Recombinant Lactobacillus plantarum Threonine--tRNA ligase (thrS), partial

What is Threonine--tRNA ligase (thrS) and what is its function in Lactobacillus plantarum?

Threonine--tRNA ligase (thrS) catalyzes the attachment of threonine to tRNA(Thr) in a two-step reaction: L-threonine is first activated by ATP to form Thr-AMP and then transferred to the acceptor end of tRNA(Thr). In L. plantarum, thrS also plays a role in editing incorrectly charged L-seryl-tRNA(Thr) . This enzyme is essential for protein translation and belongs to the class-II aminoacyl-tRNA synthetase family. The thrS protein in L. plantarum strain ATCC BAA-793/NCIMB 8826/WCFS1 consists of 654 amino acids with a molecular mass of approximately 73.8 kDa .

What methodologies are used to express recombinant thrS in L. plantarum?

Expression of recombinant proteins in L. plantarum typically employs shuttle vector systems. Researchers can use expression plasmids like pSIP411 that contain inducible promoters . For optimal expression:

• Design gene constructs with codon optimization for L. plantarum

• Include appropriate signal peptides (e.g., endogenous signal peptide 1320)

• Transform the construct into competent L. plantarum cells via electroporation

• Induce expression using specific inducers (e.g., SppIP at concentrations around 50 ng/mL)

• Maintain cultures at 37°C for 6-10 hours during induction phase

Verification of expression can be performed using SDS-PAGE, western blotting with specific antibodies, and flow cytometry to determine the percentage of cells expressing the recombinant protein .

How can researchers evaluate immune responses induced by recombinant L. plantarum expressing thrS or thrS-fused antigens?

Evaluating immune responses requires a systematic approach analyzing both humoral and cellular immunity. Based on multiple studies with recombinant L. plantarum, researchers should:

For cellular immunity assessment:

• Collect immune tissues (spleen, mesenteric lymph nodes, Peyer's patches) after immunization

• Prepare single-cell suspensions and stimulate with specific antigens

• Analyze T cell responses using flow cytometry to identify CD4+IFN-γ+ and CD8+IFN-γ+ cells

• Measure T cell proliferation using CFSE staining followed by flow cytometric analysis

For humoral immunity assessment:

• Collect serum, bile, and intestinal lavage samples

• Measure specific antibody levels (IgG, IgG1, IgG2a, IgA) using ELISA

• Calculate sample-to-positive (S/P) ratios: [(mean OD450nm of sample – mean OD450nm of negative control)]/[(mean OD450nm of positive control – mean OD450nm of negative control)]

• Analyze B cell activation in Peyer's patches using flow cytometry (B220+IgA+ cells)

Representative data example:

Statistical significance: ** P<0.01; *** P<0.001 (compared to control groups)

What factors influence the stability and activity of recombinant thrS in L. plantarum?

Several factors affect the stability and activity of recombinant thrS in L. plantarum:

• pH tolerance: Recombinant proteins expressed in L. plantarum can maintain stability at pH values as low as 1.5, making them suitable for oral administration and gastric passage

• Temperature resistance: Many recombinant proteins in L. plantarum remain stable at temperatures up to 50°C

• Salt concentration: High salt concentrations typically do not significantly affect protein stability in L. plantarum expression systems

• Expression location: Surface-displayed proteins using anchoring motifs (e.g., pgsA) generally show better stability than secreted proteins

• Culture conditions: Growth phase, media composition, and induction timing significantly affect recombinant protein yield and stability

How does thrS contribute to antibiotic resistance mechanisms, and what implications does this have for research?

Threonyl-tRNA synthetases serve as targets for certain antibiotics, and mutations or modifications in thrS can confer resistance. Research findings indicate:

• Natural product ThrRS inhibitors like borrelidin and obafluorin target thrS activity

• Biosynthetic gene clusters (BGCs) for these inhibitors encode secondary ThrRSs (e.g., BorO, ObaO) that provide self-resistance mechanisms

• The resistance mechanism involves structural modifications that prevent inhibitor binding while maintaining aminoacylation activity

• Specific amino acid residues (e.g., Y462, S463) are critical for inhibitor interactions - mutations at these sites can confer resistance

For researchers studying antibiotic development:

• Consider thrS as a potential target for novel antimicrobials

• Evaluate potential resistance mechanisms by analyzing structural features of thrS variants

• Screen for secondary thrS genes in microbial genomes as indicators of potential natural product ThrRS inhibitors

• Design inhibitors that target conserved residues essential for thrS function but difficult to mutate without loss of activity

What methodologies are most effective for studying thrS autoregulation at the translational level?

