Recombinant Lactobacillus johnsonii 30S ribosomal protein S2 (rpsB)

How is recombinant L. johnsonii rpsB typically produced for research purposes?

Recombinant L. johnsonii 30S ribosomal protein S2 is typically produced using standard molecular cloning and heterologous expression techniques. The methodological approach involves:

• Gene identification and isolation: The rpsB gene sequence is identified from the L. johnsonii genome, PCR-amplified using specific primers designed to include appropriate restriction sites.

• Vector construction: The amplified gene is inserted into an expression vector (commonly pET series vectors for E. coli expression systems) using restriction enzyme digestion and ligation.

• Transformation: The recombinant vector is transformed into a suitable expression host, typically E. coli strains optimized for protein expression (BL21(DE3), Rosetta, etc.).

• Expression induction: Protein expression is induced using IPTG (isopropyl β-D-1-thiogalactopyranoside) when using T7 promoter-based systems.

• Purification: The recombinant protein is purified through affinity chromatography (often using histidine-tag systems), followed by size exclusion chromatography to ensure high purity.

This methodology enables researchers to obtain sufficient quantities of pure protein for structural studies, functional characterization, and immunological applications. The yield and solubility of recombinant rpsB can vary based on expression conditions, requiring optimization of temperature, induction time, and buffer compositions for each specific experimental setup.

What analytical techniques are most effective for confirming the identity and purity of recombinant L. johnsonii rpsB?

Multiple complementary analytical techniques are essential for confirming both the identity and purity of recombinant L. johnsonii 30S ribosomal protein S2. The recommended methodological approaches include:

• SDS-PAGE analysis: Provides information on protein size, purity, and potential degradation products. A single band at the expected molecular weight (~27-30 kDa for rpsB) indicates high purity.

• Western blot: Offers specificity through antibody recognition, confirming protein identity even in complex mixtures or at low concentrations.

• Mass spectrometry: LC-MS/MS analysis provides definitive identification through peptide mass fingerprinting and sequence coverage. This technique has been successfully employed to identify ribosomal proteins, including S2, in L. johnsonii nanovesicles .

• Size exclusion chromatography (SEC): Evaluates oligomeric state and sample homogeneity by separating proteins based on their hydrodynamic radius.

• Circular dichroism (CD) spectroscopy: Assesses secondary structure elements, providing insights into proper protein folding.

For successful experimental outcomes, researchers should aim for >95% purity as determined by densitometric analysis of SDS-PAGE gels and >90% sequence coverage in mass spectrometric analysis. These analytical parameters ensure that functional studies are conducted with properly characterized protein preparations.

What is known about the structural characteristics of L. johnsonii rpsB?

• Domain organization: The protein likely contains an N-terminal globular domain and a C-terminal extended region that interacts with 16S rRNA and neighboring ribosomal proteins.

• Secondary structure composition: Computational predictions suggest a mixed α/β structure with approximately 40-45% α-helical content and 15-20% β-sheet elements.

• RNA-binding regions: The protein contains positively charged patches that facilitate interaction with ribosomal RNA, particularly in regions that contact the 16S rRNA helices.

• Surface accessibility: When incorporated into nanovesicles, certain epitopes of the S2 protein appear to be surface-exposed, as evidenced by immunogold labeling techniques used in microscopy studies of L. johnsonii-derived nanovesicles .

The protein has been identified as one of several ribosomal components present in L. johnsonii nanovesicles, suggesting it maintains a stable fold that allows for packaging and potential extracellular functions. Structural analysis through electron microscopy revealed that these nanovesicles exhibit spherical structures averaging 558.0 ± 67.1 nm in width, providing a physical context for rpsB localization .

How does rpsB contribute to the immunomodulatory properties of L. johnsonii?

The 30S ribosomal protein S2 from L. johnsonii appears to contribute to immunomodulatory properties through several sophisticated mechanisms:

• Nanovesicle association: rpsB has been identified as a significant component of L. johnsonii-derived nanovesicles (NV), which serve as delivery vehicles for bacterial components to distal host tissues. These NV facilitate host-microbe interactions without requiring bacterial colonization of target tissues .

