Recombinant Lactobacillus johnsonii Holo-[acyl-carrier-protein] synthase (acpS)

What is Lactobacillus johnsonii and why is it significant for recombinant protein expression?

Lactobacillus johnsonii is a gram-positive bacterial species that has garnered significant research interest due to its status as a generally recognized as safe (GRAS) organism. This safety profile makes it particularly attractive as a vehicle for oral vaccination and probiotic applications. L. johnsonii partially survives simulated gastric conditions in vitro, suggesting its potential effectiveness as an oral delivery vehicle for proteins of interest . The bacterium can be genetically modified to express foreign proteins either intracellularly or on its cell surface, making it a versatile platform for recombinant protein expression. L. johnsonii strains have been successfully used in various experimental settings, including as potential probiotics in animal feed additives and as modulators of host immune responses in respiratory infections .

What is the function of Holo-[acyl-carrier-protein] synthase (acpS) in bacterial systems?

Holo-[acyl-carrier-protein] synthase (acpS) plays a critical role in bacterial fatty acid biosynthesis. This enzyme catalyzes the post-translational modification of apo-acyl carrier protein (apoACP) by transferring the 4'-phosphopantetheine moiety from coenzyme A (CoA) to the conserved serine 36 residue of apoACP . This conversion transforms apoACP into its active form, holo-acyl carrier protein (holoACP), which is essential for fatty acid biosynthesis. The acpS gene has been identified as essential for bacterial growth, and mutations in this gene can lead to growth deficiencies, as demonstrated in Escherichia coli studies. For example, the E. coli MP4 strain, which contains a G4D mutation in AcpS (termed AcpS1), exhibits approximately 5-fold reduction in catalytic efficiency compared to wild-type AcpS, resulting in an elevated growth requirement for CoA .

How do vector systems for L. johnsonii recombinant expression work?

Vector systems for L. johnsonii recombinant expression are specialized constructs designed to introduce and maintain foreign DNA in this probiotic bacterium. These vectors typically include promoters compatible with Lactobacillus transcriptional machinery, appropriate selection markers, and elements for either cytoplasmic expression or surface display of target proteins. Research has demonstrated successful implementation of vector systems for expressing proteins on the surface of L. johnsonii, such as proteinase PrtB and a tetanus toxin mimotope integrated into proteinase PrtB (TTmim-PrtB fusion protein) . These systems incorporate necessary elements for appropriate protein folding, translocation, and anchoring to the cell wall. The effectiveness of the vector system significantly impacts expression levels, stability of the recombinant strain, and ultimately the functionality of the expressed protein in experimental or therapeutic applications.

What are the optimal conditions for culturing recombinant L. johnsonii expressing acpS?

Optimizing culture conditions for recombinant L. johnsonii expressing acpS requires careful consideration of medium composition and growth parameters. Research employing response surface methodology (RSM) and sequential quadratic programming (SQP) has identified optimal medium formulations that significantly enhance L. johnsonii growth. The optimal medium composition has been determined to contain 5.5% (wt/vol) soybean meal as a nitrogen source, 1.0% (wt/vol) molasses as a carbon source, and 1.0% (wt/vol) sodium acetate as a growth promoter . Under these conditions, maximum population densities of 8.90 log CFU/mL for L. johnsonii have been achieved. Incubation should typically be conducted at 37°C for approximately 16 hours to reach optimal growth. The addition of selection antibiotics may be necessary to maintain plasmid stability in recombinant strains, though this can slightly reduce growth rates. When expressing acpS specifically, supplementation with CoA precursors might be beneficial, as AcpS utilizes CoA as a substrate for the phosphopantetheinylation reaction .

How can researchers evaluate the expression and activity of recombinant acpS in L. johnsonii?

Evaluating the expression and activity of recombinant acpS in L. johnsonii requires a multi-faceted approach:

Expression Analysis:

• Western blotting with anti-AcpS antibodies to confirm protein expression

• Quantitative RT-PCR to measure acpS transcript levels

• Mass spectrometry to verify protein identity and expression levels

Activity Assessment:

• Enzymatic assays measuring the conversion of apoACP to holoACP

• Complementation studies in conditional acpS mutants

• Analysis of downstream fatty acid biosynthesis products

Functional Impacts:

• Growth curves comparing recombinant strains to wild-type controls

• Stress resistance profiling (acid, bile, temperature)

• Assessment of membrane integrity and composition

The gold standard for confirming AcpS activity involves demonstrating the transfer of 4'-phosphopantetheine from CoA to apoACP. This can be monitored through biochemical assays using purified components or through in vivo accumulation analysis of apoACP versus holoACP in the recombinant strain . Complementation experiments, wherein recombinant AcpS rescues growth in conditional acpS mutants, provide strong evidence of functional activity.

