Recombinant Lactobacillus johnsonii tRNA pseudouridine synthase B (truB)

What is Lactobacillus johnsonii and why is it significant for recombinant protein expression?

Lactobacillus johnsonii is a homofermentative lactic acid intestinal bacterium that has been utilized as a probiotic for decades to treat various illnesses . Its significance in recombinant protein expression stems from several key characteristics:

• L. johnsonii partially survives simulated gastric conditions in vitro, making it an attractive candidate for oral vaccine delivery vehicles

• The bacteria can be genetically modified to express foreign proteins on its surface, as demonstrated with proteinase PrtB and other fusion proteins

• L. johnsonii has co-evolved with different animals at the species or strain level, providing a reasonable basis for its relationship with health benefits

• The bacterium plays a crucial role in modulating the host immune system by altering macrophage, T-cell, and Th2 cytokine levels

These properties make L. johnsonii an excellent chassis for recombinant expression of proteins like tRNA pseudouridine synthase B (TruB).

What is tRNA pseudouridine synthase B (TruB) and what is its primary function?

tRNA pseudouridine synthase B (TruB) is an RNA-modifying enzyme responsible for introducing pseudouridine at position 55 of tRNAs during the early stage of tRNA maturation . Key characteristics of TruB include:

• TruB introduces pseudouridine, which was the first RNA modification found in tRNA and ribosomes

• Pseudouridylation enhances RNA structure by improving base-to-base stacking

• TruB functions as both an RNA-modifying enzyme and an RNA chaperone, with the latter function being independent of its enzymatic activity

• Similar to other RNA-binding proteins (like ADAR1/2, METTL3, METTL16), TruB possesses dual functions beyond its intrinsic enzymatic activity

Of particular interest, research has revealed that the human ortholog TruB1 has unexpected functions in miRNA biogenesis with let-7 specificity, presenting a new aspect of miRNA regulation by RNA-binding proteins .

How are recombinant Lactobacillus johnsonii strains expressing foreign proteins constructed?

The construction of recombinant L. johnsonii strains expressing foreign proteins typically follows this methodology:

• Plasmid Construction:

  • The gene of interest is first artificially synthesized or amplified from a source organism

  • The gene is then inserted into an expression vector (e.g., pPG-612) using appropriate restriction sites

  • The recombinant plasmid is verified through restriction enzyme digestion and sequencing

• Transformation into L. johnsonii:

  • L. johnsonii competent cells are prepared and thawed in an ice-water slurry

  • The recombinant plasmid is mixed with competent cells and incubated on ice

  • Electroporation is performed (typically at 2.1 kV for 3 ms)

  • Cells are immediately chilled after electroporation

  • Transformed cells are transferred into MRS broth containing sucrose and incubated anaerobically

  • Cells are then plated on selective media containing an appropriate antibiotic (e.g., chloramphenicol)

• Verification of Transformation:

  • PCR is used to confirm the presence of the insert in transformed colonies

  • Western blotting confirms expression of the recombinant protein

  • Genetic stability testing through multiple generations ensures reliable expression

This methodology has been successfully implemented for various recombinant proteins in L. johnsonii, including fusion proteins for vaccine delivery and therapeutic applications .

What techniques are used to verify the enzymatic activity of recombinant TruB in L. johnsonii?

Verification of TruB enzymatic activity in recombinant L. johnsonii involves several complementary techniques:

• In vitro Enzyme Assays:

  • Using purified recombinant TruB protein with a tRNA substrate (e.g., tRNA^Phe)

  • Measuring pseudouridylation activity by detecting modified nucleosides

  • Comparing enzymatic activity of wild-type TruB versus mutant versions (e.g., with inactivated enzyme activity)

• CMC-Primer Extension Assay:

  • Treatment of RNA with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC)

  • Primer extension using specific primers and ddATP

  • Presence of pseudouridine is indicated by bands at specific positions in CMC-treated RNA that are not present in untreated samples

• Electrophoretic Mobility Shift Assay (EMSA):

  • Testing the physical interaction between recombinant TruB and tRNA substrates

  • Comparing binding abilities of wild-type and mutant TruB proteins

• Analysis of RNA Modifications:

  • High-performance liquid chromatography (HPLC) or mass spectrometry to detect pseudouridine modifications

  • Quantification of modified versus unmodified nucleosides in tRNA samples

These techniques can distinguish between the enzymatic pseudouridylation activity and the non-enzymatic RNA chaperone function of TruB, which is particularly important given the dual functionality observed with this enzyme .

