Recombinant Treponema denticola 50S ribosomal protein L27 (rpmA)

What is the role of L27 protein in bacterial ribosomes?

L27 is one of the smallest and most basic polypeptides in bacterial ribosomes, playing crucial roles in both ribosome assembly and function. In bacteria like Escherichia coli, L27 deletion severely perturbs the assembly of the 50S ribosomal subunit, resulting in the accumulation of a 40S precursor particle that is deficient in several key proteins including L16, L20, and L21 . Completed subunits lacking L27 demonstrate impaired peptidyltransferase activity, likely due to defects in aminoacyl-tRNA binding to the A site . L27 has been localized to the base of the central protuberance near the peptidyltransferase center, where it appears to participate directly in the protein synthesis process . Multiple lines of evidence, including affinity labeling with peptidyltransferase inhibitors such as chloramphenicol and puromycin, support L27's presence at the active site of peptide bond formation in bacterial ribosomes .

How can recombinant T. denticola L27 protein be expressed and purified?

Based on approaches used for related ribosomal proteins, recombinant T. denticola L27 can be expressed using established prokaryotic expression systems with important modifications. The protein can be cloned into expression vectors such as pQE70 or pET series vectors, which provide inducible promoters and fusion tags to facilitate purification . When designing expression constructs, researchers should pay particular attention to:

• Codon optimization: T. denticola, like A. aeolicus, may contain rare codons (especially for arginine) that limit expression in E. coli. Co-expression with additional tRNA genes like argU can significantly improve protein yields .

• Fusion tags: C-terminal 6×His tags have been successfully used for L27 homologs, allowing purification via nickel affinity chromatography . The tag position should be chosen carefully to avoid interfering with protein folding.

• Expression conditions: Induction with IPTG at early-log phase is typical, though growth conditions may require optimization since overexpression of ribosomal proteins can slow bacterial growth .

The purification protocol would likely involve cell lysis followed by affinity chromatography using the engineered tag, with additional purification steps such as ion exchange or size exclusion chromatography as needed for experimental requirements.

What structural characteristics distinguish T. denticola L27 from homologs in other bacterial species?

While the search results don't provide direct structural information about T. denticola L27, comparative analysis with other bacterial L27 proteins suggests several important characteristics. Ribosomal proteins from extremophiles such as Aquifex aeolicus demonstrate significantly higher structural stability compared to mesophilic homologs like those from E. coli . For example, circular dichroism analysis and proton nuclear magnetic resonance spectroscopy revealed that A. aeolicus L27 readily adopts a stable structure in solution, whereas E. coli L27 remains largely unstructured under identical conditions .

T. denticola L27 likely exhibits intermediate structural properties reflecting its adaptation to the oral microenvironment. Key structural differences may include:

• Amino acid composition variations that affect thermal stability

• Potential post-translational modifications specific to Treponema species

• Differences in surface residues that mediate interactions with rRNA and neighboring proteins in the assembled ribosome

These structural differences may contribute to functional specialization in the T. denticola translation machinery and could potentially be exploited for therapeutic targeting.

What methodologies are recommended for studying the interaction between T. denticola L27 and immune cells?

Studying interactions between T. denticola L27 and immune cells requires a multifaceted approach. Based on methodologies used for other T. denticola proteins, researchers could employ:

• Fluorescent labeling of purified recombinant L27 protein to track uptake by immune cells such as macrophages using immunofluorescence assays (IFA) . This approach allows quantification of protein internalization and cellular localization.

• Real-time PCR to quantify uptake and processing of L27 by macrophages under different conditions, similar to methods used for tracking whole T. denticola .

• Opsonization experiments using specific antibodies against L27 to assess whether antibody binding enhances immune recognition and phagocytosis . This is particularly relevant as opsonization with specific antibodies has been shown to considerably improve the uptake of T. denticola by macrophages under both aerobic and anaerobic conditions .

• Cytokine production assays to measure inflammatory responses (e.g., TNF-α) elicited by recombinant L27 protein under different experimental conditions .

These methodologies should be performed under both aerobic and anaerobic conditions since T. denticola is an anaerobe, but interactions with host cells may occur in microaerophilic environments at tissue interfaces.

How can cross-species functional complementation experiments with L27 proteins inform our understanding of ribosomal evolution?

Cross-species complementation experiments, where L27 from one bacterial species is expressed in a strain lacking its endogenous L27, provide powerful insights into ribosomal evolution and functional conservation. The search results describe a fascinating experiment where A. aeolicus L27 was expressed in an E. coli L27 deletion mutant . This approach revealed that while the foreign L27 was incorporated into E. coli ribosomes and partially restored growth rate, it failed to promote 50S subunit assembly .

