Recombinant Enterococcus faecalis 50S ribosomal protein L10 (rplJ)

What is the function of the 50S ribosomal protein L10 in Enterococcus faecalis?

The 50S ribosomal protein L10 in E. faecalis serves as a crucial component of the large ribosomal subunit, contributing to ribosomal assembly and protein synthesis. Based on comparative studies with E. coli, L10 likely forms part of the ribosomal stalk, interacting with the ribosomal protein L7/L12 complex. This interaction appears essential for stabilizing L10, as evidence suggests free L10 is susceptible to rapid proteolytic degradation, while L10 complexed with L7/L12 remains stable .

The protein participates in translational regulation mechanisms, likely binding to specific mRNA target regions upstream of its own coding sequence (rplJ). Through this autoregulatory feedback loop, L10 helps maintain appropriate stoichiometric ratios of ribosomal proteins. Additionally, L10 may be involved in the binding of translation factors during protein synthesis and potentially contributes to the peptidyl transferase center function, making it an important target for antibiotics that inhibit protein synthesis.

How are the rplJ and rplL genes organized and regulated in E. faecalis?

In E. faecalis, similar to what has been observed in E. coli, the genes encoding ribosomal proteins L10 and L7/L12 (rplJ and rplL, respectively) are likely arranged in an operon structure. These genes are cotranscribed and subject to translational coupling, meaning the translation of the downstream gene (rplL) depends on the translation of the upstream gene (rplJ) .

The regulation of this operon involves a complex feedback mechanism where L10 or the L10-L7/L12 complex binds to a specific target in the mRNA leader region upstream of rplJ. This binding inhibits translation initiation of the operon, effectively creating an autoregulatory circuit . When conducting recombinant expression studies, researchers must consider this natural regulatory mechanism, as overexpression of one protein without the other may disrupt the natural stoichiometry and potentially lead to instability of the expressed proteins.

Research methodology for studying this regulation typically involves:

• Primer extension analysis to map transcription start sites

• RNA footprinting to identify protein binding sites

• Translational reporter fusions to quantify expression levels

• Protein stability assays comparing free L10 versus complexed L10

What expression systems are most efficient for producing recombinant E. faecalis L10 protein?

The expression of recombinant E. faecalis L10 protein presents several challenges due to the protein's natural regulatory mechanisms and potential instability when expressed alone. Based on current methodologies, several expression systems can be considered:

• E. faecalis native expression systems: The pCIE expression vector utilizing the PQ pheromone-responsive promoter of plasmid pCF10 offers tight repression and large dynamic range for controlled expression in E. faecalis . This system responds to nanogram quantities of peptide pheromone (cCF10) and provides regulatory control that may better accommodate the expression of potentially toxic proteins .

• E. coli-E. faecalis shuttle vectors: These vectors allow for initial cloning in E. coli followed by expression in E. faecalis. The pCIE vector serves as an E. coli-E. faecalis shuttle vector based on the lactococcal rolling-circle replicon from plasmid pCI372, encoding a chloramphenicol resistance gene functional in both hosts .

• Co-expression strategies: Given the rapid degradation of free L10 observed in E. coli , co-expression of L10 with L7/L12 proteins should be considered to enhance stability of the recombinant L10 protein. This approach addresses the natural stoichiometric requirements of these interacting proteins.

When designing expression constructs, researchers should include:

• Appropriate affinity tags positioned to minimize interference with protein function

• Inducible promoters for controlled expression

• Consideration of the protein's native context to prevent misfolding or degradation

What protocols are recommended for purification of recombinant E. faecalis L10 protein?