What vector design considerations are critical for successful acpS expression in L. johnsonii?

Successful expression of acpS in L. johnsonii requires careful vector design addressing several key considerations:

Promoter Selection:

• Strong constitutive promoters for consistent expression

• Inducible promoters for controlled expression timing

• Promoters optimized for Lactobacillus (e.g., those derived from lactobacilli housekeeping genes)

Codon Optimization:

• Adapting the acpS coding sequence to L. johnsonii codon usage bias

• Avoiding rare codons that could impede translation

Signal Peptides and Targeting:

• Including appropriate signal peptides if secretion is desired

• Cell wall anchoring domains (like those used for PrtB expression) if surface display is the goal

Selection Markers:

• Antibiotic resistance genes compatible with L. johnsonii

• Complementation markers for auxotrophic strains

Origin of Replication:

• Using origins recognized by L. johnsonii

• Considering copy number effects on expression and metabolic burden

Research has demonstrated that vector systems can be successfully employed in L. johnsonii for expressing proteins either intracellularly or on the cell surface . When designing vectors for acpS expression specifically, researchers should consider whether native regulation elements should be included to ensure appropriate integration with endogenous fatty acid biosynthesis pathways.

What is the relationship between acpS functionality and bacterial growth in recombinant L. johnsonii?

The relationship between acpS functionality and bacterial growth is fundamentally important as AcpS catalyzes an essential step in fatty acid biosynthesis. Studies in E. coli have demonstrated that acpS is an essential gene, and mutations affecting AcpS function can significantly impact bacterial growth . For example, the E. coli MP4 strain, containing the AcpS1 protein with a G4D mutation, exhibits a 5-fold reduction in catalytic efficiency and an elevated growth requirement for CoA .

In recombinant L. johnsonii expressing acpS, several scenarios could occur:

• Enhanced fatty acid biosynthesis: If functional acpS is overexpressed, it might increase the rate of apoACP to holoACP conversion, potentially enhancing fatty acid biosynthesis capacity.

• Metabolic burden: Expression of recombinant proteins generally creates a metabolic burden on bacterial cells, which could counteract any benefits from enhanced AcpS activity.

• Altered membrane composition: Changes in fatty acid biosynthesis could alter the membrane lipid composition, affecting membrane fluidity, permeability, and stress resistance.

• Regulatory imbalances: Overexpression of acpS might disrupt the balance of fatty acid biosynthesis pathway components, potentially leading to accumulation of intermediates or pathway dysregulation.

How effective is L. johnsonii as a mucosal vaccine delivery system?

L. johnsonii has demonstrated promising characteristics as a mucosal vaccine delivery system. Research indicates that L. johnsonii can partially survive simulated gastric conditions in vitro, suggesting it can reach intestinal mucosal surfaces after oral administration . When used to express antigens on its surface, L. johnsonii can induce both systemic and mucosal immune responses. Specifically, oral immunization of mice with recombinant L. johnsonii expressing proteinase PrtB fusion protein induced a systemic IgG response against both L. johnsonii and the recombinantly expressed proteinase PrtB . Importantly, this immunization also elicited a proteinase PrtB-specific fecal IgA response, demonstrating the ability of L. johnsonii to induce local mucosal immunity .

For recombinant L. johnsonii expressing acpS as a potential vaccine component, the immunogenicity would depend on whether acpS is recognized as foreign by the host immune system and whether it is presented in a manner that effectively engages immune surveillance mechanisms.

How do extracellular vesicles from L. johnsonii influence host-microbe interactions, and could acpS be delivered via this mechanism?

Extracellular vesicles (EVs) derived from L. johnsonii represent an emerging area of research with significant implications for host-microbe interactions. Research has demonstrated that the SH3b2 domain contained in L. johnsonii N6.2, which is enriched in L. johnsonii-derived EVs, functions as an effector molecule that can orchestrate the control of viral infections . These EVs can influence host cellular responses at various concentrations (10^8, 10^9, 10^10 EVs) and timing schedules (pre-treatment, co-inoculation, or post-inoculation relative to viral challenge) .