For T. denticola L27, similar experiments could:

• Determine whether T. denticola L27 can functionally replace E. coli L27 in ribosome assembly and/or function

• Identify species-specific regions of L27 essential for ribosome assembly by creating chimeric proteins

• Reveal evolutionary constraints on ribosomal protein function

The experimental design would involve:

• Creating an expression construct containing T. denticola rpmA with appropriate regulatory elements

• Transforming this construct into an E. coli L27 deletion mutant (similar to strain IW312)

• Assessing growth rates, ribosome profiles, and peptidyltransferase activity

• Analyzing the incorporation of T. denticola L27 into E. coli ribosomes via Western blotting and mass spectrometry

Such cross-species complementation studies provide unique insights into the co-evolution of ribosomal proteins with their rRNA partners and neighboring proteins.

What role might T. denticola L27 play in antibiotic resistance and how can this be experimentally investigated?

Given L27's proximity to the peptidyltransferase center, it potentially influences antibiotic sensitivity in T. denticola. L27 has been affinity-labeled by several peptidyltransferase inhibitors including chloramphenicol, carbomycin, tylosin, spiramycin, and puromycin , suggesting it may play a role in antibiotic binding or resistance.

To investigate this experimentally:

• Mutagenesis studies could be performed on recombinant T. denticola L27, targeting residues predicted to interact with antibiotics based on structural models.

• In vitro translation assays using ribosomes containing wild-type or mutant L27 proteins could assess changes in sensitivity to various antibiotics.

• Complementation of L27-deleted E. coli with T. denticola L27 variants could determine whether species-specific differences in L27 contribute to differential antibiotic sensitivity profiles.

• Structural studies (X-ray crystallography or cryo-EM) of T. denticola ribosomes in complex with antibiotics could directly visualize L27's role in antibiotic binding.

These approaches would help determine whether T. denticola L27 contributes to the bacterium's intrinsic resistance to certain antibiotics, potentially identifying novel targets for antimicrobial development against this periodontal pathogen.

How does the integration of recombinant T. denticola L27 into host cell models advance our understanding of periodontal disease pathogenesis?

Recombinant T. denticola L27 can serve as a valuable tool for investigating host-pathogen interactions in periodontal disease. Studies could address:

• Immunogenic properties: Purified L27 could be used to stimulate human gingival fibroblasts, epithelial cells, and immune cells to assess inflammatory responses relevant to periodontal disease .

• Potential moonlighting functions: Beyond its ribosomal role, L27 might have extracellular functions during infection. Experiments using labeled recombinant L27 could track potential interactions with host extracellular matrix components or cell surface receptors.

• Role in immune evasion: Similar to studies with whole T. denticola, experiments could determine whether L27 affects phagocytosis or killing by neutrophils and macrophages . If L27 is exposed on the bacterial surface or released during infection, it might directly interact with immune cells.

• Antibody responses: Recombinant L27 could be used to detect and quantify anti-L27 antibodies in patient serum, potentially correlating antibody levels with disease severity .

These approaches would help determine whether T. denticola L27 contributes directly to periodontal pathogenesis beyond its canonical role in protein synthesis, potentially identifying it as a virulence factor or diagnostic marker.

What post-translational modifications occur in native T. denticola L27 and how do they impact protein function?

Post-translational modifications (PTMs) can significantly alter protein function, and ribosomal proteins are known to undergo various modifications. For T. denticola L27, several lines of evidence suggest potential PTMs:

• The search results indicate that PrcB, another T. denticola protein, migrates at 22 kDa despite a predicted size of 17 kDa, and its N-terminus is unavailable for Edman sequencing, suggesting acylation . Similar modifications might occur in L27.

• The inability to sequence the N-terminus could indicate N-terminal processing or modification, which is common in bacterial ribosomal proteins.

To investigate PTMs in T. denticola L27:

• Compare native L27 (isolated from T. denticola ribosomes) with recombinant L27 expressed in E. coli using mass spectrometry to identify mass differences indicative of modifications.

• Perform targeted mass spectrometry approaches such as multiple reaction monitoring (MRM) to detect specific modifications (methylation, acetylation, etc.).

• Site-directed mutagenesis of potential modification sites to assess functional consequences in ribosome assembly and activity.

• Structural studies comparing modified and unmodified proteins to determine how PTMs affect L27 conformation and interactions.

Understanding these modifications could provide insights into species-specific regulation of ribosome assembly and function in T. denticola, potentially revealing unique aspects of protein synthesis in this oral pathogen.