Purification of recombinant E. faecalis 50S ribosomal protein L10 requires careful consideration of the protein's properties and potential instability. A recommended purification protocol includes:

Step 1: Expression optimization

• Co-express L10 with L7/L12 to enhance stability

• Use controlled induction systems like the pCIE vector with pheromone-responsive promoter

• Grow cultures at lower temperatures (16-25°C) during induction to improve proper folding

Step 2: Cell lysis and initial clarification

• Lyse cells in buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 1 mM DTT

• Include protease inhibitors to prevent degradation

• Clarify lysate by centrifugation at 20,000 × g for 30 minutes

Step 3: Affinity chromatography

• For His-tagged constructs, use Ni-NTA resin with imidazole gradient elution

• For GST-tagged constructs, use glutathione-sepharose with reduced glutathione elution

Step 4: Ion exchange chromatography

• Apply sample to a cation exchange column (SP Sepharose) equilibrated with 50 mM HEPES pH 7.0, 50 mM NaCl

• Elute with a linear gradient of 50-500 mM NaCl

Step 5: Size exclusion chromatography

• Final polishing step using Superdex 75 or 200 column

• Buffer: 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

Throughout the purification process, monitor protein stability and consider the addition of stabilizing agents if necessary. Verification of purified protein can be performed using SDS-PAGE, Western blot, and mass spectrometry analyses.

How does the differential stability of free L10 versus L10-L7/L12 complex impact experimental design?

The differential stability between free L10 and the L10-L7/L12 complex presents significant implications for experimental design when working with recombinant E. faecalis L10 protein. Research in E. coli has demonstrated that free L10 undergoes rapid proteolytic degradation, while L10 complexed with L7/L12 remains stable . This phenomenon likely exists in E. faecalis as well, given the conservation of ribosomal protein functions.

Experimental Considerations:

• Co-expression strategies: When designing expression vectors, researchers should consider co-expressing L10 with L7/L12 to enhance stability. Evidence from E. coli studies shows that overproduction of L7/L12 in trans stabilizes overproduced L10 protein .

• Protein-protein interaction studies: The stability difference can be exploited to study the interaction interface between L10 and L7/L12. Mutations that disrupt this interaction would be expected to decrease L10 stability, providing a functional readout for interaction studies.

• Protein half-life determination: Methods to measure protein stability such as pulse-chase experiments should be modified to account for the rapid degradation of free L10. Shorter time intervals and more sensitive detection methods may be required.

• Protease inhibitor requirements: Experimental buffers may require specific protease inhibitor cocktails optimized for the proteases responsible for L10 degradation. Identification of these specific proteases in E. faecalis would be a valuable research direction.

• Temperature considerations: Protein stability assays should be conducted at physiologically relevant temperatures (37°C), as degradation rates are temperature-dependent.

This differential stability represents an important regulatory mechanism that controls the stoichiometry of ribosomal proteins. Researchers should design experiments that either leverage this property or account for it as a potential confounding variable.

What are the implications of ribosomal protein L10 in antibiotic resistance mechanisms targeting the 50S subunit?

Ribosomal protein L10 in E. faecalis has significant implications for antibiotic resistance mechanisms targeting the 50S ribosomal subunit. Antibiotics like linezolid inhibit protein synthesis by binding to the peptidyl transferase center of the 50S subunit , making components of this subunit, including L10, potential factors in resistance development.

Key Resistance Mechanisms Involving the 50S Subunit:

• Target Site Modifications:

  • Mutations in 23S rRNA and ribosomal proteins can alter the binding site for antibiotics

  • Although not specifically identified in result , mutations in L10 could potentially affect the conformation of the 50S subunit and indirectly influence antibiotic binding

• Acquired Resistance Genes:

  • The cfr gene encodes an rRNA methyltransferase that modifies adenosine at position A2503 in 23S rRNA, conferring resistance to multiple antibiotic classes targeting the 50S subunit

  • The optrA gene encodes an ABC-F protein that confers resistance to phenicols and oxazolidinones

• Ribosomal Protection Mechanisms:

  • ABC-F proteins like OptrA may interact with components of the 50S subunit, including potentially L10, to protect the ribosome from antibiotic binding

Research Approaches:

• Structure-function analysis: Site-directed mutagenesis of recombinant L10 protein followed by antibiotic susceptibility testing to identify residues important for antibiotic sensitivity.

