitative analysis

• Evaluate microbe-host interactions:

  • Intestinal epithelial cell adhesion assays

  • Mucin binding capacity

  • Biofilm formation potential

Expected Colonization Phenotypes:

Impact on Microbiome Interactions:
B. adolescentis exhibits negative correlations with potentially harmful bacteria like Citrobacter species and certain Enterobacter species in the human gut . Fmt mutations may alter these ecological relationships by:

• Changing competitive metabolic capabilities

• Affecting bacteriocin or antimicrobial compound production

• Modifying cross-feeding relationships with other commensals

When studying these interactions, metagenomic analysis of experimental communities should be performed, focusing on shifts in relative abundance between wild-type and fmt-mutant conditions.

How can we assess the impact of recombinant fmt on protein synthesis rates in B. adolescentis?

Assessing the impact of recombinant fmt on protein synthesis rates in Bifidobacterium adolescentis requires specialized techniques that can differentiate global translation effects from specific initiation changes:

Radioisotope Incorporation Methods:

• Pulse-labeling with ³⁵S-methionine:

  • Culture B. adolescentis (wild-type and recombinant) under identical conditions

  • Add ³⁵S-methionine for a brief period (1-5 minutes)

  • Measure incorporation into TCA-precipitable material

  • Compare incorporation rates normalized to cell number or total protein

• Puromycin incorporation assay:

  • Treat cells with puromycin (which gets incorporated into nascent peptides)

  • Detect puromycin-labeled peptides using anti-puromycin antibodies

  • Quantify via western blotting or flow cytometry

Ribosome Profiling:
This technique provides genome-wide information on translation with nucleotide resolution:

• Treat cells with translation inhibitors that freeze ribosomes on mRNAs

• Isolate and sequence ribosome-protected mRNA fragments

• Analyze ribosome density at start codons to assess initiation efficiency

• Compare profiles between wild-type and fmt-overexpressing strains

Key parameters to analyze include:

• Changes in translation initiation site (TIS) utilization

• Alterations in ribosome density at start codons

• Shifts in the ratio of ribosomes at initiation vs. elongation phases

Polysome Profiling:
This approach assesses the translation status of mRNAs:

• Separate polysomes (multiple ribosomes on a single mRNA) by sucrose gradient centrifugation

• Analyze the distribution of ribosomes between non-translating (monosomes) and actively translating (polysomes) fractions

• Compare polysome/monosome ratios between strains

Proteomics Approaches:
Quantitative proteomics can reveal the global impact on the proteome:

• Stable isotope labeling (SILAC or similar approaches adapted for B. adolescentis)

• Label-free quantitative proteomics

• Targeted proteomics of key initiation factors and their interacting partners

Data Integration Framework:

For comprehensive analysis, researchers should combine at least two orthogonal methods and include appropriate controls such as catalytically inactive fmt mutants to distinguish effects due to protein overexpression from those due to enzymatic activity.

How does the use of alternative formyl donors affect the efficiency of fmt in recombinant B. adolescentis?

The efficiency of methionyl-tRNA formyltransferase (fmt) in recombinant Bifidobacterium adolescentis is significantly influenced by the availability and type of formyl donors. Recent research has revealed that fmt can utilize both 10-formyl-tetrahydrofolate (10-CHO-THF) and 10-formyl-dihydrofolate (10-CHO-DHF) as formyl group donors .

Comparative Efficiency Analysis:

Methodological Approach to Study Alternative Donors:
To investigate the impact of different formyl donors on fmt efficiency in recombinant B. adolescentis:

• Prepare recombinant fmt from B. adolescentis in a purified system

• Conduct in vitro formylation assays with:

  • Met-tRNAfMet substrate prepared from deacylated tRNAfMet charged with methionine by MetRS

  • Varying concentrations of 10-CHO-THF and 10-CHO-DHF

  • Reaction conditions mimicking the B. adolescentis cytoplasmic environment

• Analyze reaction products by:

  • Acid urea PAGE and Northern blotting for formylated Met-tRNAfMet

  • LC-MS/MS detection of reaction by-products (DHF)

  • Kinetic analysis to determine Km and Vmax for each donor

Metabolic Engineering Implications:
Understanding alternative formyl donor usage has important implications for metabolic engineering of recombinant B. adolescentis:

• In folate-limited environments (such as the inflamed intestine), fmt activity may be maintained through 10-CHO-DHF utilization

• Co-expression of folate metabolism enzymes could enhance fmt efficiency

• Strains with enhanced 10-CHO-DHF utilization capacity might show improved performance under stress conditions

Impact on Antibiotic Sensitivity:
Trimethoprim (TMP) inhibits dihydrofolate reductase, affecting folate metabolism. Research shows that:

• FolD-deficient mutants are more sensitive to TMP

• Fmt-overexpressing strains also show increased TMP sensitivity

This indicates a complex relationship between fmt activity, folate metabolism, and antibiotic sensitivity that should be considered when developing recombinant B. adolescentis strains for therapeutic applications.

How do regulatory mechanisms control fmt expression in native versus recombinant systems?