• Dendritic cell modulation: L. johnsonii supplementation significantly alters dendritic cell (DC) function in respiratory tissues. Bone marrow-derived dendritic cells (BMDCs) from L. johnsonii-supplemented mice show decreased production of innate cytokines (IL-6, IL-1β, TNFα) and increased IFNβ expression when challenged with respiratory syncytial virus (RSV) . While the specific contribution of rpsB to this effect remains to be fully elucidated, its presence in nanovesicles suggests it may play a role in this immunomodulation.

• T-cell response modification: BMDCs from L. johnsonii-supplemented mice exhibited altered capacity to stimulate T-cell responses, resulting in reduced IL-4 production and increased IFNγ. This indicates a shift from Th2 to Th1 immune responses, which is beneficial in respiratory viral infections .

• Systemic metabolic reprogramming: L. johnsonii supplementation alters the host metabolic profile, including enrichment of docosahexanoic acid (DHA), which itself possesses immunomodulatory properties. These metabolic changes correlate with altered immune cell function and may involve bacterial proteins, potentially including rpsB, that are delivered systemically via nanovesicles .

Research exploring these mechanisms could provide valuable insights for developing novel immunomodulatory strategies for respiratory infections and allergic conditions.

What experimental approaches can be used to study the interaction between recombinant rpsB and host immune cells?

Several sophisticated experimental approaches can be employed to investigate interactions between recombinant L. johnsonii rpsB and host immune cells:

• Surface plasmon resonance (SPR): Enables real-time, label-free quantification of protein-protein interactions between rpsB and potential immune cell receptors, providing association and dissociation rate constants. Experimental setup should include proper negative controls (irrelevant proteins) and positive controls (known immunomodulatory proteins).

• Dendritic cell stimulation assays: Treatment of bone marrow-derived dendritic cells with purified recombinant rpsB, followed by assessment of:

  • Maturation markers (MHC class II, CD80, CD86) by flow cytometry

  • Cytokine production (IL-6, IL-1β, TNFα, IFNβ) by ELISA or qRT-PCR

  • T cell stimulation capacity through co-culture experiments

• Fluorescently-labeled protein tracking: Conjugating recombinant rpsB with fluorescent markers enables visualization of cellular uptake, subcellular localization, and trafficking using confocal microscopy.

• Pull-down assays coupled with mass spectrometry: Identifying specific host cell receptors or binding partners for rpsB through affinity purification followed by proteomic analysis.

• Transcriptomic analysis: RNA-seq of immune cells treated with recombinant rpsB can reveal global changes in gene expression patterns and activated signaling pathways.

Experimental evidence indicates that BMDCs from L. johnsonii-supplemented mice show significantly decreased expression of maturation markers (MHC class II, CD80, CD86) when infected with RSV, compared to controls . These findings suggest that rpsB or other L. johnsonii components may modulate dendritic cell maturation and function, warranting further investigation using the methodologies described above.

How can researchers distinguish between the direct effects of rpsB and those mediated through L. johnsonii nanovesicles?

Distinguishing between direct rpsB effects and those mediated through L. johnsonii nanovesicles requires methodical experimental design and comparative analyses:

• Differential purification protocols:

  • Isolate highly purified recombinant rpsB (>95% purity)

  • Purify intact nanovesicles containing native rpsB

  • Generate rpsB-depleted nanovesicles through immunoprecipitation or genetic approaches

  • Create reconstituted nanovesicles with controlled rpsB content

• Comparative functional assays:

  • Parallel testing of each preparation in identical immune cell stimulation experiments

  • Dose-response analyses to identify potential threshold effects

  • Time-course studies to determine kinetics of response

• Receptor blocking experiments:

  • Pretreatment of target cells with antibodies against potential receptors

  • Competitive inhibition assays with known ligands

  • Receptor knockout or knockdown in cell lines

• Subcellular tracking approaches:

  • Differential labeling of rpsB and nanovesicle membranes

  • Live-cell imaging to track internalization and trafficking pathways

  • Co-localization with endosomal/lysosomal markers

Research has demonstrated that plasma from L. johnsonii-supplemented mice can recapitulate the immunomodulatory effects observed with direct bacterial supplementation. When BMDCs were treated with plasma (2% in culture media) from L. johnsonii-supplemented animals and then infected with RSV, they exhibited decreased production of inflammatory cytokines (IL-6, IL-1β, TNFα) and increased type I interferon (IFNβ) . This suggests that systemic factors, possibly including nanovesicles containing rpsB, mediate these effects rather than requiring direct bacterial contact.

What genomic and proteomic approaches can be used to understand the evolutionary conservation and variation of rpsB among different Lactobacillus species?

Understanding the evolutionary conservation and variation of rpsB among Lactobacillus species requires integrated genomic and proteomic methodologies:

• Comparative genomic analysis:

  • Whole-genome sequencing of multiple Lactobacillus strains

  • Identification of rpsB orthologs through BLAST and hidden Markov model searches

  • Synteny analysis to examine genomic context conservation

  • Calculation of selection pressure metrics (dN/dS ratios) to identify conserved functional domains versus variable regions

• Phylogenetic studies:

  • Construction of phylogenetic trees based on rpsB sequences

  • Comparison with species trees based on 16S rRNA or whole-genome data

  • Analysis of horizontal gene transfer events using phylogenetic incongruence methods

• Structural proteomics:

  • Homology modeling of rpsB proteins from different species

  • Identification of conserved surface patches potentially involved in RNA binding

  • Prediction of species-specific structural elements

• Functional proteomics:

  • Cross-species comparison of post-translational modifications

  • Conservation analysis of protein-protein interaction networks

  • Experimental validation of functional differences through heterologous expression

The evolutionary analysis of Lactobacillus species has revealed interesting patterns in protein domain architecture. For example, while most Lactobacillus species contain two or fewer SH3b domains in surface proteins, L. taiwanensis strains have four to six SH3b domains, and L. gasseri 224-1 has six SH3b domains . Similar comparative analyses focused specifically on rpsB could reveal evolutionary patterns relevant to its functional diversity.

What are the optimal conditions for expressing and purifying recombinant L. johnsonii rpsB?

The optimization of expression and purification conditions for recombinant L. johnsonii 30S ribosomal protein S2 involves multiple factors that must be systematically evaluated:

Expression Optimization:

• Expression system selection:

  • E. coli BL21(DE3) typically yields high protein levels

  • Rosetta strains may improve expression if rare codons are present

  • Lactobacillus-based expression systems may provide native folding environment

• Temperature optimization:

  • Standard induction at 37°C often leads to inclusion body formation

  • Reduced temperatures (16-25°C) improve solubility but decrease expression rate

  • Optimal results typically achieved at 18°C for 16-18 hours post-induction

• Induction parameters:

  • IPTG concentration: Titrate between 0.1-1.0 mM (optimal usually 0.2-0.5 mM)

  • Cell density at induction: OD600 between 0.6-0.8 for balanced yield and solubility

Purification Protocol:

• Cell lysis buffer composition:

  • 50 mM Tris-HCl or phosphate buffer (pH 7.5-8.0)

  • 300-500 mM NaCl to maintain solubility

  • 5-10% glycerol as stabilizing agent

  • 1-5 mM β-mercaptoethanol or DTT to prevent oxidation

  • Protease inhibitor cocktail to prevent degradation

• Affinity purification:

  • His-tagged variants: Ni-NTA chromatography with 20-40 mM imidazole in wash buffer

  • GST-tagged variants: Glutathione-Sepharose with PBS washing

• Secondary purification:

  • Ion-exchange chromatography (typically Q-Sepharose)

  • Size-exclusion chromatography for final polishing

• Quality control metrics:

  • 95% purity by SDS-PAGE

  • Single peak by size-exclusion chromatography

  • Correct mass by mass spectrometry

  • Functional activity in RNA binding assays

The protein should be stored in buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, and 10% glycerol at -80°C for long-term stability. Avoid repeated freeze-thaw cycles, which can lead to aggregation and loss of activity.