• Protein-antibiotic interaction studies: Techniques such as surface plasmon resonance or isothermal titration calorimetry to detect direct interactions between purified L10 and antibiotics.

• Cryo-EM studies: Structural analysis of ribosomes containing wild-type versus mutant L10 in the presence of antibiotics to visualize conformational changes.

• Comparative genomics: Analysis of L10 sequences from resistant versus susceptible strains to identify naturally occurring variants.

Understanding the role of L10 in antibiotic resistance could provide insights into novel therapeutic strategies targeting multi-drug resistant E. faecalis.

How can recombinant L10 protein be utilized to study translational regulation in E. faecalis?

Recombinant E. faecalis L10 protein serves as a valuable tool for investigating translational regulation mechanisms. Based on findings from E. coli, L10 likely plays a key role in autoregulation of the rplJL operon by binding to a specific target in the mRNA leader region upstream of rplJ . This regulatory mechanism can be studied using recombinant L10 through several sophisticated approaches:

1. RNA-Protein Interaction Studies:

• Electrophoretic Mobility Shift Assays (EMSA) using purified recombinant L10 and in vitro transcribed mRNA fragments containing the putative binding site

• RNA footprinting techniques (e.g., hydroxyl radical probing, RNase protection assays) to precisely map the binding site

• Fluorescence-based assays such as fluorescence anisotropy to determine binding kinetics and affinities

2. Structural Analysis of L10-RNA Complexes:

• X-ray crystallography or cryo-EM of L10 bound to its target RNA

• NMR spectroscopy for dynamic analysis of the interaction

• Hydrogen-deuterium exchange mass spectrometry to identify regions of conformational change upon binding

3. In Vivo Translational Regulation:

• Reporter gene constructs containing the rplJ leader region fused to reporter genes like gfp or lacZ

• Site-directed mutagenesis of both L10 protein and its RNA target to identify critical residues and nucleotides

• Ribosome profiling experiments comparing wild-type and L10-depleted conditions

4. Competitive Binding Assays:

• Analysis of whether L10 alone or the L10-L7/L12 complex is the primary regulatory factor

• Competition assays between binding to mRNA versus incorporation into ribosomes

These approaches provide a comprehensive toolkit for understanding how L10 functions in translational regulation, potentially revealing mechanisms that could be targeted for antimicrobial development or exploited for biotechnological applications.

What analytical techniques are most effective for characterizing L10-L7/L12 interactions in E. faecalis?

Characterizing the interactions between L10 and L7/L12 proteins in E. faecalis requires sophisticated analytical techniques that can provide insights into binding kinetics, interaction interfaces, and complex stability. The following methodologies are particularly effective:

Biophysical Characterization Techniques:

• Surface Plasmon Resonance (SPR)

  • Immobilize purified L10 on a sensor chip and flow L7/L12 at various concentrations

  • Determine association (ka) and dissociation (kd) rate constants

  • Calculate equilibrium dissociation constant (KD)

  • Typical KD values for ribosomal protein interactions range from 10^-7 to 10^-9 M

• Isothermal Titration Calorimetry (ITC)

  • Provides thermodynamic parameters (ΔH, ΔS, ΔG) of the interaction

  • Determines stoichiometry of binding (how many L7/L12 molecules bind to each L10)

  • No immobilization required, proteins interact in solution

• Microscale Thermophoresis (MST)

  • Measures changes in molecular movement in temperature gradients

  • Requires small sample volumes and can detect interactions in complex backgrounds

  • Good for difficult-to-immobilize proteins

Structural Characterization Techniques:

• X-ray Crystallography

  • Provides atomic-level details of the complex structure

  • Identifies specific residues at the interaction interface

  • Challenges include obtaining diffraction-quality crystals

• Cryo-Electron Microscopy

  • Can visualize the complex in near-native conditions

  • Particularly useful for larger assemblies including L10-L7/L12 within the ribosomal context

