Recombinant Lactobacillus plantarum CCA-adding enzyme (cca)

What is Lactobacillus plantarum and why is it used as a recombinant expression system?

Lactobacillus plantarum is a lactic acid bacterium widely used in recombinant protein expression, particularly for vaccine development and therapeutic applications. It offers several advantages as an expression system:

• Safe for human and animal consumption with GRAS (Generally Recognized As Safe) status

• Inherent adjuvanticity that enhances immune responses

• Suitability for oral administration, facilitating mucosal immunity

• Ability to survive gastric passage and colonize intestinal mucosa

• Capacity to display foreign proteins on its surface

L. plantarum has been successfully used to express various antigens including influenza virus HA1, avian leukosis virus gp85, and Borrelia burgdorferi OspA, demonstrating its versatility as a recombinant expression platform .

What is the CCA-adding enzyme and what is its biological role?

The CCA-adding enzyme (tRNA nucleotidyltransferase) is responsible for synthesizing and maintaining the conserved CCA sequence at the 3' end of all mature tRNA molecules. This sequence is critical as it serves as the site for amino acid attachment during protein synthesis.

Key characteristics of CCA-adding enzymes include:

• Stepwise addition of nucleotides C74, C75, and A76 to tRNA

• Template-independent polymerase activity (unusual for RNA polymerases)

• Ubiquitous presence in all living organisms

• Essential role in tRNA maturation and functionality

The enzyme performs either de novo synthesis of CCA in organisms where tRNA genes don't encode this sequence or repair of damaged 3' ends in organisms where tRNA genes include the CCA sequence .

What are the structural classes of CCA-adding enzymes?

CCA-adding enzymes are divided into two structurally distinct classes:

Class I enzymes (primarily in archaea):

• Define nucleotide specificity through dynamic conformational changes

• Bases of CTP and ATP interact with phosphate backbones of the RNA primer's 3' region

• Template for CCA addition is the dynamic RNA-protein complex

• Undergo significant conformational shifts between nucleotide addition steps

Class II enzymes (in bacteria and eukaryotes):

• Use a protein-based template mechanism

• Specific amino acid residues mimic complementary nucleotide bases

• Share conserved catalytic core with Class I but employ different strategies for nucleotide selection

• Display more rigid protein-based templating for nucleotide selection

Both classes achieve the same remarkable feat of precise CCA synthesis without a nucleic acid template .

What are the methods for constructing recombinant L. plantarum expressing foreign proteins?

Based on published protocols, the following methodological approach is typically used:

• Gene design and synthesis:

  • Codon optimization for L. plantarum expression

  • Addition of appropriate restriction sites

  • Inclusion of tags or fusion partners as needed

• Vector construction:

  • Selection of appropriate shuttle vectors (e.g., pWCF, pMG36e)

  • Incorporation of strong promoters for efficient expression

  • Addition of signal sequences for secretion or surface display

• Cloning and verification:

  • Restriction enzyme digestion and ligation of target gene into vector

  • Transformation into E. coli for plasmid propagation

  • Plasmid extraction and verification by restriction analysis and sequencing

• Transformation into L. plantarum:

  • Electroporation (typically at 1.5-2.5 kV, 25 μF, 400 Ω)

  • Selection on appropriate antibiotic media

  • Verification of positive transformants

• Expression verification:

  • Western blotting using specific antibodies

  • Flow cytometry for surface-displayed proteins

  • Immunofluorescence analysis for protein localization

How can the surface display of recombinant proteins be achieved in L. plantarum?

Surface display is a valuable approach for enhancing immunogenicity of recombinant proteins expressed in L. plantarum. The following methods have proven effective:

• Anchoring motifs:

  • pgsA surface-display motif (derived from Bacillus subtilis)

  • Dendritic cell-targeting peptide (DCpep) for enhanced immune responses

  • Signal peptides for efficient translocation

• Construct design considerations:

  • Direct fusion of target protein to anchoring motifs

  • Incorporation of flexible linkers between protein and anchor

  • Optimization of signal sequence for efficient export

• Verification methods:

  • Western blotting of cell wall fractions

  • Flow cytometry with specific antibodies

  • Indirect immunofluorescence microscopy

For example, researchers successfully displayed the gp85 protein of avian leukosis virus on L. plantarum using the pgsA motif (demonstrated by SDS-PAGE, western blotting, and flow cytometry) . Similarly, HA1-DCpep fusion proteins were effectively displayed on L. plantarum surface, verified by immunoblotting and flow cytometry .

