Recombinant Lactobacillus johnsonii Elongation factor G (fusA), partial

What is the function of Elongation factor G (fusA) in Lactobacillus johnsonii?

Elongation factor G (EF-G), encoded by the fusA gene, is a GTPase that facilitates the translocation of tRNAs and mRNA by one codon during protein synthesis in prokaryotes like L. johnsonii. EF-G catalyzes this movement by utilizing energy liberated through GTP hydrolysis. The protein undergoes significant conformational changes between its GTP and GDP-bound forms, which are critical for its function .

How does L. johnsonii adapt to survive in host environments, and how might this impact recombinant protein expression?

L. johnsonii has evolved several mechanisms to survive the challenging conditions of the gastrointestinal tract, which researchers must consider when developing recombinant strains:

• Acid and bile resistance: L. johnsonii has demonstrated partial survival under simulated gastric conditions in vitro , suggesting inherent resistance mechanisms that make it suitable as an oral delivery vehicle.

• Structural adaptations: The bacterium possesses a protective S-layer (outer protein shell), extracellular peptidoglycan, teichoic acids, and polysaccharides that maintain cellular integrity and facilitate host adherence .

• Stress sensing and export systems: These complement the stress resistance machinery and allow the bacterium to respond to environmental challenges .

• Metabolic flexibility: L. johnsonii's genome encodes numerous phosphotransferase system (PTS) and ATP-binding cassette (ABC) transporters along with amino acid proteases and peptidases, enabling utilization of various sugars and amino acids available in the host GI tract .

These adaptations must be considered when designing recombinant expression systems, as they may influence protein yield, stability, and functionality in experimental and therapeutic applications.

What characteristics make L. johnsonii suitable for recombinant protein expression in vaccine development?

L. johnsonii possesses several attributes that make it particularly attractive for recombinant protein expression in vaccine development:

• Safety profile: Lactobacilli are generally recognized as safe organisms, making them attractive vehicles for oral vaccination without significant safety concerns .

• Gastrointestinal survival: L. johnsonii can partially survive gastric conditions, allowing it to reach intestinal tissues where immune interactions occur .

• Surface display capability: The bacterium can express heterologous proteins on its cell surface, as demonstrated with proteinase PrtB and mimotope peptides derived from tetanus toxin .

• Immunomodulatory properties: L. johnsonii can induce both systemic (IgG) and local mucosal (IgA) immune responses, critical for effective mucosal vaccination strategies .

• Genetic tractability: Researchers have successfully developed vector systems for L. johnsonii that enable reliable expression of recombinant proteins .

These properties collectively support L. johnsonii's potential as a vaccine delivery platform, particularly for antigens targeting mucosal immunity.

How does the expression of recombinant proteins affect the probiotic properties of L. johnsonii?

The expression of recombinant proteins in L. johnsonii creates a complex interplay between heterologous protein production and native probiotic functions:

• Metabolic burden: Expression of recombinant proteins diverts cellular resources from natural metabolic processes, potentially affecting the production of beneficial metabolites like short-chain fatty acids (SCFAs) and lactic acid, which are important for L. johnsonii's probiotic effects .

• Immune response modulation: Recombinant strains may alter the bacterium's immunomodulatory properties. For example, when L. johnsonii expressed the TTmim-PrtB fusion protein, it induced specific systemic IgG and fecal IgA responses, demonstrating that recombinant expression can modulate host immune interactions .

• Colonization and persistence: Genetic modifications may affect cell surface structures involved in adhesion and colonization, potentially changing the bacterium's residence time in the host.

• Antagonistic activities: L. johnsonii naturally exhibits antagonistic properties against pathogens like H. pylori . Recombinant protein expression could enhance or diminish these capabilities depending on the protein's nature and metabolic requirements.

• Stress response: Recombinant protein production can trigger stress responses that might alter the bacterium's resistance to environmental challenges, affecting its survival in the gastrointestinal tract.

Researchers should evaluate these parameters when developing recombinant L. johnsonii strains to ensure that beneficial probiotic properties are maintained alongside the desired recombinant protein expression.

What mechanisms drive translocation during protein synthesis, and how might partial expression of EF-G affect this process?

The translocation process during protein synthesis involves a sophisticated mechanism combining power-stroke and Brownian ratchet elements, as revealed by recent research:

• Initial power stroke: After GTP hydrolysis, EF-G undergoes a small (~10°) global rotational motion relative to the ribosome that generates force to "unlock" the ribosomal complex .

• Domain-specific movements: Following the initial movement, larger rotations occur within domain III of EF-G before its dissociation from the ribosome .

• Hybrid mechanism: The initial steps of translocation involve ribosome unlocking driven by EF-G-dependent GTP hydrolysis (power stroke), while subsequent steps are primarily driven by the energetics of the ribosome itself (Brownian ratchet) .