  • Recent advances allow near-atomic resolution

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

  • Maps regions protected from solvent upon complex formation

  • Identifies binding interfaces and conformational changes

  • Provides dynamic information complementary to static structures

Computational and Mutational Analysis:

• Alanine Scanning Mutagenesis

  • Systematically replace interface residues with alanine

  • Measure effects on binding affinity and complex stability

  • Identify "hot spots" critical for the interaction

• Molecular Dynamics Simulations

  • Model the dynamics of the interaction over time

  • Predict effects of mutations or environmental changes

  • Visualize conformational changes not captured by static structures

These analytical approaches should be applied in combination to build a comprehensive understanding of the L10-L7/L12 interaction, which is critical given the importance of complex formation for L10 stability and function .

What are the key considerations when designing expression constructs for E. faecalis rplJ?

When designing expression constructs for E. faecalis rplJ, researchers must navigate several important considerations to ensure successful expression and functional studies:

Promoter Selection:

• For regulated expression in E. faecalis, the PQ pheromone-responsive promoter of plasmid pCF10 offers tight repression, nanogram sensitivity to peptide pheromone, and a large dynamic range

• Consider the pCIE expression vector, which utilizes this promoter system and includes the coresident repressor prgX from E. faecalis plasmid pCF10

• The vector contains the iCF10 precursor whose mature peptide competitively inhibits cCF10, counteracting endogenous pheromone

Vector Backbone:

• The pCIE vector functions as an E. coli-E. faecalis shuttle vector, facilitating initial cloning in E. coli

• Based on the lactococcal rolling-circle replicon from plasmid pCI372, it contains a chloramphenicol resistance gene functional in both hosts

• Consider compatibility with existing plasmids if co-expression is planned

Expression Strategy:

• Co-expression with L7/L12 is recommended to enhance stability, as free L10 is subject to rapid proteolytic decay in E. coli

• For co-expression, consider either a polycistronic construct or separate compatible plasmids

• If using separate plasmids, ensure balanced expression through appropriate promoter strength selection

Tag Selection and Placement:

• N-terminal tags may interfere with binding to L7/L12 or RNA targets

• C-terminal tags may affect incorporation into ribosomes

• Consider removable tags with protease cleavage sites

• TEV protease cleavage sites offer efficient removal with minimal residual amino acids

Codon Optimization:

• Analyze the codon usage in rplJ against the preferred codons in the expression host

• Optimize rare codons while preserving regulatory elements if present in the coding sequence

• Consider the impact of mRNA secondary structure on translation efficiency

Cloning Sites:

• In the pCIE vector, restriction sites for cloning are approximately 400 nucleotides downstream of the PQ transcriptional start site and downstream of the IRS1 terminator

• Ensure the cloning strategy preserves the reading frame and includes appropriate translation initiation signals

Careful consideration of these factors will significantly improve the likelihood of successful expression and purification of functional recombinant E. faecalis L10 protein.

How can researchers effectively study the role of L10 in antibiotic resistance development?

Investigating the role of L10 in antibiotic resistance development requires a multifaceted approach combining genetic, biochemical, and structural techniques. The following methodological framework provides a comprehensive strategy:

Genetic Approaches:

• Gene Deletion and Complementation Studies

  • Create conditional knockdowns of rplJ using inducible antisense RNA

  • Complement with wild-type or mutant L10 variants

  • Assess changes in antibiotic susceptibility profiles

• Site-Directed Mutagenesis

  • Target conserved residues or regions proximal to known antibiotic binding sites

  • Create alanine scanning libraries to identify functional residues

  • Use saturating mutagenesis at key positions to explore all possible substitutions

• Laboratory Evolution

  • Expose E. faecalis to sub-inhibitory concentrations of antibiotics targeting the 50S subunit