Threonine--tRNA ligase has been shown to regulate its own translation through RNA binding. Based on research with E. coli thrS, effective methodologies include:

• Gene fusion approaches:

  • Construct thrS-reporter gene fusions (e.g., thrS-lac) to monitor translational regulation

  • Introduce mutations in the mRNA region upstream of the translation initiation codon

  • Assess changes in reporter expression under various conditions

• RNA structure analysis:

  • Examine primary and secondary structure homologies between thrS mRNA and tRNA(Thr)

  • Use computational methods to predict mRNA folding and potential binding sites

  • Perform RNA footprinting to identify regions protected by thrS binding

• Binding assays:

  • Develop in vitro binding assays using purified thrS and synthetic mRNA fragments

  • Measure binding affinities under different conditions (varying threonine concentration)

  • Analyze competition between tRNA(Thr) and mRNA for thrS binding

• Mutational analysis:

  • Create mutations in regions 10-40 base pairs upstream of the translation initiation codon

  • Assess effects on translational repression and derepression

  • Identify key nucleotides required for autoregulation

What experimental design is optimal for evaluating the efficacy of recombinant L. plantarum expressing thrS-fused antigens?

For robust evaluation of recombinant L. plantarum expressing thrS-fused antigens, implement this experimental design:

Animal model setup:

• Use appropriate animal models (e.g., mice for initial studies, target species for application-specific research)

• Include at least 4 groups: PBS control, empty vector L. plantarum control, recombinant L. plantarum, and positive control (conventional vaccine if available)

• Use minimum 8-10 animals per group for statistical power

• Ensure ethical approval and appropriate housing conditions

Immunization protocol:

• Administer via appropriate route (typically oral for L. plantarum)

• Establish proper dosing schedule (e.g., primary immunization followed by 2-3 boosters at 2-week intervals)

• Standardize dose based on bacterial CFU (typically 10^9-10^10 CFU)

Sample collection timeline:

• Collect serum samples weekly to monitor antibody development

• Obtain tissue samples (intestinal, respiratory, or target tissues) at key timepoints

• Perform terminal collection of lymphoid tissues for cellular immunity assessment

Challenge studies:

• Challenge with appropriate pathogen or toxin after complete immunization schedule

• Monitor protection parameters (survival, clinical scores, pathogen shedding)

• Collect samples to assess immune correlates of protection

Data analysis:

• Use appropriate statistical methods (ANOVA for multiple comparisons)

• Present complete datasets including all controls

• Calculate protection rates and relative risk reductions

What are the technical challenges in analyzing gene expression of L. plantarum thrS in complex biological environments such as the gastrointestinal tract?

Analyzing gene expression of L. plantarum thrS in complex environments like the gastrointestinal tract presents several challenges:

• Sample collection and RNA preservation:

  • Intestinal samples contain RNA-degrading enzymes requiring rapid processing

  • Differentiate between luminal and mucosal-attached bacteria

  • Preserve RNA integrity during extraction from intestinal samples

• Bacterial RNA isolation from complex matrices:

  • Separate bacterial RNA from host RNA and food components

  • Obtain sufficient RNA quantity from relatively low bacterial numbers

  • Minimize contamination with host DNA and RNA

• Specificity of detection methods:

  • Design primers/probes specific to L. plantarum thrS that don't cross-react with host or other microbial genes

  • Use appropriate controls to confirm specificity

  • Validate detection limits in complex matrices

• Quantification approaches:

  • Apply quantitative RT-PCR for targeted gene expression analysis

  • Use RNA-seq or microarrays for global gene expression profiling

  • Include appropriate housekeeping genes for normalization

• Data interpretation:

  • Account for variation between subjects and intestinal regions

  • Compare gene expression between different intestinal compartments (ileum vs. colon)

  • Distinguish between gene expression of attached versus luminal bacteria

Research has successfully employed these approaches by:

• Using perfusion of intestinal segments with L. plantarum followed by RNA extraction

• Analyzing samples from ileostomy patients who consumed L. plantarum in food matrices

• Comparing gene expression in different intestinal regions of the same subject

How should researchers interpret conflicting immune response data when evaluating recombinant L. plantarum expressing thrS-fused antigens?

When faced with conflicting immune response data, researchers should:

• Examine strain-specific variations:

  • Different L. plantarum strains may induce varying immune responses

  • Check if strain backgrounds (NC8Δ, WCFS1, etc.) differ between studies

• Analyze antigen presentation context:

  • Surface-displayed vs. secreted antigens typically yield different immune profiles

  • Fusion partners (like DCpep) can significantly alter immunogenicity

  • Compare expression systems and display motifs used (pgsA, etc.)

• Consider dosing and administration variables:

  • Vaccination schedules influence immune response kinetics

  • Dose-dependent effects may explain threshold-dependent responses

  • Route of administration affects mucosal vs. systemic immunity balance

• Evaluate measurement methodologies:

  • Different assay sensitivities and specificities between studies

  • Timing of sample collection relative to immunization

  • Normalization methods for immune cell counts or antibody titers

• Assess host factors:

  • Age, sex, and health status of experimental animals

  • Genetic background differences between animal models

  • Pre-existing immunity or microbiota composition variations

Comparative analysis approach:

What bioinformatic approaches can researchers use to identify and characterize novel thrS variants in bacterial genomes?

