Recombinant Enterococcus faecalis Peptidyl-tRNA hydrolase (pth)

What is Peptidyl-tRNA hydrolase (Pth) and what is its essential function in bacteria?

Peptidyl-tRNA hydrolase (Pth, E.C. 3.1.1.29) is a ubiquitous and essential bacterial enzyme that prevents the harmful accumulation of peptidyl-tRNA and sequestration of tRNA molecules. Functionally, Pth acts as an esterase that cleaves the ester bond between the nascent peptide and tRNA that has been prematurely released from stalled ribosomes. This enzymatic activity is crucial for maintaining available tRNA pools, thereby ensuring continuous protein synthesis within bacterial cells. Without Pth activity, bacteria would experience rapid depletion of free tRNA resources, leading to translation inhibition and eventual cell death .

The importance of Pth in bacterial physiology is underscored by studies showing that when Pth levels decline, peptidyl-tRNA accumulates to such an extent that usable tRNA pools drop significantly. This effect is particularly notable for prolyl-tRNAs, indicating that Pth is essential for maintaining normal tRNA levels across different tRNA species . The enzyme's essentiality makes it an attractive target for antibacterial drug development, especially against multidrug-resistant pathogens like Enterococcus faecalis.

How is the structure of Pth characterized in Enterococcus species?

The crystal structure of Peptidyl-tRNA hydrolase from Enterococcus faecium (EfPth) has been resolved at a high resolution of 1.92 Å, providing valuable insights into the structural features of this enzyme in Enterococcus species. The structural analysis revealed two molecules in the asymmetric unit with differences in the orientation of the sidechain of N66, a conserved residue within the catalytic site. This structural detail is particularly important as N66 plays a critical role in the enzyme's catalytic function .

While the search results primarily focus on E. faecium Pth, the high degree of conservation among Pth enzymes suggests that E. faecalis Pth likely shares similar structural features. The detailed structural information provides a foundation for understanding catalytic mechanisms and structure-based drug design targeting Pth enzymes in Enterococcus species. This structural knowledge is essential for researchers attempting to express and purify functional recombinant Pth for crystallographic studies or enzymatic characterization.

What enzymatic parameters characterize Pth activity, and how are they determined experimentally?

The enzymatic activity of Peptidyl-tRNA hydrolase can be characterized using standard Michaelis-Menten kinetic parameters. For example, in the case of Enterococcus faecium Pth (EfPth), enzymatic hydrolysis of the substrate α-N-BODIPY-lysyl-tRNALys (BLT) was characterized by a KM of 163.5 nM and Vmax of 1.9 nM/s . These parameters provide quantitative measures of the enzyme's substrate affinity and catalytic efficiency.

To determine these parameters experimentally, researchers typically use fluorescently labeled substrates like BLT, which allow real-time monitoring of the hydrolysis reaction. The experimental approach involves:

• Preparing recombinant Pth protein through expression and purification

• Incubating various concentrations of substrate with a fixed amount of enzyme

• Measuring initial reaction rates at each substrate concentration

• Fitting the data to the Michaelis-Menten equation to derive KM and Vmax values

This methodology provides crucial information about enzyme kinetics and can be employed to evaluate the effects of potential inhibitors on Pth activity, as demonstrated by the identification of the pyrrolinone scaffold compound 1040-C, which exhibits an IC50 of 180 nM against EfPth .

What are the barriers to recombinant expression of E. faecalis Pth, and how can they be overcome?

Recombinant expression of E. faecalis proteins, including Pth, faces significant barriers primarily related to the natural defense systems of Enterococci. The main obstacles include:

• Restriction-Modification (RM) Systems: Enterococci possess diverse RM systems that lead to host-specific DNA methylation signatures. These systems recognize and degrade foreign DNA lacking appropriate methylation patterns. E. faecium strains have been shown to contain both type I and type II RM systems, associated with 6-methyl adenine and 5-methyl cytosine DNA methylation patterns, respectively .

• Physical Barriers: The cell wall structure of Enterococci, including capsular polysaccharides and enterococcal polysaccharide antigen (EPA), can significantly reduce DNA uptake efficiency .

To overcome these barriers, researchers can employ several strategies:

• Pre-methylation of Plasmid DNA: Using methyltransferases that match the RM system specificity of the target strain to protect foreign DNA from restriction.

• Cell Wall Weakening Protocols: Treatment with glycine (0.5-2%) to weaken peptidoglycan, with sucrose as an osmotic stabilizer. For E. faecalis, supplementing media with glycine and keeping all reagents on ice during electroporation has improved transformation efficiency up to 1 × 10^6 transformants per μg of DNA .