  • Sequence rplJ in evolved resistant strains

  • Introduce identified mutations into clean genetic backgrounds to confirm causality

Biochemical and Structural Approaches:

• Ribosome Binding Assays

  • Purify ribosomes containing wild-type or mutant L10

  • Measure antibiotic binding using equilibrium dialysis or fluorescently labeled antibiotics

  • Determine binding constants and compare between variants

• Translation Assays

  • Use in vitro translation systems with purified components

  • Compare activity and antibiotic sensitivity of ribosomes with different L10 variants

  • Measure specific steps of translation (initiation, elongation, termination)

• Structural Analysis

  • Cryo-EM of ribosomes containing wild-type versus mutant L10

  • Focus on structural changes near antibiotic binding sites

  • Co-crystallize L10 with interacting partners in the presence of antibiotics

Comparative Genomics:

• Analysis of Clinical Isolates

  • Sequence rplJ in resistant clinical isolates of E. faecalis

  • Correlate sequence variations with resistance phenotypes

  • Verify the role of identified variants through genetic reconstruction

• Cross-Species Comparison

  • Compare L10 sequences from naturally resistant versus susceptible species

  • Identify residues that correlate with intrinsic resistance

  • Test these residues through site-directed mutagenesis

Data Integration:

This comprehensive approach will provide insights into how L10 contributes to antibiotic resistance mechanisms, potentially revealing novel targets for combination therapies to overcome resistance.

What are the common challenges in purifying stable and active recombinant E. faecalis L10 protein?

Purifying stable and active recombinant E. faecalis L10 protein presents several technical challenges that researchers should anticipate and address. Based on the evidence from E. coli studies, free L10 is particularly susceptible to rapid proteolytic degradation when not complexed with L7/L12 , suggesting similar challenges may exist for the E. faecalis ortholog.

Common Challenges and Solutions:

• Protein Instability

  • Challenge: Free L10 undergoes rapid proteolytic decay

  • Solutions:

    • Co-express with L7/L12 to form a stable complex

    • Purify under conditions that minimize protease activity (4°C, protease inhibitor cocktails)

    • Add stabilizing agents such as glycerol (10-20%) or specific ions

    • Consider fusion partners that enhance stability (e.g., MBP, SUMO)

• Solubility Issues

  • Challenge: Ribosomal proteins often have charged surfaces that can lead to aggregation

  • Solutions:

    • Optimize buffer conditions (pH, ionic strength)

    • Include solubility enhancers (0.1-0.5 M arginine, low concentrations of non-ionic detergents)

    • Express at lower temperatures (16-20°C) to slow folding and prevent aggregation

    • Use solubility-enhancing tags (GST, MBP)

• Co-purification of Nucleic Acids

  • Challenge: Ribosomal proteins naturally bind RNA, leading to contamination

  • Solutions:

    • Include high salt washes (500 mM - 1 M NaCl)

    • Treat with nucleases (RNase A, Benzonase)

    • Use ion exchange chromatography to separate protein from nucleic acids

    • Add polyethyleneimine (0.05-0.1%) to precipitate nucleic acids

• Maintaining Functional Activity

  • Challenge: Ensuring purified L10 retains its ability to bind partners and integrate into ribosomes

  • Solutions:

    • Validate function through binding assays with known partners (L7/L12, rRNA)

    • Compare secondary structure profiles (circular dichroism) with native protein

    • Minimize freeze-thaw cycles by aliquoting purified protein

    • Include stabilizing cofactors if known

• Purity Assessment

  • Challenge: Distinguishing between L10 and similarly sized contaminating proteins

  • Solutions:

    • Use mass spectrometry for definitive identification

    • Western blotting with specific antibodies

    • 2D gel electrophoresis to separate based on both size and charge

Optimization Strategy Table:

By anticipating these challenges and implementing appropriate solutions, researchers can increase their chances of obtaining pure, stable, and functionally active recombinant E. faecalis L10 protein for downstream applications.