How can researchers design experiments to evaluate the immunomodulatory effects of recombinant rpsB in respiratory viral infection models?

Designing robust experiments to evaluate immunomodulatory effects of recombinant rpsB in respiratory viral infection models requires multi-level approaches spanning in vitro to in vivo systems:

In Vitro Experimental Design:

• Cell culture models:

  • Primary human bronchial epithelial cells (air-liquid interface)

  • BMDC cultures from wild-type mice

  • Human peripheral blood mononuclear cells (PBMCs)

• Treatment protocol:

  • Pre-treatment with purified rpsB (5-50 μg/ml) for 6-24 hours

  • Viral challenge with RSV (MOI 0.1-1.0)

  • Appropriate controls: heat-denatured rpsB, irrelevant protein, vehicle

• Readout parameters:

  • Viral replication (plaque assay, qPCR)

  • Cytokine/chemokine profiles (multiplex assay)

  • Gene expression changes (RNA-seq)

  • Cell surface activation markers (flow cytometry)

Ex Vivo Systems:

• Precision-cut lung slices from naive mice:

  • Treatment with rpsB followed by viral challenge

  • Assessment of viral spread and tissue inflammatory response

• BMDC-T cell co-culture system:

  • BMDCs pre-treated with rpsB then infected with RSV

  • Co-culture with CD4+ T cells from lungs of RSV-infected mice

  • Measurement of T cell cytokine production and proliferation

In Vivo Models:

• Administration routes:

  • Intranasal delivery of rpsB (10-100 μg per mouse)

  • Systemic (intraperitoneal) administration

  • Comparison with L. johnsonii supplementation

• Timing variations:

  • Prophylactic (pre-infection) administration

  • Therapeutic (post-infection) intervention

• Analysis parameters:

  • Clinical disease scoring

  • Viral load in respiratory tissues

  • Pulmonary inflammation (histopathology)

  • Airway hyperreactivity

  • Immune cell populations (flow cytometry)

  • Local and systemic cytokine profiles

Previous research has demonstrated that BMDCs from L. johnsonii-supplemented mice exhibited significantly decreased production of inflammatory cytokines (IL-6, IL-1β, TNFα) and increased IFNβ expression when challenged with RSV . Furthermore, these BMDCs showed reduced expression of maturation markers (MHC class II, CD80, CD86) and altered capacity to stimulate T cell responses, with decreased IL-4 and increased IFNγ production . These findings provide valuable baseline comparisons for experiments specifically evaluating recombinant rpsB effects.

What are the most effective methods for incorporating recombinant rpsB into experimental nanovesicles for delivery studies?

Creating experimental nanovesicles incorporating recombinant rpsB requires sophisticated methodologies that balance protein stability, nanovesicle integrity, and functional delivery. The following approaches represent current best practices:

Reconstitution Approaches:

• Direct membrane protein incorporation:

  • Detergent-mediated reconstitution: Solubilize purified rpsB in mild detergents (n-dodecyl-β-D-maltoside or CHAPS), then incorporate into preformed liposomes followed by detergent removal via dialysis

  • Detergent-free incorporation: Mix rpsB with lipids in aqueous solution, followed by freeze-thaw cycles and extrusion

• Surface conjugation methods:

  • Maleimide chemistry: Add a C-terminal cysteine to rpsB for covalent linking to maleimide-functionalized lipids in preformed vesicles

  • Click chemistry: Incorporate azide-modified lipids into vesicles and alkyne-modified rpsB for bioorthogonal conjugation

• Encapsulation techniques:

  • Passive encapsulation during vesicle formation (typically yields 5-15% efficiency)

  • Active loading using pH or ion gradients (if applicable to rpsB properties)

Characterization Parameters:

• Physical characterization:

  • Size distribution: Dynamic light scattering (optimal range: 50-200 nm)

  • Morphology: Transmission electron microscopy with negative staining

  • Zeta potential: Surface charge measurement (typically -5 to -30 mV)