In experimental models, oral administration of L. johnsonii N6.2 EVs (1x10^10) for seven days protected mice against subsequent viral challenge . The immunomodulatory mechanisms appear to involve both NF-κB-dependent pathways and interferon signaling responses .

Regarding acpS delivery via EVs, several considerations emerge:

• Packaging potential: Whether acpS protein or its encoding mRNA could be naturally or artificially packaged into L. johnsonii EVs

• Functional delivery: If packaged, whether the delivered acpS would maintain functionality in recipient cells

• Targeted engineering: Possibilities for engineering L. johnsonii EVs specifically for enhanced acpS delivery

• Therapeutic applications: Potential applications of acpS-containing EVs for modulating host lipid metabolism or immune responses

This represents an advanced research direction that could expand our understanding of both L. johnsonii biology and the therapeutic potential of bacterial EVs as delivery vehicles.

What metabolic reprogramming occurs in host cells exposed to L. johnsonii, and how might acpS expression influence these effects?

L. johnsonii supplementation induces significant metabolic reprogramming in host systems, with implications for immune responses and disease outcomes. Research demonstrates that L. johnsonii supplementation is associated with a reprogrammed circulating metabolic environment, including docosahexanoic acid (DHA) enrichment . This metabolic shift appears functionally significant, as bone marrow-derived dendritic cells (BMDCs) from L. johnsonii-supplemented mice exhibit altered cytokine production profiles and reduced expression of co-stimulatory molecules when challenged with respiratory syncytial virus (RSV) .

Intriguingly, these immunomodulatory effects could be reproduced by treating wild-type BMDCs with either plasma from L. johnsonii-supplemented mice or directly with DHA . This suggests that metabolites produced or induced by L. johnsonii, rather than direct bacterial interaction, mediate some of the observed immunological effects.

Regarding recombinant acpS expression, several hypotheses emerge:

• Altered lipid metabolism: Enhanced acpS activity might modify the fatty acid composition of L. johnsonii, potentially altering the profile of lipid metabolites that interact with host cells.

• Bacterial metabolite production: Changes in fatty acid biosynthesis could influence the production of bacterial metabolites that affect host metabolism.

• Immune signaling effects: Modified bacterial surface lipids resulting from altered acpS activity might change how pattern recognition receptors recognize the bacterium.

• EV composition changes: acpS-mediated changes in lipid biosynthesis could alter the composition and bioactivity of L. johnsonii-derived extracellular vesicles.

Understanding these metabolic interactions represents an important frontier in probiotic research and could inform the development of next-generation therapeutic approaches.

What challenges exist in maintaining genetic stability of recombinant L. johnsonii expressing acpS?

Maintaining genetic stability in recombinant L. johnsonii expressing acpS presents several significant challenges:

Plasmid Stability Issues:

• Without selection pressure, plasmids carrying the acpS gene can be lost over generations

• Metabolic burden of acpS expression may select for plasmid-free populations

• Recombination events can disrupt expression cassettes

Expression Level Variability:

• Promoter activity may vary under different growth conditions

• Copy number fluctuations can cause inconsistent expression levels

• Protein degradation rates may affect steady-state acpS levels

Metabolic Interference:

• Overexpression of acpS might interfere with endogenous fatty acid metabolism

• Altered membrane composition due to modified fatty acid synthesis could affect cell viability

• Feedback inhibition mechanisms might counteract consistent expression

Genetic Drift:

• Mutations accumulating in the acpS gene or regulatory elements

• Selection for compensatory mutations to counterbalance metabolic effects

• Potential for horizontal gene transfer in non-sterile applications

Strategies to address these challenges include chromosomal integration rather than plasmid-based expression, use of stabilization elements, codon optimization, selection of appropriate promoters with consistent activity in Lactobacillus, and regular monitoring of genetic stability through sequencing and functional assays across multiple generations.

How can researchers address variable expression levels of recombinant proteins in L. johnsonii?