Understanding the regulatory mechanisms controlling fmt expression is essential for optimizing recombinant systems. While specific data on fmt regulation in Bifidobacterium adolescentis is limited, comparative analysis with other bacterial systems provides valuable insights.

Native Regulatory Mechanisms:

In bacteria, fmt expression is typically regulated through:

• Nutritional Status Sensing:

  • Response to amino acid availability

  • Integration with folate metabolism pathways

  • Coordination with tRNA aminoacylation systems

• Growth Phase-Dependent Regulation:

  • Higher expression during exponential growth

  • Downregulation during stationary phase

  • Integration with stringent response

• Potential Regulatory Elements:

  • Promoter structures responsive to metabolic regulators

  • Ribosome binding site accessibility modulation

  • Potential riboswitches sensitive to formylated amino acids or folate derivatives

Recombinant System Optimization:

When expressing fmt in recombinant B. adolescentis, several regulatory approaches can be considered:

Experimental Assessment Methodology:

To characterize fmt regulation in both native and recombinant contexts:

• Transcriptional analysis:

  • RT-qPCR across growth phases and nutrient conditions

  • Promoter-reporter fusion assays (e.g., using luciferase)

  • RNA-seq for global regulatory context

• Translational efficiency:

  • Ribosome profiling specifically targeting fmt mRNA

  • Western blotting to correlate mRNA and protein levels

  • Polysome association analysis

• Protein stability determination:

  • Pulse-chase experiments with labeled amino acids

  • Protease sensitivity assays

  • Half-life determination under various conditions

For recombinant systems, testing different promoter-RBS combinations is essential to identify optimal expression parameters that balance fmt activity with minimal metabolic burden.

How can recombinant B. adolescentis expressing fmt be used to study translation mechanisms in probiotics?

Recombinant Bifidobacterium adolescentis expressing methionyl-tRNA formyltransferase (fmt) provides a valuable model system for investigating translation mechanisms in probiotics, offering several distinct research advantages:

Translation Initiation Studies:

Fmt catalyzes a critical step in bacterial translation initiation by formylating Met-tRNAfMet. Recombinant strains with modified fmt enable researchers to:

• Investigate the impact of translation initiation efficiency on:

  • Adaptation to intestinal environmental stresses

  • Rapid response to nutrient availability changes

  • Expression of colonization factors

• Study specialized initiation mechanisms by:

  • Creating reporter systems with alternative start codons

  • Analyzing leaderless mRNA translation efficiency

  • Evaluating non-canonical initiation pathways

Methodological Approaches:
To leverage this system effectively:

• Develop B. adolescentis strains with:

  • Titratable fmt expression using inducible promoters

  • Tagged fmt variants for affinity purification

  • Catalytically altered fmt mutants based on human disease variants

• Implement specialized assays:

  • Ribosome profiling to map initiation sites genome-wide

  • Selective ribosome profiling targeting initiating ribosomes

  • Translational fidelity reporters to assess start codon recognition accuracy

Stress Response Mechanism Investigation:

Probiotic effectiveness depends on stress adaptation capabilities. Recombinant fmt systems allow researchers to:

• Evaluate translation initiation under relevant stresses:

  • Acid stress (stomach passage simulation)

  • Bile salt exposure (small intestine conditions)

  • Oxidative stress (inflammatory environments)

• Compare wild-type and modified fmt strains for:

  • Differential gene expression under stress

  • Selective translation of stress-response genes

  • Recovery rates after stress exposure

Potential Research Applications:

These studies can reveal fundamental aspects of translation regulation in probiotic bacteria while providing insights for optimizing therapeutic applications of B. adolescentis.

What are the implications of fmt-enhanced B. adolescentis for treating inflammatory bowel diseases?

The potential of fmt-enhanced Bifidobacterium adolescentis for treating inflammatory bowel diseases (IBD) represents a promising frontier in microbiome-based therapeutics, building on established evidence of B. adolescentis's anti-inflammatory properties.

Rationale for B. adolescentis in IBD Treatment:

Multiple studies have demonstrated that:

• B. adolescentis is depleted in IBD patients compared to healthy individuals

• B. adolescentis strain AF91-08b2A significantly enhances disease activity index (DAI) and reduces colonic damage in DSS-induced colitis models

• B. adolescentis effectively modulates immune responses by reducing pro-inflammatory cytokines (IL-6, IL-1β, IL-17A, IFN-γ, TNF-α) while promoting anti-inflammatory cytokines (IL-4, IL-10, TGF-β1)

• B. adolescentis helps restore tight junction proteins (ZO-1, occludin, claudin-2), safeguarding intestinal barrier function

Added Value of fmt Enhancement:

Recombinant fmt expression in B. adolescentis could potentiate these effects through:

• Improved Stress Adaptation:

  • Enhanced translation initiation efficiency during intestinal transit

  • Faster recovery and adaptation in inflammatory environments

  • Potentially improved colonization persistence

• Optimized Protein Expression:

  • More efficient expression of anti-inflammatory factors

  • Enhanced production of enzymes involved in beneficial metabolite generation

  • Improved expression of adhesion factors for better epithelial interaction

Experimental Evaluation Framework:

To assess the therapeutic potential of fmt-enhanced B. adolescentis:

Integration with FMT Approaches:

Recent research on fecal microbiota transplantation (FMT) has shown that:

• B. adolescentis is a key beneficial microbe in successful FMT donors

• Donor microbiota with high levels of B. adolescentis achieves better engraftment (67% rate)

This suggests that fmt-enhanced B. adolescentis could potentially:

• Serve as a defined component in next-generation FMT formulations

• Enhance engraftment success of other beneficial microbes

• Provide a more standardized alternative to donor-dependent FMT

These approaches align with the emerging paradigm of using defined microbial consortia rather than complete FMT for treating conditions like C. difficile infection and potentially IBD.