What methods can be used to evaluate the immune responses induced by recombinant L. plantarum?

A comprehensive immunological assessment typically includes:

• Humoral immune responses:

  • Measurement of specific antibodies (IgG, IgG1, IgG2a) in serum by ELISA

  • Detection of secretory IgA in mucosal secretions (feces, bile, intestinal lavage)

  • Hemagglutination inhibition (HI) assays for functional antibody assessment

• Cellular immune responses:

  • Flow cytometric analysis of CD4+ and CD8+ T cell activation

  • Quantification of IFN-γ producing T cells (CD4+IFN-γ+ and CD8+IFN-γ+)

  • T cell proliferation assays using CFSE staining and antigenic stimulation

• Mucosal immunity assessment:

  • Detection of B220+IgA+ cells in Peyer's patches

  • Immunofluorescence staining for IgA in mucosal tissues

  • Measurement of IgA levels in different intestinal segments

• Dendritic cell activation:

  • Analysis of surface markers (CD80, CD86, MHC-II) on dendritic cells

  • Evaluation of dendritic cell maturation in Peyer's patches

• Challenge studies:

  • In vivo challenge with pathogens following immunization

  • Viremia detection to assess protection efficacy

  • Survival rate and clinical symptom evaluation

The table below shows representative data for dendritic cell activation markers following exposure to recombinant L. plantarum:

Values represent mean fluorescence intensity as measured by flow cytometry .

How does the CCA-adding enzyme contribute to tRNA quality control?

Recent kinetic analysis has revealed that CCA-adding enzymes play a previously unrecognized role in tRNA quality control:

• The E. coli CCA enzyme can discriminate against tRNAs with backbone damage

• Intact tRNAs rapidly receive the CCA tag while damaged tRNAs experience delays

• This quality control mechanism prevents damaged tRNAs from completing maturation

• The discrimination occurs at each step of CCA synthesis, with the enzyme using different determinants to control the rate of addition

This quality control function is particularly relevant under cellular stress conditions that can damage tRNA and may represent a mechanism to prevent compromised tRNAs from entering protein synthesis machinery. The function appears to be an innate property of the enzyme and not dependent on additional factors, suggesting it operates as a first-line quality control mechanism .

What are the kinetic properties of CCA-adding enzymes?

Detailed kinetic studies, particularly of the E. coli class II CCA enzyme, have revealed:

• Maximum rate constant (kpot) of ~170 s⁻¹ for all three steps of CCA addition

• Comparable to the kpol of template-dependent T7 RNA polymerase

• Apparent equilibrium binding constants (Kd) for tRNA substrate in the range of 1-3 μM

• Binding constants similar to physiological tRNA concentrations available in vivo

• Conformational transitions before nucleotidyl transfer ensure fidelity

• Uniform rate constant for chemistry of nucleotide addition at each step

These kinetic properties allow the enzyme to operate efficiently under physiological conditions while maintaining high fidelity in the absence of a nucleic acid template. The uniformity of rate constants across all three nucleotide addition steps suggests a conserved catalytic mechanism despite different nucleotide specificities .

How can mutations in CCA-adding enzymes affect their specificity and activity?

Structural and functional studies have revealed critical insights into how mutations affect CCA-adding enzyme function:

• A small deletion in a flexible region near the catalytic core converts CCA-adding enzymes to CC-adding enzymes

• Introducing this region from a CCA-adding enzyme into a CC-adding enzyme restores full activity

• This region is located outside the conserved catalytic motifs yet plays a decisive role in A76 addition

• The presence of this deletion can be used to predict CC-adding activity in uncharacterized enzymes

In evolutionary terms, CC-adding enzymes appear to have descended from CCA-adding enzymes through the occurrence of this deletion. The discovery enabled researchers to successfully predict and identify previously uncharacterized CC-adding enzymes in Thermus thermophilus and Bacillus clausii based on sequence analysis .

What are the implications of CCA-adding enzyme quality control in stress response?

