Recombinant Lactobacillus johnsonii Enolase 2 (eno2)

What is Lactobacillus johnsonii Enolase 2 and what cellular functions does it perform?

Enolase 2 (eno2) is a glycolytic enzyme found in Lactobacillus johnsonii that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolysis pathway. Beyond its metabolic role, enolase in L. johnsonii may serve moonlighting functions when enriched in extracellular vesicles (EVs). Research indicates that proteins enriched in L. johnsonii EVs, including enolase, may contribute to host-microbe interactions and potentially modulate immune responses. In experimental studies, purified enolase from L. johnsonii showed small but significant induction of immune signaling pathways, suggesting it may play a role in the bacterium's immunomodulatory effects .

How does L. johnsonii Enolase 2 differ from enolases in other bacterial species?

L. johnsonii Enolase 2 shares structural similarities with enolases from other lactic acid bacteria but may contain unique sequence motifs that contribute to strain-specific functions. While the core catalytic domain is likely conserved, variations in surface-exposed regions may influence its ability to interact with host cells or components. Comparative genomic analyses of different L. johnsonii strains have revealed variations in their protein repertoire that correlate with distinct host adaptations and functional properties. These strain-specific differences extend to proteins like enolase that may be involved in host-microbe communication and contribute to the specific health benefits associated with different L. johnsonii strains .

What purification methods are recommended for isolating recombinant L. johnsonii Enolase 2?

For the purification of recombinant L. johnsonii Enolase 2, nickel affinity chromatography has proven effective. Research protocols have successfully employed Ni+ affinity columns to purify enolase and other L. johnsonii proteins (Eno3, P1875, P8875, P1390) . The general methodology involves:

• Cloning the eno2 gene into an expression vector with a His-tag

• Transforming into a suitable expression host (typically E. coli BL21)

• Inducing protein expression with IPTG

• Cell lysis (usually by sonication or French press)

• Clarification of lysate by centrifugation

• Loading the supernatant onto a Ni+ affinity column

• Washing with increasing imidazole concentrations

• Eluting with high imidazole buffer

• Dialysis to remove imidazole

• Concentration determination using Bradford or BCA assay

Researchers should include appropriate controls in their purification workflow, such as mock preparations from E. coli BL21 (DE3) not containing an expression vector, to control for potential contaminants from the expression host .

What cell culture models are most appropriate for studying the immunomodulatory effects of recombinant L. johnsonii Enolase 2?

Based on current research with L. johnsonii proteins, macrophage cell lines with reporter systems are highly recommended for studying immunomodulatory effects. Specifically, RAW-Dual™ KO-TLR4 macrophage cells have been successfully employed to evaluate the immune-stimulating capacity of L. johnsonii proteins while excluding potential lipopolysaccharide contamination effects. These cells contain dual reporter genes that allow simultaneous quantification of:

• NF-κB activation (via MIP-2 promoter fusion to secreted alkaline phosphatase)

• Interferon signaling (via ISRE promoter fusion to Lucia luciferase)

This dual-reporter system enables comprehensive assessment of different immune signaling pathways activated by recombinant proteins. For optimal results, proteins should be tested at concentrations around 1.5 μg/mL, with appropriate controls including mock preparations from expression hosts .

How can researchers assess the interaction between recombinant L. johnsonii Enolase 2 and host epithelial cells?

To assess interactions between recombinant L. johnsonii Enolase 2 and host epithelial cells, researchers should employ a multi-faceted approach:

• Binding assays: Use fluorescently-labeled recombinant Enolase 2 to quantify binding to epithelial cell lines (e.g., Caco-2, HT-29 for intestinal studies).

• Transepithelial electrical resistance (TEER): Measure changes in epithelial barrier integrity using polarized epithelial monolayers exposed to recombinant Enolase 2.

• Immunofluorescence microscopy: Track the localization of Enolase 2 after exposure to epithelial cells to determine if it remains surface-bound or becomes internalized.

• Gene expression analysis: Assess changes in epithelial cell gene expression following Enolase 2 exposure using qRT-PCR for genes involved in barrier function and immune signaling.

• Cytokine profiling: Measure secreted cytokines and chemokines using ELISA or multiplex assays to characterize the epithelial response.

Research with L. johnsonii has demonstrated that its components can modulate intestinal epithelial barrier function and enhance protection against pathogens, suggesting similar experimental approaches would be valuable for studying Enolase 2 specifically .

What protein-protein interaction methods are suitable for identifying binding partners of L. johnsonii Enolase 2?