How stable is fmt expression in recombinant B. adolescentis during gastrointestinal transit?

The stability of fmt expression in recombinant Bifidobacterium adolescentis during gastrointestinal (GI) transit is a critical consideration for therapeutic applications. Several methodological approaches can address this question comprehensively:

In Vitro Gastrointestinal Simulation:

Sequential exposure to simulated gastrointestinal conditions allows for controlled assessment of expression stability:

• Simulated gastric fluid exposure:

  • pH 2.0-3.0 with pepsin

  • Timed sampling (0-120 minutes)

  • Analysis of cell viability and fmt expression

• Small intestinal transition:

  • Bile salt exposure (0.3-1.0%)

  • Pancreatic enzymes

  • pH adjustment to 6.5-7.5

• Colonic environment simulation:

  • Anaerobic conditions

  • Presence of short-chain fatty acids

  • Competition with other microbiota members

Expression Stability Assessment Methods:

In Vivo Transit Studies:

For definitive assessment of stability during actual GI transit:

• Animal model studies:

  • Oral administration of recombinant B. adolescentis to mice

  • Timed sacrifices to sample different GI compartments

  • Recovery of bacteria from stomach, small intestine, cecum, and colon

• Non-invasive monitoring approaches:

  • Co-expression of reporter genes (e.g., luciferase) with fmt

  • Bioluminescence imaging to track bacterial colonization

  • Fecal recovery and analysis for expression stability

Stability Enhancement Strategies:

If stability issues are identified, several approaches can improve expression maintenance:

• Genetic stabilization:

  • Chromosomal integration rather than plasmid-based expression

  • Use of theta-replicating plasmids (e.g., pMB1, pBC1) which offer greater stability than rolling circle plasmids

  • Addition of plasmid addiction systems

• Physiological protection:

  • Microencapsulation technologies

  • Co-administration with buffering agents

  • Enteric coating for targeted delivery

• Strain hardening:

  • Adaptive laboratory evolution under GI stress conditions

  • Selection for enhanced stress resistance

The combined data from these approaches will provide a comprehensive profile of fmt expression stability throughout the GI tract, enabling rational optimization of recombinant B. adolescentis for therapeutic applications.

Can recombinant B. adolescentis expressing modified fmt be used as a live vaccine vector?

Recombinant Bifidobacterium adolescentis expressing modified methionyl-tRNA formyltransferase (fmt) presents a compelling platform for development as a live vaccine vector, building on emerging evidence of bifidobacteria's potential in this domain.

Rationale for B. adolescentis as a Vaccine Vector:

Several characteristics support the use of B. adolescentis as a live vaccine vector:

• Safety profile:

  • Generally Recognized as Safe (GRAS) status

  • Long history of safe use as probiotics

  • Low risk of adverse effects when administered orally

• Mucosal immunogenicity:

  • Ability to interact with intestinal epithelium and immune cells

  • Potential to stimulate both mucosal and systemic immunity

  • Natural adjuvant properties through microbe-associated molecular patterns

• Genetic manipulation capabilities:

  • Established transformation protocols

  • Available E. coli/Bifidobacterium shuttle vectors

  • Demonstrated ability to express heterologous proteins

Role of Modified fmt in Vaccine Development:

Modified fmt could enhance vaccine efficacy through:

• Improved antigen expression:

  • Enhanced translation initiation efficiency

  • Optimized expression of formylated peptides that stimulate immune responses

  • Controlled timing of antigen production

• Immunomodulatory effects:

  • Formylated peptides are recognized by formyl peptide receptors on immune cells

  • Potential to enhance innate immune activation

  • Adjuvant-like effects to boost adaptive responses

Methodological Approach for Development:

Precedent in Literature:

Previous studies support this approach:

• Bifidobacteria have been successfully used to express antigens from pathogenic bacteria as live vaccines, with promising results in animal models

• Specific examples include expression of E. coli antigens in B. longum subsp. infantis

• Research has shown bifidobacteria can translocate from the gastrointestinal tract to systemic circulation and target specific tissues without severe side effects

Potential Applications:

• Enteric pathogen vaccines:

  • Protection against C. difficile (leveraging B. adolescentis antagonism against this pathogen)

  • Vaccines against enteropathogenic E. coli

  • Salmonella and other food-borne pathogens

• Mucosal immunity enhancement:

  • Respiratory pathogen protection through trained immunity

  • Adjuvant for conventional vaccines

• Therapeutic vaccines:

  • Cancer immunotherapy applications

  • Autoimmune disease modulation