• Protein incorporation verification:

  • Protein:lipid ratio determination

  • Orientation analysis (inside vs. outside) using protease protection assays

  • Functionality verification through binding assays

• Stability assessment:

  • Storage stability at 4°C and 37°C

  • Serum stability testing

  • Freeze-thaw resistance evaluation

Delivery Optimization:

• Surface modifications:

  • PEGylation for extended circulation

  • Targeting ligands for cell-specific delivery

  • Cell-penetrating peptides for enhanced uptake

• Administration routes:

  • Intranasal delivery for respiratory applications

  • Intravenous administration for systemic distribution

  • Intraperitoneal injection for immunological studies

Research on natural L. johnsonii nanovesicles has revealed they contain various proteins including ribosomal proteins like S2 . These natural nanovesicles appear as spherical structures budding off the bacterial cell with an average width of 558.0 ± 67.1 nm . Experimental nanovesicles should aim to recapitulate the key features of these natural structures while allowing for controlled incorporation of recombinant rpsB.

How should researchers interpret contradictory results between in vitro and in vivo studies of rpsB immunomodulatory effects?

Interpreting contradictory results between in vitro and in vivo studies of rpsB immunomodulatory effects requires systematic analysis of multiple factors that could contribute to these discrepancies:

Methodological Considerations:

• Concentration disparities:

  • In vitro systems often use non-physiological protein concentrations

  • Calculate effective concentrations reached in target tissues in vivo and match in vitro conditions accordingly

  • Perform dose-response studies in both systems

• Temporal dynamics:

  • In vitro systems typically examine short-term effects (hours to days)

  • In vivo effects may involve adaptive responses over longer timeframes

  • Implement time-course experiments in both systems

• Cellular complexity:

  • In vitro models often use homogeneous cell populations

  • In vivo responses involve complex cellular interactions

  • Consider using co-culture systems or ex vivo tissue explants as intermediate models

Biological Factors:

• Microenvironmental differences:

  • Create a comparison table documenting differences in pH, oxygen tension, nutrient availability, and cytokine milieu between systems

  • Test the impact of these variables systematically

• Protein modifications:

  • Verify protein integrity in both systems

  • Examine post-translational modifications or proteolytic processing

  • Consider using tagged variants for tracking protein fate

• Indirect effects:

  • In vivo effects may be mediated through intermediary cell types absent in vitro

  • Metabolites induced by rpsB may contribute to in vivo effects

  • Design experiments to identify potential mediators

Reconciliation Strategies:

• Mechanistic hypothesis generation:

  • Develop hypotheses that could explain both sets of observations

  • Design targeted experiments to test these hypotheses

  • Consider whether contradictory results represent different aspects of a complex response

• Systems biology approach:

  • Apply computational modeling to integrate in vitro and in vivo data

  • Identify potential feedback loops or compensatory mechanisms present only in vivo

  • Use network analysis to predict additional factors

Research with L. johnsonii has demonstrated that while direct administration affects local immune responses, systemic effects are also observed that could be mediated by circulating factors. For example, plasma from L. johnsonii-supplemented mice could recapitulate the immunomodulatory effects on dendritic cells observed with direct bacterial supplementation . This suggests that reconciling in vitro and in vivo observations requires consideration of both direct protein effects and secondary mediators that may arise in the complex in vivo environment.

What bioinformatic tools and approaches are most valuable for analyzing the structural and functional relationships of rpsB?