For identifying binding partners of L. johnsonii Enolase 2, researchers should consider these methodologies:

• Pull-down assays: Immobilize His-tagged recombinant Enolase 2 on nickel resin and incubate with host cell lysates to capture interacting proteins.

• Co-immunoprecipitation: Use antibodies against Enolase 2 to precipitate the protein along with its binding partners from mixtures containing host cell components.

• Surface plasmon resonance (SPR): Determine binding kinetics and affinities between purified Enolase 2 and candidate interacting proteins.

• Cross-linking mass spectrometry: Identify proximity-based interactions by chemically cross-linking Enolase 2 with potential partners before mass spectrometric analysis.

• Yeast two-hybrid screening: Screen for interactions between Enolase 2 and host proteins in a high-throughput manner.

These approaches can help elucidate the molecular mechanisms through which L. johnsonii proteins like Enolase 2 might contribute to health benefits observed in studies, such as immune modulation and pathogen antagonism .

How does recombinant L. johnsonii Enolase 2 contribute to the activation of innate immune pathways?

The contribution of L. johnsonii Enolase 2 to innate immune pathway activation appears to be modest compared to other L. johnsonii components. Experimental evidence shows that while Enolase from L. johnsonii showed "small but significant induction" of immune signaling pathways, it was not as potent as other bacterial components such as the SH3b2 domain of Sdp . Specifically:

What role might L. johnsonii Enolase 2 play in protection against viral infections?

While L. johnsonii has demonstrated antiviral properties, the specific contribution of Enolase 2 to this effect remains to be fully elucidated. Research has shown that L. johnsonii N6.2 extracellular vesicles (EVs) can stimulate antiviral responses by inducing the 2',5'-oligoadenylate synthetase (OAS) pathway and expression of antiviral genes including IFI44L, MX1, MX2, and DDX60 . This response has been shown to inhibit murine norovirus (MNV-1) replication both in vitro and in vivo.

For researchers investigating Enolase 2's potential antiviral role, experiments comparing viral replication in cells treated with purified recombinant Enolase 2 versus other L. johnsonii components would help clarify its specific contribution to the observed antiviral effects of this probiotic bacterium.

How can researchers investigate the potential role of L. johnsonii Enolase 2 in pathogen antagonism?

To investigate L. johnsonii Enolase 2's potential role in pathogen antagonism, researchers should implement a comprehensive experimental approach:

• Growth inhibition assays: Test purified recombinant Enolase 2 against various pathogens (bacteria, fungi) in different pH conditions, as L. johnsonii's antimicrobial effects are often pH-dependent .

• Adhesion interference studies: Examine whether Enolase 2 can block pathogen adhesion to epithelial cells by:

  • Pre-treating epithelial cells with Enolase 2 before pathogen exposure

  • Simultaneously exposing cells to Enolase 2 and pathogens

  • Quantifying adhered pathogens through colony counting or fluorescence microscopy

• Biofilm inhibition analysis: Assess the impact of Enolase 2 on pathogen biofilm formation using crystal violet staining or confocal microscopy to visualize biofilm architecture.

• Enzymatic activity measurements: Determine if Enolase 2 possesses any direct antimicrobial enzymatic activities that could affect pathogen viability.

• Metabolic pathway analysis: Investigate whether Enolase 2 interferes with pathogen metabolism through competitive inhibition or other mechanisms.

Research has shown that L. johnsonii can antagonize various pathogens including enteropathogenic E. coli, Salmonella typhimurium, and Candida albicans through multiple mechanisms including pH modulation, H₂O₂ production, antimicrobial metabolites, and competitive exclusion . Investigating whether Enolase 2 contributes to these activities would provide valuable insights into its potential as an antimicrobial agent.

What are common challenges in expressing and purifying functionally active recombinant L. johnsonii Enolase 2?

Researchers working with recombinant L. johnsonii Enolase 2 should be aware of several common challenges and their solutions:

• Protein solubility issues:

  • Challenge: Enolase 2 may form inclusion bodies when overexpressed in E. coli.

  • Solution: Optimize expression conditions by lowering incubation temperature (16-25°C), reducing IPTG concentration, or using solubility-enhancing fusion tags like SUMO or MBP.

• Endotoxin contamination:

  • Challenge: Preparations from E. coli may contain lipopolysaccharide that can confound immunological studies.

  • Solution: Use endotoxin removal columns or TLR4 knockout cell lines (like RAW-Dual™ KO-TLR4) for immunology experiments, as demonstrated in studies with other L. johnsonii proteins .

• Protein activity loss:

  • Challenge: Purification steps may reduce enzymatic activity.