How does TruB1 regulate microRNA biogenesis independently of its pseudouridylation activity?

Research has revealed that TruB1 (a mammalian homolog of bacterial TruB) can regulate microRNA biogenesis through a mechanism independent of its pseudouridylation activity. This represents an unexpected function that has significant implications for understanding RNA regulation .

Mechanism of let-7 regulation by TruB1:

• Direct RNA Binding:

  • TruB1 selectively and specifically interacts with the stem-loop structure of pri-let-7 microRNA

  • High-throughput sequencing crosslinking immunoprecipitation (HITS-CLIP) and biochemical analyses confirmed direct binding between endogenous TruB1 and pri-let-7

• Enhanced Microprocessor Interaction:

  • TruB1 selectively enhances the interaction between pri-let-7 and the microprocessor DGCR8

  • This interaction promotes the maturation steps of let-7 microRNA

• Enzymatic Independence:

  • Using mutant versions of TruB1 (mt1 with inactivated enzyme activity and mt2 with suppressed RNA-binding ability), researchers demonstrated that:

    • Wild-type and mt1 TruB1 significantly increased let-7a maturation

    • Mt2 (lacking RNA binding) did not increase let-7a maturation

    • This confirms that promotion of let-7 maturation is independent of enzyme activity but dependent on RNA binding

• Absence of Pseudouridylation in let-7:

  • In vitro enzyme assays showed that pri-let-7 could not be pseudouridylated by recombinant TruB1

  • CMC-primer extension assays indicated no pseudouridine in endogenous let-7

This mechanism distinguishes TruB1 from other pseudouridine synthases like PUS10, which non-specifically increases maturation of various miRNAs, while TruB1 shows let-7 specificity .

What are the potential applications of recombinant L. johnsonii expressing therapeutic proteins?

Recombinant L. johnsonii expressing therapeutic proteins has shown promise in several biomedical applications:

• Mucosal Vaccine Delivery:

  • L. johnsonii can partially survive gastric conditions, making it suitable for oral vaccination

  • Surface expression of antigens (e.g., proteinase PrtB and fusion proteins) can induce both systemic IgG and mucosal IgA immune responses

  • This approach has been tested with mimotope peptides derived from tetanus toxin integrated into the sequence of proteinase PrtB

• Treatment of Respiratory Infections:

  • Supplementation with L. johnsonii significantly reduced RSV-induced pulmonary responses via immunomodulatory metabolites

  • Administration improved lung development in hyperoxia-exposed neonatal mice

  • It reduced inflammatory cytokines (IL-4, IL-5, IL-13, IL-6, IL-1b, TNFα) while increasing beneficial factors (IFNβ, DHA, AcedoPC)

• Treatment of Bovine Postpartum Endometritis:

  • Recombinant L. johnsonii expressing bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been developed

  • The expression system used pPG-612 vector with GM-CSF gene insertion

  • Successful transformation and stable expression over 40 generations was confirmed

• Other Therapeutic Applications:

  • Treatment of H. pylori-associated gastritis

  • Modulation of intestinal microflora

  • Immune system regulation

These applications leverage the natural probiotic properties of L. johnsonii combined with the targeted therapeutic effects of recombinantly expressed proteins.

How can researchers address contradictory data in TruB functional studies?

When confronted with contradictory data in TruB functional studies, researchers should implement a systematic approach to reconcile discrepancies:

• Structured Contradiction Analysis Framework:

  • Use a notation system considering three parameters (α, β, θ):

    • α: number of interdependent items

    • β: number of contradictory dependencies defined by domain experts

    • θ: minimal number of required Boolean rules to assess these contradictions

  • This structured approach helps handle the complexity of multidimensional interdependencies within datasets

• Distinguish Enzymatic vs. Non-Enzymatic Functions:

  • Create experimental controls that separate TruB's enzymatic activity (pseudouridylation) from its RNA chaperone function

  • Use mutant versions of TruB with specifically disrupted functions:

    • Enzyme-inactive mutants (e.g., D48, D90 mutations)

    • RNA-binding deficient mutants (e.g., K64 mutation)

  • Compare results across multiple experimental systems and model organisms

• Validate Through Multiple Techniques:

  • Combine in vitro biochemical assays with in vivo functional studies

  • Use both gain-of-function and loss-of-function approaches

  • Apply high-throughput techniques (HITS-CLIP, RNA-seq) alongside targeted assays

• Consider Model-Specific Differences:

  • Account for differences between bacterial TruB and mammalian TruB1

  • Compare results across different expression systems (e.g., E. coli vs. L. johnsonii)

  • Evaluate substrate specificity differences between organisms

For example, when contradictory results emerge regarding TruB's effect on miRNA maturation, researchers should systematically evaluate:

• Substrate specificity (which miRNAs are affected)

• Mechanism of action (direct binding vs. enzymatic modification)

• Contextual dependencies (cell type, developmental stage, physiological conditions)

This structured approach to contradiction analysis allows for more robust experimental design and interpretation of complex functional data .

What methodological considerations are important when measuring TruB enzyme activity in recombinant systems?

When measuring TruB enzyme activity in recombinant systems, researchers should consider several methodological factors to ensure accurate and reproducible results:

• Protein Purification and Quality Control:

  • Ensure high purity of recombinant TruB protein

  • Verify protein folding and activity through multiple methods

  • Use size exclusion chromatography to confirm monomeric state

  • Include wild-type and catalytically inactive mutants as controls

• Substrate Preparation and Specificity:

  • Use both in vitro transcribed and naturally isolated tRNA substrates

  • Consider position-specific modifications that may influence TruB activity

  • Verify that pseudouridine is specifically introduced at position 55 of tRNAs

• Assay Conditions and Controls:

  • Optimize reaction conditions (pH, temperature, buffer composition)

  • Include appropriate controls:

    • No enzyme control

    • Heat-inactivated enzyme

    • Substrate without target uridine

  • Use E. coli TruB as a reference standard due to its well-characterized activity

• Detection Methods and Validation:

  • Use multiple detection techniques for pseudouridylation:

    • CMC-primer extension assay

    • HPLC analysis of nucleosides

    • Mass spectrometry

  • Validate results through multiple independent experiments

  • Consider quantitative analysis of pseudouridylation efficiency

• Expression System Considerations:

  • Account for differences between homologous expression (in Lactobacillus) and heterologous expression (in E. coli)

  • Evaluate the impact of different promoters and expression vectors

  • Monitor stability of the recombinant protein in the expression host

For example, when evaluating TruB activity in recombinant L. johnsonii, researchers should:

• Compare activity with purified protein versus whole-cell extracts

• Consider potential competition with endogenous TruB

• Account for differences in tRNA substrates between expression hosts

• Evaluate the impact of growth conditions on enzymatic activity

These methodological considerations ensure that TruB activity measurements in recombinant systems accurately reflect the enzyme's biological function.

What statistical approaches are recommended for analyzing complex datasets in TruB functional studies?

When analyzing complex datasets in TruB functional studies, researchers should employ robust statistical approaches that account for the multifaceted nature of the data:

• Experimental Design Considerations:

  • Use factorial designs to assess multiple variables simultaneously

  • Implement appropriate blocking and randomization

  • Include technical and biological replicates (minimum n=3 for each condition)

  • Power analysis to determine sample size requirements

• Data Preprocessing and Quality Control:

  • Normalization strategies for different data types:

    • RT-qPCR data: Use reference genes (e.g., GAPDH) for normalization

    • RNA-seq: TPM/RPKM normalization with batch effect correction

  • Outlier detection and handling

  • Assessment of data distributions and transformations if needed

• Statistical Testing Framework:

  • For comparing TruB activity across conditions:

    • ANOVA with post-hoc tests for multiple comparisons

    • t-tests with appropriate corrections (e.g., Bonferroni, FDR)

  • For time-course experiments:

    • Repeated measures ANOVA

    • Mixed-effects models

  • For dose-response relationships:

    • Regression analysis

    • Non-linear curve fitting

• Advanced Analytical Methods:

  • For high-dimensional data (e.g., RNA-seq, CLIP-seq):

    • Differential expression analysis

    • Clustering algorithms (hierarchical, k-means)

    • Principal component analysis

  • For binding site analysis:

    • Motif discovery algorithms

    • Structural prediction and analysis

• Reporting and Visualization:

  • Present data as mean ± SD with appropriate significance indicators

  • Use significance thresholds (typically p < 0.05)

  • Create comprehensive visualizations that capture:

    • Main effects

    • Interaction effects

    • Temporal dynamics

For example, when analyzing the differential effects of wild-type versus mutant TruB on multiple miRNA targets, researchers should:

• Perform multivariate analysis to capture correlated responses

• Use hierarchical clustering to identify patterns in miRNA regulation

• Implement statistical models that account for both direct and indirect effects

What are promising research avenues for exploring the dual functionality of TruB in different biological systems?