• Enzymatic Treatment: Application of lysozyme or a combination of lysozyme and mutanolysin to log-phase cells (OD600 0.5-1.0) has been effective in weakening the peptidoglycan layer and improving DNA uptake for both E. faecalis and E. faecium .

• Optimized Electroporation Conditions: Using ice-cold reagents and adding ice-cold broth immediately after electroporation, followed by incubation on ice to delay the closure of membrane pores, has been shown to improve transformation efficiency .

• Alternative Delivery Methods: When electroporation is challenging, conjugation using appropriate donor strains (like E. faecalis CK111 for RepA-dependent conjugative plasmid delivery) or bacteriophage transduction can be considered as alternative approaches .

What expression systems are most effective for producing active recombinant E. faecalis Pth?

For expressing active recombinant E. faecalis Pth, researchers should consider the following optimized expression systems and conditions:

• E. coli Expression Systems:

  • BL21(DE3) or its derivatives are commonly used for recombinant protein expression

  • pET vector systems with T7 promoter control offer high-level expression

  • Addition of a His-tag facilitates purification via nickel affinity chromatography

  • Expression at lower temperatures (16-25°C) after induction often improves solubility

• Homologous Expression in Enterococcus:

  • For studies requiring native post-translational modifications

  • Requires overcoming the genetic barriers discussed previously

  • Nisin-inducible expression systems can be effective for controlled expression

  • May yield lower protein quantities but with potentially higher specific activity

• Expression Conditions Optimization:

  • Induction at mid-log phase (OD600 ~0.6-0.8)

  • IPTG concentration between 0.1-0.5 mM for E. coli systems

  • Supplementation with appropriate cofactors if needed

  • Post-induction expression time of 4-16 hours depending on temperature

• Solubility Enhancement Strategies:

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

  • Addition of solubility-enhancing tags (MBP, SUMO, TrxA)

  • Inclusion of low concentrations of non-ionic detergents in lysis buffers

The choice between these systems depends on the specific research goals, required protein yields, and downstream applications. For structural studies requiring high purity and yield, E. coli systems are typically preferred, while studies of native function might benefit from homologous expression.

How can crystal structures of E. faecalis Pth inform antimicrobial drug design?

Crystal structures of Pth enzymes provide critical insights that can guide structure-based antimicrobial drug design through several mechanisms:

• Catalytic Site Targeting: The crystal structure of EfPth revealed important details about the catalytic site, including the orientation of the N66 sidechain, a conserved residue essential for catalytic activity . This information allows for the rational design of inhibitors that can specifically interact with the catalytic residues, blocking substrate binding or hydrolysis.

• Substrate Binding Pocket Analysis: Structural analysis of the substrate binding pocket can identify unique features that can be exploited to design highly specific inhibitors. The understanding of how peptidyl-tRNA interacts with the enzyme enables the development of competitive inhibitors that mimic the natural substrate.

• Allosteric Site Identification: Beyond the catalytic site, crystal structures can reveal potential allosteric sites that could be targeted by non-competitive inhibitors. These sites might not be immediately obvious from sequence analysis alone.

• Structure-Activity Relationship Studies: The success of the pyrrolinone scaffold compound 1040-C, with an IC50 of 180 nM against EfPth, demonstrates the potential of structure-guided inhibitor design . Further optimization of these compounds can be guided by understanding their binding mode through co-crystallization studies.

• Species Selectivity Engineering: Structural comparisons between bacterial and human Pth enzymes can highlight differences that enable the design of inhibitors selective for bacterial enzymes, minimizing potential side effects.

The high-resolution structure of EfPth (1.92 Å) provides a solid foundation for computational approaches such as molecular docking, virtual screening, and molecular dynamics simulations to identify and optimize potential inhibitors. This structure-based approach has already yielded promising results with the identification of 1040-C, which shows potent antimicrobial activity against both susceptible and resistant strains of S. aureus and Enterococcus .

What methodologies are most effective for assessing Pth enzymatic activity in vitro?