To identify and characterize novel thrS variants in bacterial genomes, researchers should implement these bioinformatic approaches:

• Sequence-based identification:

  • Use BLAST or HMMER with known thrS sequences as queries

  • Apply profile hidden Markov models based on conserved thrS domains

  • Search for additional copies beyond the primary thrS gene that may indicate specialized functions

• Structural prediction and analysis:

  • Generate homology models using solved thrS structures as templates

  • Identify catalytic residues and substrate binding pockets

  • Compare structural features between primary and secondary thrS copies

• Genomic context analysis:

  • Examine neighboring genes for biosynthetic gene clusters (BGCs)

  • Identify potential regulatory elements upstream of thrS genes

  • Look for co-localization with antibiotic resistance or production genes

• Phylogenetic analysis:

  • Construct phylogenetic trees to classify thrS variants

  • Identify horizontal gene transfer events

  • Determine evolutionary relationships between thrS variants across species

• Functional prediction:

  • Analyze amino acid substitutions at key functional sites

  • Predict substrate specificity based on binding pocket composition

  • Assess potential for antibiotic resistance based on known resistance-conferring mutations

• Transcriptional regulation:

  • Identify potential regulatory elements in the promoter region

  • Look for autoregulatory features similar to those identified in E. coli thrS

  • Predict RNA secondary structures that may participate in translational regulation

This approach has successfully identified secondary thrS genes in biosynthetic gene clusters for natural product ThrRS inhibitors, revealing their role in self-resistance mechanisms .

What are promising research avenues for utilizing recombinant L. plantarum expressing thrS-fusion proteins for vaccine development?

Based on current research trends, several promising directions for vaccine development using recombinant L. plantarum expressing thrS-fusion proteins include:

• Multi-epitope vaccine design:

  • Fuse multiple protective epitopes from different pathogens to thrS

  • Create polyvalent vaccines addressing multiple diseases

  • Design epitope arrangements that optimize presentation to the immune system

• Enhanced mucosal targeting:

  • Incorporate mucosal targeting peptides like DCpep to enhance dendritic cell activation

  • Develop tissue-specific targeting to direct immune responses to desired mucosal sites

  • Optimize antigen release at specific intestinal locations

• Immune response modulation:

  • Co-express immunomodulatory molecules with thrS-antigen fusions

  • Balance Th1/Th2/Th17 responses for optimal protection

  • Engineer strains that selectively activate specific immune cell subsets

• Improved antigen stability:

  • Design thrS fusions that protect antigens from degradation in the GI tract

  • Develop pH-responsive display systems that release antigens at target sites

  • Create temperature-sensitive expression systems for controlled antigen delivery

• Novel delivery formulations:

  • Develop freeze-dried formulations for improved shelf stability

  • Create microencapsulation systems to protect live bacteria during passage

  • Design delayed-release systems for targeted intestinal delivery

• Applications beyond infectious diseases:

  • Explore applications for autoimmune disease therapy

  • Develop cancer immunotherapy approaches

  • Create tolerance-inducing vaccines for allergic conditions

Research on recombinant L. plantarum expressing SARS-CoV-2 spike protein and avian influenza virus antigens has already demonstrated the feasibility of this approach for addressing emerging infectious diseases .

How might thrS research contribute to the development of novel antibiotics or antibiotic resistance countermeasures?

Research on thrS presents significant opportunities for antibiotic development and combating resistance:

• Structure-based drug design:

  • Utilize thrS crystal structures to design specific inhibitors

  • Target conserved catalytic residues essential for aminoacylation

  • Develop allosteric inhibitors that bind sites distinct from the active site

• Resistance mechanism elucidation:

  • Characterize how secondary thrS proteins confer resistance to natural inhibitors

  • Identify critical mutations that enable resistance without compromising function

  • Map the evolutionary pathways to resistance to predict future resistance mechanisms

• Combination therapy approaches:

  • Design inhibitors targeting both primary and secondary thrS variants

  • Develop adjuvants that sensitize resistant bacteria to existing antibiotics

  • Create molecules that block resistance mechanisms while preserving antibiotic activity

• Natural product mining:

  • Screen for novel BGCs containing secondary thrS genes as indicators of potential antibiotics

  • Identify new natural ThrRS inhibitors beyond the known examples (borrelidin, obafluorin)

  • Engineer improved variants of natural ThrRS inhibitors with enhanced specificity

• Bacterial communication disruption:

  • Investigate thrS roles in bacterial adaptation to environmental challenges

  • Target non-canonical functions of thrS in bacterial physiology

  • Develop approaches to selectively inhibit thrS function under specific conditions

• Cross-species comparison:

  • Analyze structural differences between bacterial and human thrS

  • Exploit these differences to design selective antibacterials

  • Create broad-spectrum inhibitors targeting conserved bacterial thrS features

These approaches could help address the growing challenge of antibiotic resistance by developing novel compounds with unique mechanisms of action that are less susceptible to existing resistance mechanisms.