Recombinant Lactobacillus johnsonii 30S ribosomal protein S4 (rpsD)

Basic Research Questions

• What is the functional role of 30S ribosomal protein S4 in Lactobacillus johnsonii?

The 30S ribosomal protein S4 (rpsD) in L. johnsonii plays a crucial role in translational accuracy during protein synthesis. Similar to its homologs in other bacteria, it interacts directly with the 16S rRNA and is essential for proper ribosome assembly and function . The protein works in conjunction with other ribosomal proteins like S5 and S12 to ensure accuracy during the translation process .

In L. johnsonii, rpsD is part of the universal ribosomal protein uS4 family and functions in maintaining the structural integrity of the small ribosomal subunit. Unlike the arrangement in E. coli where rpsD is located in the alpha operon, in Bacillus subtilis (a model Gram-positive bacterium similar to Lactobacillus), rpsD exists as a monocistronic gene with its expression subject to autogenous control .

• How is recombinant L. johnsonii 30S ribosomal protein S4 typically expressed and purified?

Recombinant L. johnsonii 30S ribosomal protein S4 can be expressed in various host systems, with E. coli being the most common for initial characterization studies. The expression and purification process typically follows this methodology:

For L. johnsonii proteins specifically, codon optimization considering the organism's high AT content is essential for successful heterologous expression. When expressing in Lactobacillus hosts, the pPG612 vector system has been successfully employed, as demonstrated in studies with other recombinant proteins .

• What are the immunological properties of L. johnsonii ribosomal proteins including S4?

L. johnsonii ribosomal proteins, including the 30S ribosomal protein S4, display significant immunoreactivity in host systems. Studies have identified several ribosomal proteins from L. johnsonii with immunogenic properties:

While S4-specific immunological data is less abundant, its structural homology to S7 suggests potential similar immunomodulatory roles. The immunogenicity of ribosomal proteins makes them interesting candidates for vaccine development and immunomodulatory applications. These proteins can trigger antibody responses, including both IgG and IgA production, and may participate in bacterial adhesion and stress adaptation mechanisms .

• How does the genetic diversity among L. johnsonii strains affect rpsD structure and function?

Genetic diversity among L. johnsonii strains significantly impacts genomic architecture and potentially the expression and function of essential genes like rpsD. Key findings include:

• Comparative genomic analysis among 12 strains of L. johnsonii (including 8 porcine, 3 human, and 1 poultry isolate) revealed significant intra-species genomic rearrangements .

• A large symmetrical inversion of approximately 750 kb was observed when comparing the human isolate L. johnsonii NCC 533 with the porcine isolate DPC6026, potentially affecting gene expression patterns .

• The genome architecture found in the porcine isolate DPC6026 appears to be more common within the species than that of the human isolate NCC 533 .

• These structural variations may influence the regulation and expression of essential genes like rpsD, although direct evidence of rpsD sequence variation is limited in the available data.

This genetic diversity suggests that strain-specific optimizations may be necessary when designing recombinant expression systems for L. johnsonii rpsD .

Advanced Research Questions

• What are the optimal conditions for expressing recombinant L. johnsonii rpsD in different host systems?

The optimal conditions for expressing recombinant L. johnsonii rpsD vary depending on the host system. Based on existing research with similar proteins:

In E. coli expression systems:

• Use of T7-based expression vectors with IPTG induction

• Growth at lower temperatures (16-25°C) following induction to enhance solubility

• Supplementation with additional zinc ions may improve folding

• Typical yield: 0.1-1.0 mg/mL (post-IMAC purification)

In Lactobacillus expression systems:

• pSIP vectors with inducible promoters show high efficiency for Gram-positive targets

• For L. plantarum hosts, xylose-inducible promoters demonstrate good regulation

• Optimal induction at mid-log phase (OD600 0.5-0.8)

• Incubation at 37°C for 20 hours post-induction

• Addition of Sec-type signal peptides (e.g., Lp_2145) enhances secretion

In Baculovirus/insect cell systems:

• Better for complex proteins requiring post-translational modifications

• Lower yields but potentially better folding for complex proteins

The selection of appropriate host strain is critical; L. plantarum WCFS1 has been shown to be particularly effective for expressing Gram-positive bacterial proteins, while E. coli remains the most time-efficient system for initial studies.

• How can L. johnsonii rpsD be engineered for enhanced therapeutic properties?

Engineering L. johnsonii rpsD for enhanced therapeutic properties can be approached through several strategies based on current research:

Fusion protein strategies:

• Integration of bioactive peptides or mimotopes into the rpsD sequence

• An example from related research shows that fusion proteins like TTmim-PrtB (tetanus toxin mimotope fused to proteinase PrtB) can be successfully expressed on the surface of L. johnsonii

• Such fusion constructs can induce both systemic IgG and local mucosal IgA responses

Expression optimization:

• Implementing codon optimization for the target host

• Using inducible promoter systems, such as the xylose-inducible system used in L. johnsonii GM-CSF expression studies

• Selection of appropriate signal peptides for optimal localization (cytoplasmic, membrane-associated, or secreted)

Cell surface anchoring:

• Using dedicated anchoring domains for cell wall attachment

• Surface-exposed ribosomal proteins have been shown to mediate bacterial adhesion and stress adaptation, properties that could be enhanced through directed engineering

Validation methods:

• Employing Western blotting to confirm expression (as shown in studies with recombinant L. johnsonii expressing bovine GM-CSF)

• Stability testing over multiple generations (40+ generations) to ensure consistent inheritance of the recombinant plasmid

• How does stress response affect rpsD expression in L. johnsonii, particularly under bile salt exposure?