How can researchers optimize expression systems for studying antibiotic resistance mechanisms involving L10?

Optimizing expression systems for studying antibiotic resistance mechanisms involving L10 requires careful consideration of several factors to ensure physiologically relevant results. The following strategies provide a comprehensive approach for researchers:

Vector Selection and Design:

• Controlled Expression Levels

  • Use the pCIE vector system with the PQ pheromone-responsive promoter for tight regulation

  • This promoter is tightly repressed in the absence of exogenous cCF10, sensitive to nanogram quantities of pheromone, and has a large dynamic range

  • Ensure expression levels approximate physiological conditions to avoid artifacts

• Genomic Context Preservation

  • Consider maintaining the natural operon structure if studying regulatory effects

  • For E. faecalis, the ribosomal protein genes may be arranged similarly to E. coli, where rplJ and rplL are cotranscribed and subject to translational coupling

• Resistance Gene Considerations

  • Select antibiotic resistance markers that do not interfere with the resistance mechanisms being studied

  • The chloramphenicol resistance marker in pCIE may be suitable for studying oxazolidinone resistance

Expression Strategies:

• Site-Directed Mutagenesis Platform

  • Design constructs allowing easy introduction of mutations identified in resistant strains

  • Include restriction sites or utilize Gibson Assembly for efficient mutant generation

  • Create libraries of L10 variants to screen for resistance phenotypes

• Reporter Systems

  • Incorporate luminescent or fluorescent reporters to monitor expression levels

  • Use dual reporters to simultaneously track L10 expression and ribosome activity

  • Design reporters that respond to specific antibiotics to measure resistance directly

• Co-expression Considerations

  • Express L10 with its natural partners (L7/L12) to maintain stability and function

  • For studying interactions with resistance factors like cfr or optrA , design co-expression systems

Antibiotic Resistance Testing:

• Gradient Plate Analysis

  • Create antibiotic gradient plates to visualize growth patterns

  • Compare wild-type L10 with mutant variants across concentration ranges

• MIC Determination Methodology

  • Use broth microdilution testing following CLSI guidelines

  • Include appropriate controls (e.g., E. faecalis ATCC 29212)

  • Test multiple antibiotics targeting the 50S subunit (linezolid, chloramphenicol, etc.)

• Time-Kill Kinetics

  • Measure bacterial killing over time to assess resistance dynamics

  • Compare strains expressing different L10 variants

Ribosome Activity Assays:

• In vivo Translation Measurement

  • Use reporters like luciferase to measure ongoing translation

  • Compare activity in the presence of antibiotics between L10 variants

• In vitro Translation Systems

  • Develop reconstituted translation systems with purified components

  • Replace wild-type L10 with recombinant variants to assess direct effects

Data Analysis Framework:

By optimizing these aspects of expression systems, researchers can effectively study the role of L10 in antibiotic resistance mechanisms, potentially leading to new strategies for combating resistant E. faecalis infections.

What emerging technologies could advance our understanding of L10's role in ribosome assembly and function?

Emerging technologies offer unprecedented opportunities to deepen our understanding of L10's role in ribosome assembly and function in E. faecalis. These innovative approaches can overcome limitations of traditional methods and provide new insights into this critical ribosomal protein:

Cryo-Electron Microscopy Advances:

• Time-resolved cryo-EM can capture intermediate states of ribosome assembly, revealing the precise timing and structural changes associated with L10 incorporation

• High-throughput cryo-EM enables screening of multiple conditions and L10 variants simultaneously

• Correlative light and electron microscopy (CLEM) allows tracking of fluorescently tagged L10 followed by structural analysis of the same samples

Advanced Genetic Manipulation:

• CRISPR interference (CRISPRi) provides tunable repression of rplJ expression without permanently altering the genome

• Base editing technologies enable precise single nucleotide changes to study specific residues without double-strand breaks