Comprehensive analysis of rpsB structural and functional relationships requires an integrated bioinformatic workflow combining sequence analysis, structural prediction, evolutionary studies, and functional annotation:

Sequence Analysis Tools:

• Multiple sequence alignment:

  • MUSCLE or MAFFT for accurate alignment of rpsB sequences across species

  • T-Coffee for incorporating structural information into alignments

  • Jalview for visualization and analysis of conservation patterns

• Motif and domain identification:

  • InterProScan for comprehensive domain annotation

  • MEME Suite for novel motif discovery

  • ConSurf for identifying functionally important residues based on evolutionary conservation

• Post-translational modification prediction:

  • NetPhos for phosphorylation sites

  • GlycoEP for glycosylation sites

  • NetAcet for acetylation prediction

Structural Analysis Pipeline:

• Secondary structure prediction:

  • PSIPRED for accurate secondary structure element identification

  • JPred4 for consensus prediction approach

• 3D structure modeling:

  • AlphaFold2 or RoseTTAFold for de novo structure prediction

  • SWISS-MODEL for homology modeling using related ribosomal proteins

  • PyMOL for visualization and structural analysis

• Molecular dynamics simulations:

  • GROMACS for analyzing conformational flexibility

  • Normal mode analysis for identifying functional movements

Functional Annotation Methods:

• Protein-protein interaction prediction:

  • STRING database for known interactions

  • PRISM for structure-based interaction prediction

  • Coevolution analysis using methods like GREMLIN

• RNA-binding prediction:

  • BindN+ for nucleic acid binding site prediction

  • catRAPID for RNA-protein interaction propensity

• Immunological epitope analysis:

  • IEDB analysis tools for B-cell and T-cell epitope prediction

  • EpiPred for antibody epitope prediction

Evolutionary Analysis:

• Selection pressure analysis:

  • PAML for detecting positive or purifying selection

  • FEL/MEME via Datamonkey for site-specific selection analysis

• Phylogenetic studies:

  • RAxML for maximum likelihood phylogenetic tree construction

  • MrBayes for Bayesian phylogenetic inference

  • Dendroscope for visualization and comparison of phylogenetic trees

Bioinformatic analysis has already revealed that Lactobacillus species display varying patterns in protein domain architecture. For example, most Lactobacillus species contain two or fewer SH3b domains in certain surface proteins, while L. taiwanensis strains have four to six SH3b domains . Similar comparative approaches applied specifically to rpsB could provide insights into its evolutionary conservation and potential functional adaptations across species.

What statistical approaches are most appropriate for analyzing dose-dependent effects of recombinant rpsB on immune cell responses?

Analyzing dose-dependent effects of recombinant rpsB on immune cell responses requires robust statistical methodologies that account for biological variability, non-linear responses, and multiple outcome measures:

Experimental Design Considerations:

• Dose selection:

  • Minimum of 5-7 concentrations spanning at least 3 orders of magnitude

  • Include zero-dose control and positive control

  • Use logarithmic spacing between doses (e.g., 0.1, 1, 10, 100 μg/ml)

• Replication strategy:

  • Minimum of 3-4 biological replicates per dose

  • 2-3 technical replicates within each biological replicate

  • Consider repeated experiments for confirmation

• Response variables:

  • Define primary and secondary endpoints in advance

  • Include both quantitative (cytokine levels) and categorical (activation status) outcomes

  • Consider time-dependent measurements

Basic Statistical Approaches:

• Dose-response curve fitting:

  • Four-parameter logistic regression (4PL) for sigmoidal responses

  • EC50/IC50 determination with 95% confidence intervals

  • Comparison of curve parameters between experimental conditions

• ANOVA-based methods:

  • One-way ANOVA with post-hoc tests (Tukey or Dunnett's) for single-factor analysis

  • Two-way ANOVA for examining interaction effects (e.g., dose × cell type)

  • Mixed-effects models for repeated measures designs

• Non-parametric alternatives:

  • Kruskal-Wallis with Dunn's post-hoc test for non-normally distributed data

  • Jonckheere-Terpstra test for detecting monotonic trends across ordered doses

Advanced Statistical Methods:

• Multivariate approaches:

  • Principal Component Analysis (PCA) to identify patterns across multiple cytokines

  • Partial Least Squares (PLS) regression for relating dose to multiple responses

  • MANOVA for simultaneous analysis of multiple dependent variables

• Hierarchical and mixed modeling:

  • Nested design analysis for complex experimental structures

  • Random-effects models to account for inter-experimental variability

  • Repeated measures approaches for time-course experiments

• Bayesian methods:

  • Bayesian hierarchical modeling for integration of prior knowledge

  • Markov Chain Monte Carlo (MCMC) for parameter estimation

  • Credible intervals for more intuitive interpretation of uncertainty

Reporting Standards:

Previous research with L. johnsonii has shown dose-dependent effects on dendritic cell responses, with significant reductions in inflammatory cytokines (IL-6, IL-1β, TNFα) and increases in IFNβ observed in response to stimulation . Statistical approaches for analyzing rpsB-specific effects should build upon these observations while implementing rigorous control conditions and appropriate statistical methods for the specific experimental design employed.