Several promising research avenues exist for further exploring the dual functionality of TruB in different biological systems:

• Comparative Studies Across Species:

  • Investigate TruB homologs from diverse bacteria, including other Lactobacillus species and probiotic strains

  • Compare bacterial TruB with eukaryotic counterparts (e.g., human TruB1)

  • Examine evolutionary conservation of enzymatic versus RNA chaperone functions

  • Create chimeric proteins to identify functional domains responsible for specific activities

• Expanded RNA Target Identification:

  • Apply HITS-CLIP and related techniques to identify the complete repertoire of TruB RNA targets

  • Investigate whether bacterial TruB, like human TruB1, can regulate specific miRNAs

  • Explore potential roles in regulating other non-coding RNAs

  • Develop high-throughput screening methods for RNA-protein interactions

• Structural Biology Approaches:

  • Determine crystal structures of TruB in complex with different RNA substrates

  • Use cryo-EM to visualize TruB interactions with larger ribonucleoprotein complexes

  • Perform molecular dynamics simulations to understand conformational changes during RNA binding

  • Develop structure-based design of TruB variants with enhanced or modified activities

• Therapeutic Applications Development:

  • Engineer L. johnsonii expressing modified TruB variants with enhanced RNA chaperone activity

  • Investigate potential applications in modulating miRNA expression in disease models

  • Develop TruB-based RNA targeting systems for gene therapy

  • Explore combinatorial approaches with other RNA-modifying enzymes

• Systems Biology Integration:

  • Map the impact of TruB activity on the global RNA modification landscape

  • Integrate transcriptomics, proteomics, and epitranscriptomics data

  • Model the regulatory networks influenced by TruB activity

  • Investigate context-dependent functions in different physiological states

These research directions will provide deeper insights into the multifunctional nature of TruB and potentially reveal novel applications in biotechnology and medicine.

How might recombinant L. johnsonii expressing TruB be utilized to study RNA modification in probiotic-host interactions?

Recombinant L. johnsonii expressing TruB offers a unique platform to study RNA modification in probiotic-host interactions:

• In vivo RNA Modification Tracking:

  • Develop tagged versions of TruB that allow tracking of modified RNAs in vivo

  • Create reporter systems to monitor pseudouridylation events during host colonization

  • Compare wild-type versus catalytically inactive TruB to distinguish enzymatic versus non-enzymatic effects

  • Examine transfer of bacterial RNA modifications to host cells

• Host-Microbe RNA Communication Studies:

  • Investigate whether bacterial TruB can modify host RNAs during probiotic colonization

  • Examine potential horizontal transfer of modified RNAs between bacteria and host cells

  • Study the impact of bacterial RNA modifications on host immune responses

  • Determine if TruB-modified RNAs serve as microbe-associated molecular patterns (MAMPs)

• Disease Model Applications:

  • Test recombinant L. johnsonii expressing TruB in models of:

    • Respiratory infections (e.g., RSV models)

    • Inflammatory bowel diseases

    • H. pylori infections

    • Endometritis models

  • Compare effects of wild-type versus modified TruB on disease progression

  • Evaluate impact on host immune responses and microbiome composition

• Multi-omics Integration:

  • Combine RNA-seq, epitranscriptomics, and metatranscriptomics to create comprehensive maps of RNA modifications

  • Correlate TruB activity with changes in host and microbiome gene expression

  • Develop computational methods to predict functional consequences of RNA modifications

  • Create models of RNA modification networks in host-microbe ecosystems

• Technological Innovations:

  • Develop methods for site-specific labeling of pseudouridylated RNAs in complex biological samples

  • Create biosensors for real-time monitoring of RNA modification events

  • Establish in situ visualization techniques for RNA modifications in host tissues

  • Design controllable expression systems for temporal regulation of TruB activity

This research approach would provide unprecedented insights into the role of RNA modifications in probiotic-host interactions and potentially reveal new mechanisms underlying the health benefits of L. johnsonii.