  • Solution: Include glycerol (10-20%) and reducing agents in buffers, minimize freeze-thaw cycles, and validate enzymatic activity through standard enolase assays measuring conversion of 2-phosphoglycerate to phosphoenolpyruvate.

• Protein yield variability:

  • Challenge: Expression yields may vary between batches.

  • Solution: Standardize growth conditions, OD at induction, and optimize codon usage for E. coli expression.

• Protein aggregation during storage:

  • Challenge: Purified protein may aggregate during storage.

  • Solution: Store at appropriate protein concentration (typically 1-5 mg/mL), add stabilizers like trehalose, and store in small aliquots at -80°C.

Implementing these strategies can help ensure consistent production of high-quality, functionally active recombinant L. johnsonii Enolase 2 for research applications.

How can researchers differentiate between the metabolic and moonlighting functions of L. johnsonii Enolase 2?

Differentiating between the metabolic and potential moonlighting functions of L. johnsonii Enolase 2 requires specialized experimental approaches:

Recent research with L. johnsonii extracellular vesicles has shown that bacterial proteins can perform different functions when released into the host environment compared to their intracellular roles , making these approaches valuable for understanding the multifunctional nature of Enolase 2.

How might L. johnsonii Enolase 2 contribute to the development of novel bacterial therapeutics?

L. johnsonii Enolase 2 could contribute to bacterial therapeutics development through several research avenues:

• Engineered probiotics: Researchers could develop L. johnsonii strains with modulated Enolase 2 expression to enhance specific beneficial effects. This approach requires understanding how Enolase 2 levels correlate with probiotic functions, particularly in the context of immune modulation and pathogen antagonism reported for L. johnsonii .

• Protein-based therapeutics: If Enolase 2 demonstrates significant immune-modulating or antimicrobial properties, it could be developed as a standalone therapeutic protein. This approach would build upon findings that specific L. johnsonii proteins, particularly those found in extracellular vesicles, can independently elicit biological responses in host cells .

• Targeted delivery systems: Encapsulating recombinant Enolase 2 in liposomal or nanoparticle delivery systems could enhance its stability and targeting to specific tissues. This strategy mirrors successful approaches using liposome-encapsulated L. johnsonii proteins like Sdp-SH3b2, which significantly decreased murine norovirus titers in the distal ileum in mouse models .

• Adjuvant development: If Enolase 2 shows consistent immunomodulatory properties, it could be investigated as a potential adjuvant for vaccines or immunotherapies, leveraging the ability of certain L. johnsonii components to enhance immune responses.

• Combination therapies: Research could explore synergistic effects between Enolase 2 and other L. johnsonii components or conventional therapeutics, potentially enhancing efficacy against pathogens or in managing inflammatory conditions.

Each of these approaches requires rigorous validation of Enolase 2's specific functions and mechanisms of action before clinical applications can be pursued.

What advanced techniques could help elucidate the structural determinants of L. johnsonii Enolase 2 functions?

Advanced structural biology techniques can provide crucial insights into the molecular basis of L. johnsonii Enolase 2 functions:

By integrating structural insights with functional studies, researchers can identify key structural determinants that differentiate L. johnsonii Enolase 2 from enolases in other species and potentially explain its unique contributions to the probiotic properties of L. johnsonii .

How can systems biology approaches enhance our understanding of L. johnsonii Enolase 2 in the context of host-microbe interactions?

Systems biology approaches offer powerful frameworks for understanding L. johnsonii Enolase 2's role within complex host-microbe interactions:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data from both host cells and L. johnsonii following their interaction can reveal how Enolase 2 fits into broader regulatory networks. This approach can help identify unexpected connections between Enolase 2 and host pathways beyond its known functions.

• Network analysis: Constructing protein-protein interaction networks involving Enolase 2 and host proteins can identify central nodes and pathways affected by this bacterial protein. Research has shown that L. johnsonii can modulate immune signaling networks and metabolic pathways in the host , and network analysis could clarify Enolase 2's specific contributions.

• In silico predictive modeling: Developing computational models that simulate the effects of Enolase 2 on host cell signaling can generate testable hypotheses about its mechanisms of action. This is particularly valuable for complex processes like immune modulation, where multiple pathways may be affected simultaneously.

• Single-cell analyses: Advanced single-cell technologies can reveal heterogeneity in host cell responses to Enolase 2, potentially identifying specific cell populations that are particularly responsive to this bacterial protein.

• Microbiome interaction studies: Investigating how Enolase 2 affects the broader gut microbiome composition and function can provide insights into indirect mechanisms by which it might influence host health. L. johnsonii has been shown to modulate the intestinal microbiota in various contexts , and Enolase 2 may contribute to these effects.