• Multiplex genome editing allows simultaneous modification of L10 and interacting partners

Single-Molecule Approaches:

• Single-molecule FRET can measure dynamic interactions between L10 and other ribosomal components in real-time

• Optical tweezers combined with fluorescence microscopy can probe mechanical forces involved in L10 incorporation

• Nanopore sequencing adapted for protein analysis could provide insights into L10 conformational states

Synthetic Biology Approaches:

• Minimal synthetic ribosomes with defined components can test the necessity and sufficiency of L10 for specific functions

• Orthogonal translation systems can evaluate L10 function isolated from cellular context

• Unnatural amino acid incorporation allows site-specific probes to be introduced into L10 for mechanistic studies

Mass Spectrometry Innovations:

• Cross-linking mass spectrometry (XL-MS) can map interaction networks of L10 within the ribosome

• Native mass spectrometry preserves non-covalent interactions, revealing binding partners and complex stoichiometry

• Protein painting combined with MS identifies solvent-accessible regions that change upon complex formation

Computational Methods:

• AlphaFold and RoseTTAFold can predict structures of L10 variants and their complexes

• Molecular dynamics simulations at extended timescales reveal conformational changes relevant to function

• Machine learning approaches can identify patterns in L10 sequence variations associated with antibiotic resistance

These technologies, especially when applied in combination, promise to revolutionize our understanding of how L10 contributes to ribosome assembly, function, and antibiotic resistance in E. faecalis. The integration of structural, genetic, and functional approaches will provide a comprehensive picture that could inform the development of novel antimicrobial strategies.

What are the potential applications of engineered L10 variants in combating antibiotic resistance?

Engineered variants of the 50S ribosomal protein L10 represent an innovative frontier in addressing antibiotic resistance in E. faecalis. By strategically modifying this crucial component of the ribosomal machinery, researchers can potentially develop novel approaches to overcome resistance mechanisms or create new antimicrobial strategies.

Diagnostic Applications:

• Biosensor Development

  • Engineered L10 variants with fluorescent tags that respond to specific antibiotics

  • L10-based detection systems for monitoring ribosomal targeting antibiotic levels in clinical samples

  • Rapid screening tools to identify resistance mechanisms affecting the 50S subunit

• Resistance Mechanism Identification

  • L10 variant libraries to characterize unknown resistance mechanisms

  • Comparative binding studies with different L10 variants to map resistance determinants

  • Structural probes to detect conformational changes associated with resistance

Therapeutic Applications:

• Antibiotic Adjuvants

  • Modified L10 peptides that sensitize resistant bacteria to existing antibiotics

  • L10 fragments that compete with resistance factors like OptrA or Cfr

  • Delivery systems targeting modified L10 to bacterial cells to disrupt ribosome assembly

• Novel Drug Development

  • Identification of L10 interaction sites as targets for new antimicrobials

  • Structure-based design of compounds that stabilize L10 in non-functional conformations

  • Development of peptide mimetics that interfere with L10-L7/L12 complex formation

• Combination Therapies

  • L10-targeting compounds that synergize with existing antibiotics

  • Multi-target approaches addressing both L10 and other ribosomal components

  • Strategies to overcome specific resistance mechanisms while preserving antibiotic efficacy

Research Tool Applications:

• Structure-Function Analysis

  • Creation of chimeric L10 proteins to map functional domains

  • Site-specific incorporation of photo-crosslinkable amino acids to capture transient interactions

  • Development of L10 variants with altered specificity for mechanistic studies

• Ribosome Engineering

  • Modified L10 to create ribosomes with altered translation properties

  • Orthogonal ribosome systems for synthetic biology applications

  • Specialized ribosomes for production of difficult-to-express proteins

Implementation Challenges and Solutions:

The development of engineered L10 variants represents a promising approach that could complement traditional antibiotic development pipelines, particularly as resistance to existing drugs continues to emerge in clinical isolates of E. faecalis.