Variable expression levels represent a common challenge in recombinant protein production using L. johnsonii. Researchers can employ several strategies to address this variability:

Optimizing Growth Conditions:

• Use defined medium compositions optimized for L. johnsonii growth, such as media containing specific ratios of soybean meal (5.5% w/v), molasses (1.0% w/v), and sodium acetate (1.0% w/v)

• Maintain consistent temperature (37°C), pH, and incubation times (16h) across experiments

• Control for growth phase effects by standardizing harvest points

Vector Design Improvements:

• Test multiple promoters to identify those with consistent activity in L. johnsonii

• Optimize ribosome binding sites for efficient translation initiation

• Include transcriptional terminators to prevent read-through

• Consider codon optimization tailored to L. johnsonii preferences

Expression Monitoring Systems:

• Incorporate reporter genes (e.g., fluorescent proteins) to track expression levels

• Develop real-time monitoring systems for culture optimization

• Implement inline process analytical technologies for large-scale production

Selection Strategies:

• Screen multiple transformants to identify high-producing clones

• Use antibiotic selection to maintain plasmid presence

• Consider integrative approaches for stable expression

Statistical Approach:

• Employ Design of Experiments (DoE) methodologies similar to the Box-Behnken design used for optimizing growth media

• Develop predictive models for expression based on multiple variables

• Establish robust quality control metrics for consistent protein production

By systematically addressing these factors, researchers can significantly reduce variability in recombinant protein expression, leading to more reliable and reproducible experimental outcomes.

What analytical methods can distinguish between native and recombinant acpS activity?

Distinguishing between native and recombinant acpS activity requires sophisticated analytical approaches that can specifically detect and quantify each form. Researchers can employ several complementary methods:

Molecular Tagging Strategies:

• Express recombinant acpS with affinity tags (His6, FLAG) for selective purification and detection

• Use epitope tags that allow selective immunoprecipitation of recombinant acpS

• Incorporate fluorescent protein fusions if protein function is preserved

Activity-Based Approaches:

• Develop assays using differentially labeled substrates specific to each form

• Measure enzyme kinetics parameters (Km, Vmax) to detect differences between native and recombinant forms

• Perform inhibition studies using form-specific inhibitors

Genetic Complementation:

• Test whether recombinant acpS can rescue conditional acpS mutants under non-permissive conditions

• Compare complementation efficiency between native and recombinant forms

• Analyze in vivo accumulation of apoACP versus holoACP as a functional readout

Structural Analysis:

• Use mass spectrometry to distinguish between native and recombinant forms based on mass differences

• Employ protein footprinting methods to detect structural differences

• Conduct thermal stability assays to identify functional variations

Substrate Specificity Profiling:

• Examine preference for different ACP isoforms

• Test activity with modified CoA analogs

• Evaluate phosphopantetheine transfer to non-native substrates

These methods can be combined in a comprehensive analytical framework to provide robust discrimination between native and recombinant acpS activity, essential for understanding the functional consequences of recombinant expression.

How can researchers resolve data inconsistencies in immunological studies using L. johnsonii?

Immunological studies using L. johnsonii can yield inconsistent results due to multiple variables affecting host-microbe interactions. Researchers can implement several strategies to improve reproducibility and resolve data inconsistencies:

Standardization of Bacterial Preparations:

• Define precise growth conditions using optimized media compositions

• Characterize bacterial cultures by growth phase, cell count, and viability

• Standardize preparation methods (harvesting, washing, storage)

• Verify strain identity through molecular methods

Comprehensive Immune Profiling:

• Analyze multiple immune parameters rather than single readouts

• Examine kinetics of responses at various timepoints (e.g., 2, 4, and 8 days post-infection)

• Assess both innate and adaptive immune components

• Integrate systems biology approaches for holistic understanding

Experimental Design Considerations:

• Control for microbiome effects through consistent housing and diet

• Include appropriate positive and negative controls

• Perform dose-response studies with multiple concentrations of L. johnsonii

• Account for sex, age, and genetic background of experimental animals

Technical Controls:

• Validate antibody specificity for flow cytometry and immunoassays

• Include isotype controls for immunological assays

• Verify cytokine detection sensitivity and specificity

• Implement rigorous normalization procedures for qPCR and protein quantification

Statistical Approaches:

• Conduct power analyses to determine appropriate sample sizes

• Apply appropriate statistical tests for data distribution

• Use multiple testing corrections for large datasets

• Consider hierarchical or mixed-effects models for nested data structures

Metadata Documentation:

• Maintain detailed records of experimental conditions

• Document lot numbers of reagents and materials

• Record environmental variables (temperature, humidity)

• Implement standardized reporting guidelines

By systematically addressing these factors, researchers can significantly improve the consistency and reproducibility of immunological findings in L. johnsonii studies, advancing our understanding of this important probiotic species and its potential applications in health and disease.