When partial expression or truncated versions of EF-G are present, several effects may occur:

These considerations are crucial when studying recombinant L. johnsonii strains expressing modified EF-G proteins or when targeting fusA for genetic manipulation.

How should researchers design experiments to evaluate the impact of recombinant L. johnsonii on host immune responses?

When designing experiments to evaluate immune responses to recombinant L. johnsonii, researchers should implement a comprehensive approach:

Experimental Design Framework:

• Strain preparation:

  • Construct recombinant L. johnsonii expressing the protein of interest

  • Prepare appropriate controls (wild-type strain, vector-only strain)

  • Verify protein expression through Western blotting or flow cytometry

  • Characterize growth curves to ensure comparable viability

• In vitro evaluation:

  • Co-culture with relevant immune cells (dendritic cells, macrophages, T cells)

  • Measure cytokine production profile (e.g., IL-10, IL-12, TNF-α)

  • Assess cell surface marker expression on immune cells

  • Analyze transcriptional changes in immune-related genes

• In vivo assessment:

  • Design appropriate immunization schedule (single vs. multiple doses)

  • Consider route of administration (oral immunization has been successful)

  • Include control groups (placebo, wild-type bacteria, purified antigen)

  • Plan time points for sample collection (blood, feces, mucosal tissues)

• Immune readouts to measure:

  • Systemic antibody responses (serum IgG, as seen with PrtB-specific responses)

  • Mucosal antibody responses (fecal IgA, as demonstrated with PrtB)

  • T cell responses (proliferation, cytokine production)

  • Changes in microbiome composition

• Statistical considerations:

  • Power analysis to determine sample size

  • Appropriate statistical tests for data types

  • Multiple testing correction for high-dimensional data

  • Consideration of biological vs. technical replicates

This framework has been validated in previous studies, where oral immunization with recombinant L. johnsonii expressing TTmim-PrtB fusion protein successfully induced both systemic IgG responses against L. johnsonii and recombinant PrtB, as well as PrtB-specific fecal IgA responses .

What experimental approaches can detect and characterize rotational motions of EF-G domains during translation?

The detection and characterization of EF-G domain rotational motions during translation require sophisticated biophysical techniques:

Methodological Approaches:

• Single-molecule polarized fluorescence microscopy:

  • This technique was instrumental in capturing the three-dimensional rotational motions of individual EF-G domains during normal translocation

  • Enables real-time observation of protein dynamics without the need for translation-inhibiting antibiotics

  • Requires fluorescent labeling of specific EF-G domains with polarization-sensitive fluorophores

• Time-resolved cryo-electron microscopy (cryo-EM):

  • Captures structural snapshots at different stages of translocation

  • Provides high-resolution structural information about EF-G conformational changes

  • Samples must be rapidly frozen at defined time points after initiation of translation

• Förster resonance energy transfer (FRET):

  • Measures distances between labeled domains during conformational changes

  • Can be performed at the single-molecule level (smFRET) for detailed dynamics

  • Requires strategic placement of donor-acceptor fluorophore pairs

• Molecular dynamics simulations:

  • Complements experimental data with atomistic models of domain movements

  • Predicts energetics and conformational intermediates

  • Requires high-performance computing resources

When designing such experiments for L. johnsonii EF-G, researchers should consider the bacterium's specific growth conditions and genetic accessibility for introducing fluorescent tags or mutations.

How should researchers interpret contradictory results in studies of L. johnsonii's effects on host immunity?

Contradictory results in L. johnsonii immunological studies require systematic analysis to resolve inconsistencies:

Analytical Framework:

• Strain-specific differences:

  • Different L. johnsonii strains exhibit varying immunomodulatory properties

  • Compare genomic and proteomic profiles of strains used across studies

  • Example: L. johnsonii NCC 533 supernatants did not control H. pylori persistence in humans, while other strains showed efficacy

• Experimental conditions:

  • Viability status: Live vs. heat-killed bacteria can elicit different responses

  • Dosage: Evaluate dose-dependent effects across studies

  • Administration route: Oral vs. other routes affect immune responses

  • Treatment duration: Short-term vs. long-term administration

• Host factors:

  • Species differences: Mouse vs. human responses may vary significantly

  • Microbiome background: Pre-existing microbiota influences outcomes

  • Health status: Response in healthy vs. disease states

  • Genetic background: Host genetic variation affects immune responses

• Readout parameters:

  • Different immune parameters measured (antibodies vs. cytokines vs. cell populations)

  • Local vs. systemic immunity assessment

  • Timing of measurements relative to treatment

• Technical considerations:

  • Assay sensitivity and specificity

  • Sample processing methods

  • Statistical approaches and power

When analyzing contradictory findings, researchers should construct a comprehensive comparison table of these factors across studies. For example, while oral immunization with recombinant L. johnsonii induced specific immune responses against the PrtB protein, no antibody response was observed against the tetanus toxin mimotope in the same study, suggesting antigen-specific differences in immunogenicity .