Stress responses, particularly bile salt exposure, significantly alter protein expression patterns in L. johnsonii, potentially affecting rpsD expression and function:

L. johnsonii PF01 has been shown to be highly resistant to bile, a critical property for probiotic applications. Upon exposure to bile stress:

• Quantitative proteomic analysis using iTRAQ–LC–MS/MS technology identified 215 proteins with changed expression levels in response to bile stress (0.1%, 0.2%, and 0.3% bile salts)

• Of these, 94 proteins showed increased expression while 121 decreased

• Affected categories included stress responses, cell division, transcription, translation, nucleotide metabolism, carbohydrate transport, cell wall biosynthesis, and amino acid biosynthesis

• Both proteomic and mRNA expression data indicated increased phosphotransferase activity and cell wall biosynthesis under bile stress

While direct data on rpsD expression under stress is limited, as a ribosomal protein involved in translation, its expression is likely affected by these stress conditions. Understanding these changes is crucial for developing stable recombinant strains for gastrointestinal applications .

• What methodologies are effective for studying L. johnsonii rpsD interactions with host immune systems?

Effective methodologies for studying L. johnsonii rpsD interactions with host immune systems include:

In vitro immunological assays:

• Western blotting to detect antibody reactivity against specific bacterial proteins

• Two-dimensional gel electrophoresis followed by immunoblotting to identify immunoreactive spots

• Mass spectrometry for protein identification and characterization

• T-cell proliferation assays to assess immunostimulatory properties

Recombinant expression systems for immunological studies:

• Expression of rpsD in L. johnsonii for surface display

• Development of fusion proteins combining rpsD with immunogenic epitopes

• Oral immunization models to evaluate mucosal immune responses

In vivo models:

• Mouse models to assess systemic (IgG) and mucosal (IgA) immune responses

• Measurement of inflammatory markers (IL-6, IL-1β, TNF-α) to evaluate immunomodulatory effects

• Histological examination to assess tissue responses to recombinant L. johnsonii

Clinical evaluation:

• Analysis of serum samples from human subjects to detect antibody responses against L. johnsonii proteins

• Fecal IgA analysis to evaluate mucosal immune responses

• Monitoring of inflammatory markers to assess immunomodulatory effects

This combined approach allows for comprehensive characterization of L. johnsonii rpsD's immunological properties and its potential applications in vaccine development and immunomodulation .

• What are the challenges in maintaining stability of recombinant L. johnsonii expressing modified rpsD?

Maintaining stability of recombinant L. johnsonii expressing modified rpsD presents several challenges:

Plasmid stability issues:

• Recombinant plasmids can be lost over generations without selective pressure

• Studies with recombinant L. johnsonii have implemented stability testing over 40+ generations to ensure consistent inheritance

• Selection of appropriate antibiotic resistance markers (e.g., chloramphenicol resistance) is crucial for maintaining selective pressure

Environmental stress impacts:

• Gastrointestinal conditions (low pH, bile salts) significantly affect L. johnsonii viability and protein expression

• L. johnsonii strains exhibit variable resistance to acid stress, with strain 456 showing higher viability at lower pH compared to other tested strains

• Bile salt exposure leads to significant proteomic changes affecting 215 proteins across various functional categories

Expression system considerations:

• Inducible promoter systems (e.g., xylose-inducible) provide better control but may lack stability in vivo without the inducer

• Constitutive promoters offer more stable expression but may impose metabolic burden

• Signal peptide selection affects protein localization and potential immunogenicity

Host-specific adaptation:

• Genomic analysis indicates host-specific divergence of L. johnsonii strains with respect to genome inversion and phage exposure

• These host adaptations may impact the stability and expression of recombinant constructs in different environments

• How can antagonistic interactions between L. johnsonii and pathogens be leveraged in therapeutic applications of recombinant rpsD?

The antagonistic interactions between L. johnsonii and pathogens can be strategically leveraged in therapeutic applications of recombinant rpsD:

Understanding the natural antimicrobial mechanisms:

• L. johnsonii displays pH-dependent and pH-independent antagonistic interactions against pathogens like Candida albicans

• These interactions inhibit pathogen growth in both planktonic and biofilm states

• The antagonism is influenced by nutrient availability and the production of soluble metabolites with antimicrobial activity

• L. johnsonii can physically co-aggregate with pathogens, particularly along hyphal structures of C. albicans

Engineering enhanced antimicrobial properties:

• Recombinant rpsD could be engineered to include antimicrobial peptide domains

• The cell surface localization of ribosomal proteins can be exploited for direct interaction with pathogens

• Surface-exposed ribosomal proteins mediate bacterial adhesion and stress adaptation, properties that could be enhanced through directed engineering

Combination strategies:

• L. johnsonii with engineered rpsD could be combined with other antimicrobial agents for synergistic effects

• The natural acid production by L. johnsonii (reducing media pH from 6.5 to 3.9 in MRS broth) could complement engineered antimicrobial properties

Delivery considerations:

• L. johnsonii partially survives simulated gastric conditions in vitro, making it suitable as an oral delivery vehicle

• Recombinant L. johnsonii strains can induce both systemic IgG and local mucosal IgA responses, enhancing host defense against pathogens