How might recombinant rpsB be utilized in developing novel immunomodulatory therapeutics for respiratory diseases?

Recombinant L. johnsonii rpsB holds significant potential for development into novel immunomodulatory therapeutics for respiratory diseases through several strategic approaches:

Therapeutic Modalities:

• Direct protein administration:

  • Intranasal delivery of purified rpsB for localized respiratory effects

  • Inhalation formulations (dry powder or nebulized solutions)

  • Dose optimization to balance immunomodulation versus potential inflammatory responses

• Nanovesicle-based delivery systems:

  • Synthetic liposomes incorporating rpsB

  • Bacterial outer membrane vesicles (OMVs) engineered to contain rpsB

  • Targeted nanovesicles with respiratory epithelium-specific ligands

• Combination approaches:

  • Co-administration with other immunomodulatory molecules

  • Sequential treatment protocols (priming with rpsB followed by specific antigens)

  • Integration into existing treatment regimens

Target Respiratory Conditions:

• Viral respiratory infections:

  • RSV infection, where L. johnsonii supplementation has shown efficacy in reducing Th2 cytokines and viral load

  • Influenza infection models

  • Potential applications for COVID-19-associated inflammation

• Allergic respiratory conditions:

  • Allergic asthma, targeting the Th2-biased immune responses

  • Allergic rhinitis

  • Hypersensitivity pneumonitis

• Chronic inflammatory conditions:

  • COPD exacerbations

  • Non-CF bronchiectasis

  • Inflammatory phenotypes of interstitial lung disease

Developmental Pathway:

• Preclinical development:

  • Comprehensive toxicology assessment

  • Pharmacokinetic/pharmacodynamic modeling

  • Formulation optimization for stability and bioavailability

• Biomarker development:

  • Identification of response predictors

  • Development of companion diagnostics

  • Patient stratification strategies

• Regulatory considerations:

  • Classification pathway (biologic vs. drug)

  • Safety monitoring protocols

  • Target population definition

Research has demonstrated that L. johnsonii supplementation significantly reduces airway Th2 cytokines (IL-4, IL-5, IL-13) during RSV infection, increases regulatory T cells, and enhances viral clearance . If recombinant rpsB can recapitulate these effects, it would represent a significant advance in targeted immunotherapy for respiratory diseases, potentially offering advantages in terms of standardization, scalability, and mechanistic specificity compared to whole bacteria supplementation.

What are the potential applications of rpsB in developing research tools for studying host-microbe interactions?

Recombinant L. johnsonii rpsB offers diverse applications as a research tool for investigating host-microbe interactions, with potential to advance our understanding of fundamental immunological processes:

Molecular Probe Applications:

• Tagged rpsB variants:

  • Fluorescently labeled rpsB for tracking cellular uptake and trafficking

  • Biotinylated rpsB for pull-down experiments to identify binding partners

  • Photo-crosslinkable rpsB for capturing transient interactions

• Reporter systems:

  • RpsB-luciferase fusion proteins for real-time monitoring of protein localization

  • Split reporter complementation assays for protein-protein interaction studies

  • FRET-based biosensors to detect conformational changes upon binding

• Affinity reagents:

  • Development of anti-rpsB antibodies for immunolocalization studies

  • rpsB-based affinity columns for purifying interaction partners

  • Peptide libraries derived from rpsB sequences for epitope mapping

Cellular Biology Applications:

• Immune cell phenotyping:

  • Standardized stimulation reagent for dendritic cell maturation assays

  • Benchmark for comparing immunomodulatory properties of different bacterial components

  • Tool for investigating strain-specific differences in immune recognition

• Host-microbe interaction models:

  • Development of reporter cell lines expressing fluorescent markers in response to rpsB

  • Creation of "minimal microbiome" systems with defined bacterial components

  • Reconstitution experiments to determine sufficient components for immunomodulation

• Comparative studies:

  • Systematic comparison of rpsB from different bacterial species

  • Structure-function analysis through mutational studies

  • Cross-species conservation analysis of immune recognition

Advanced Research Applications:

• Single-cell technologies:

  • Single-cell RNA-seq of rpsB-stimulated immune populations

  • Mass cytometry (CyTOF) panel development including rpsB-responsive markers

  • Spatial transcriptomics to map tissue-level responses to rpsB

• Systems biology approaches:

  • Network analysis of rpsB-induced signaling pathways

  • Integration with metabolomic data to link immune and metabolic responses

  • Computational modeling of dose-response relationships

Studies with L. johnsonii have demonstrated that its components, potentially including rpsB, can modulate dendritic cell function, alter T cell cytokine profiles, and influence systemic metabolic patterns . Purified recombinant rpsB would enable researchers to dissect the specific contribution of this protein to these complex host-microbe interactions, facilitating more mechanistic studies than are possible with whole bacteria or crude fractions.

How might CRISPR-Cas9 gene editing be utilized to study the functional importance of rpsB in L. johnsonii?

CRISPR-Cas9 gene editing provides powerful approaches for elucidating the functional importance of rpsB in L. johnsonii through precise genetic manipulation:

CRISPR System Development for L. johnsonii:

• Vector design considerations:

  • Codon optimization of Cas9 for Lactobacillus expression

  • Selection of appropriate promoters (e.g., P23, Ppgm)

  • Temperature-sensitive replicons for transient expression

  • Single-plasmid versus dual-plasmid systems

• gRNA design strategy:

  • Target selection within rpsB gene with minimal off-target effects

  • PAM site identification (typically NGG for SpCas9)

  • Prediction of editing efficiency using algorithms like DeepCRISPR

  • Multiple gRNA approach for increased efficiency

• Delivery methods:

  • Electroporation protocols optimized for Lactobacillus (field strength 1.5-2.0 kV/cm)

  • Temperature and growth phase optimization for competent cell preparation

  • Recovery media composition for maximal transformation efficiency

Genetic Modification Strategies:

• Complete knockout approaches:

  • Since rpsB is likely essential, conditional knockout systems may be required

  • Inducible promoter replacement (tetO, nisin-inducible)

  • Degron-tagging for controlled protein degradation

• Domain-specific modifications:

  • Precise editing of functional domains using HDR templates

  • Introduction of point mutations at conserved residues

  • Creation of domain deletion variants

• Tagged variant generation:

  • C-terminal or N-terminal epitope tags (FLAG, HA)

  • Fluorescent protein fusions for localization studies

  • Insertion of cleavable purification tags

Functional Characterization Approaches:

• Growth phenotype analysis:

  • Growth curves under various conditions (stress, nutrient limitation)

  • Competition assays with wild-type strains

  • Biofilm formation capacity

• Protein expression and localization:

  • Western blot analysis of modified rpsB

  • Immunofluorescence microscopy for localization

  • Fractionation studies to determine subcellular distribution

• Nanovesicle characterization:

  • Quantitative and qualitative analysis of NV production

  • Proteomics of NV composition

  • Functional testing of modified NV in immune modulation assays

• Host interaction studies:

  • In vitro co-culture with immune cells

  • Ex vivo tissue interactions

  • In vivo colonization and immune response assessment

Research has shown that 30S ribosomal proteins, including S2, are found at high concentrations in both nanovesicles and culture medium of L. johnsonii . CRISPR-Cas9 modified strains could help determine whether rpsB is passively incorporated into nanovesicles or actively targeted for inclusion, and how alterations in rpsB structure affect nanovesicle formation, composition, and function.