What approaches can distinguish between EF-G-mediated effects and other factors in protein synthesis experiments?

Distinguishing EF-G-specific effects from other factors in protein synthesis experiments requires careful experimental design and controls:

Methodological Strategies:

• Site-directed mutagenesis:

  • Create point mutations in key functional domains of EF-G

  • Target GTP-binding site, ribosome-binding interfaces, or domain interfaces

  • Compare effects of specific mutations to wild-type EF-G

• Domain swapping and chimeric proteins:

  • Replace individual domains of EF-G with corresponding domains from other species

  • Create fusion proteins to isolate domain-specific functions

  • Analyze which chimeric constructs retain function and which fail

• Specific inhibitors:

  • Use EF-G-specific inhibitors (e.g., fusidic acid) at sub-inhibitory concentrations

  • Compare with inhibitors targeting other translation factors

  • Establish dose-response relationships for different inhibitors

• Reconstituted in vitro translation systems:

  • Use purified components to reconstruct translation machinery

  • Systematically omit or replace individual factors

  • Measure effects on specific steps of translation

• Time-resolved measurements:

  • Track translation kinetics using fluorescent reporters

  • Identify rate-limiting steps affected by EF-G modifications

  • Compare with effects of modulating other translation factors

Data analysis should include:

• Multi-factorial statistical models to partition variance among factors

• Kinetic modeling to identify steps most sensitive to EF-G function

• Structural correlations between observed functional effects and known EF-G conformational changes

This approach aligns with findings showing that EF-G has a specific role in the initial unlocking of the ribosome through a small rotational motion after GTP hydrolysis .

What strategies can overcome challenges in achieving stable expression of recombinant proteins in L. johnsonii?

Achieving stable expression of recombinant proteins in L. johnsonii requires addressing several technical challenges:

Optimization Strategies:

• Vector system optimization:

  • Use species-specific or closely related promoters for reliable expression

  • Implement inducible promoter systems to control expression timing

  • Optimize ribosome binding sites for L. johnsonii's translational machinery

  • Include transcription terminators to prevent read-through effects

  • Example: Researchers successfully used a new vector system to express proteinase PrtB and TTmim-PrtB fusion proteins on the surface of L. johnsonii

• Codon optimization:

  • Analyze L. johnsonii's codon usage bias

  • Adapt heterologous gene sequences to match preferred codons

  • Balance GC content to match the host genome

  • Avoid rare codons that might cause translational pausing

• Protein stabilization:

  • Fuse recombinant proteins to stability-enhancing tags or domains

  • Co-express chaperones to assist proper folding

  • Include protease inhibitors in growth medium

  • Consider secretion signals matched to L. johnsonii's secretion machinery

• Growth condition optimization:

  • Determine optimal temperature, pH, and media composition

  • Test different induction timing and concentration

  • Consider microaerobic or anaerobic growth conditions

  • Supplement with cofactors required for protein function

• Selection strategy:

  • Maintain selective pressure throughout cultivation

  • Implement post-segregational killing mechanisms

  • Use complementation of essential gene deletion for plasmid maintenance

  • Consider chromosomal integration for long-term stability

Implementing these strategies has enabled successful expression of functional recombinant proteins on the surface of L. johnsonii, as demonstrated in vaccine delivery studies .

How can researchers validate the functional impacts of EF-G modifications on translation in L. johnsonii?

Validating the functional impacts of EF-G modifications requires a multi-faceted approach:

Validation Framework:

• In vitro translation assays:

  • Purify modified EF-G proteins from recombinant L. johnsonii

  • Conduct reconstituted translation reactions with purified ribosomes

  • Measure translation rates of reporter mRNAs

  • Compare translocation efficiency with wild-type EF-G

• Ribosome profiling:

  • Analyze ribosome positioning on mRNAs genome-wide

  • Identify altered translation elongation rates or ribosome stalling

  • Correlate changes with specific mRNA sequence features

  • Compare profiles between modified and wild-type EF-G strains

• Growth and stress response assessments:

  • Measure growth rates under various conditions

  • Challenge with translation-specific stressors

  • Monitor expression of stress response genes

  • Assess protein aggregation and quality control activation

• Structural studies:

  • Use single-molecule techniques to track EF-G rotational movements similar to those observed in previous studies

  • Perform comparative cryo-EM of translation complexes

  • Analyze changes in EF-G domain positioning and interactions

• Complementation experiments:

  • Express wild-type EF-G in strains with modified variants

  • Test whether growth defects or translation abnormalities are rescued

  • Create chimeric EF-G proteins to map functional domains

This comprehensive approach can reveal how modifications to EF-G affect the hybrid mechanism of translation, which combines power-stroke and Brownian ratchet elements as described in recent research .