The quality control function of CCA-adding enzymes has significant implications for cellular stress response:

• Under stress conditions that damage tRNA, the discrimination function prevents damaged tRNAs from entering translation

• This mechanism potentially avoids cell death due to accumulation of damaged tRNAs

• It may give cells opportunity to mount defensive strategies against stress

• The function is relevant to both bacterial and eukaryotic systems due to shared enzyme homology

This quality control could be particularly important in clinical contexts:

• Stress-induced tRNA damage has been associated with cancer and other diseases

• In host-pathogen interactions, both bacteria and hosts respond to stress by inflicting tRNA damage

• Stress conditions often favor bacterial virulence expression

• The CCA quality control mechanism could be a potential therapeutic target

Understanding this quality control mechanism could lead to new therapeutic approaches targeting this process in pathogenic bacteria or in diseases associated with tRNA damage .

What are the challenges in expressing functional CCA-adding enzymes in L. plantarum?

Expression of functional CCA-adding enzymes in L. plantarum presents several specific challenges:

• Enzyme structure and folding:

  • Ensuring proper folding of the complex tertiary structure

  • Maintaining critical interdomain interactions needed for activity

  • Preventing interference from bacterial proteases

• Catalytic activity preservation:

  • Maintaining precise coordination of nucleotide selection

  • Preserving template-independent polymerase function

  • Ensuring proper metal ion coordination at the active site

• Expression optimization:

  • Balancing expression levels to avoid toxicity

  • Selecting appropriate promoters and regulatory elements

  • Optimizing codon usage for L. plantarum

• Functional verification:

  • Developing appropriate assays to verify enzymatic activity

  • Distinguishing recombinant enzyme activity from native L. plantarum CCA-adding enzyme

  • Measuring template-independent nucleotide addition accurately

• Stability concerns:

  • Ensuring enzyme stability during storage and delivery

  • Preserving activity through gastrointestinal passage (for oral delivery)

  • Maintaining activity in diverse physiological environments

These challenges require careful experimental design and optimization strategies to develop a functional recombinant system with therapeutic or research potential.

How might recombinant L. plantarum expressing CCA-adding enzymes be applied in research and therapeutics?

Several promising applications emerge from combining L. plantarum's benefits as a delivery vehicle with CCA-adding enzyme functions:

• Stress response modulation:

  • Delivery of engineered CCA-adding enzymes to modulate cellular stress responses

  • Potential applications in inflammatory conditions and infection

  • Development of probiotic strains with enhanced stress adaptation properties

• Novel vaccine adjuvant approach:

  • Using CCA-adding enzyme quality control to enhance antigen presentation

  • Development of recombinant vaccines with built-in adjuvant properties

  • Mucosal delivery of immunomodulatory molecules

• Targeted therapy for tRNA-related conditions:

  • Delivery of functional CCA-adding enzymes to compensate for deficiencies

  • Application in mitochondrial diseases with tRNA processing defects

  • Potential intervention in conditions with aberrant tRNA metabolism

• Research tools:

  • Creating model systems to study tRNA quality control in vivo

  • Development of biosensors for cellular stress detection

  • Investigation of host-microbe interactions through tRNA processing

• Synthetic biology applications:

  • Engineering novel tRNA processing capabilities

  • Creation of specialized translation systems with modified tRNAs

  • Development of bacterial chassis with enhanced protein production capabilities

These applications represent promising avenues for future research at the intersection of probiotic delivery systems and RNA processing machinery.

What methodological advances are needed to improve recombinant expression in L. plantarum?

To advance the field of recombinant protein expression in L. plantarum, several methodological improvements are needed:

• Enhanced expression systems:

  • Development of stronger, inducible promoters specific for L. plantarum

  • Creation of tunable expression systems with tight regulation

  • Engineering of secretion signals optimized for different protein classes

• Improved transformation methods:

  • Higher efficiency transformation protocols

  • Development of targeted integration strategies for stable expression

  • CRISPR-Cas9 systems optimized for L. plantarum genome editing

• Advanced protein engineering approaches:

  • Computational tools for predicting protein folding in L. plantarum

  • High-throughput screening methods for identifying optimal expression constructs

  • Directed evolution platforms for L. plantarum-expressed proteins

• Standardized characterization methods:

  • Unified protocols for quantifying surface display efficiency

  • Standardized assays for protein function and stability

  • Comprehensive methods for assessing immunomodulatory properties

• In vivo monitoring tools:

  • Non-invasive imaging techniques for tracking L. plantarum in vivo

  • Biosensors for real-time monitoring of protein expression

  • Methods for assessing colonization and persistence in mucosal tissues

These methodological advances would significantly enhance the utility of L. plantarum as a recombinant expression platform for both research and therapeutic applications.