Several methodologies can be employed to effectively assess Pth enzymatic activity in vitro, each with specific advantages depending on the research question:

• Fluorescence-Based Assays:

  • Using fluorescently labeled substrates such as α-N-BODIPY-lysyl-tRNALys (BLT)

  • Allows real-time monitoring of hydrolysis reactions

  • High sensitivity enabling detection of low enzymatic activity

  • Suitable for high-throughput screening of inhibitors

  • Enables precise determination of kinetic parameters (KM, Vmax, kcat)

• Radiolabeled Substrate Assays:

  • Using 32P or 14C-labeled peptidyl-tRNA substrates

  • Provides quantitative measurement of hydrolysis products

  • Particularly useful for measuring activity with native substrates

  • Requires special handling and disposal procedures

• HPLC-Based Assays:

  • Separation and quantification of reaction products

  • Allows analysis of multiple reaction products simultaneously

  • Useful for detailed mechanistic studies

  • Can be coupled with mass spectrometry for product identification

• Coupled Enzyme Assays:

  • Linking Pth activity to a secondary reaction that produces a measurable signal

  • Useful when direct detection of hydrolysis is challenging

  • Can provide continuous monitoring of reaction progress

For the assessment of inhibitors, methodologies should include:

• IC50 Determination:

  • Testing inhibitors at multiple concentrations to determine concentration required for 50% inhibition

  • Essential for initial characterization of inhibitor potency

  • Example: The pyrrolinone scaffold compound 1040-C was found to have an IC50 of 180 nM against EfPth

• Inhibition Mechanism Analysis:

  • Determining competitive, non-competitive, or uncompetitive inhibition mechanisms

  • Requires measurement of enzymatic activity at various substrate and inhibitor concentrations

  • Provides insights into binding mode and can guide inhibitor optimization

• Thermal Shift Assays (Differential Scanning Calorimetry):

  • Measurement of protein thermal stability in the presence of inhibitors

  • Can confirm direct binding of inhibitors to the target enzyme

  • Referenced as a methodology used in the characterization of EfPth

The choice of methodology should be guided by the specific research objectives, available equipment, and the nature of the inhibitors being tested.

How does Pth inhibition affect bacterial physiology and antibiotic sensitivity?

Inhibition of Peptidyl-tRNA hydrolase (Pth) has profound effects on bacterial physiology and can significantly alter antibiotic sensitivity through several mechanisms:

The relationship between Pth inhibition and antibiotic sensitivity provides a rational basis for combination therapy approaches. By targeting Pth, researchers can potentially resensitize resistant bacteria to conventional antibiotics or enhance the efficacy of current treatment regimens, addressing the growing challenge of antimicrobial resistance in pathogens like E. faecalis.

What structural features make Pth a viable target for species-specific antimicrobial development?

Peptidyl-tRNA hydrolase (Pth) exhibits several structural features that make it an attractive target for the development of species-specific antimicrobials against Enterococcus faecalis and related pathogens:

• Essential Nature:

  • Pth is essential in nearly all bacteria, including E. faecalis and related enterococcal species

  • It serves a critical function in maintaining tRNA pools that cannot be compensated by alternative pathways

  • This essentiality minimizes the development of resistance through target loss or modification

• Structural Distinctions from Eukaryotic Counterparts:

  • Bacterial Pth enzymes (Pth1) differ structurally and mechanistically from their eukaryotic counterparts (Pth2)

  • These differences allow for the development of inhibitors that selectively target bacterial enzymes without affecting human cells

  • This selectivity is crucial for minimizing toxicity in antimicrobial therapies

• Conserved Catalytic Residues with Unique Arrangements:

  • The catalytic site of E. faecium Pth includes conserved residues like N66, whose orientation varies between the two molecules in the asymmetric unit

  • These subtle structural differences in catalytic residues can be exploited to develop inhibitors specific to enterococcal Pth

  • The crystal structure at 1.92 Å resolution provides detailed insights into these unique features

• Species-Specific Substrate Binding Regions:

  • While the catalytic mechanism is conserved, the substrate binding regions may show variations between different bacterial species

  • These variations can be targeted to develop species-specific inhibitors that preferentially act against E. faecalis and related enterococci

  • This specificity could help preserve beneficial microbiota during treatment

• Demonstrated Druggability:

  • The success of pyrrolinone scaffold compounds, particularly 1040-C with an IC50 of 180 nM, demonstrates that Pth is druggable

  • These compounds show promising activity against both drug-susceptible and resistant strains (MRSA, VRSA, VSE, VRE) with MICs of 2-8 μg/mL

  • The efficacy comparable to vancomycin in reducing bacterial loads in murine infection models further validates Pth as a viable target

• Potential for Narrow-Spectrum Activity:

  • By targeting structural features unique to enterococcal Pth, it may be possible to develop narrow-spectrum antibiotics

  • Such agents would address the current need for alternatives to broad-spectrum antibiotics that disrupt the normal microbiota

These structural features, combined with the availability of high-resolution crystal structures and demonstrated efficacy of prototype inhibitors, position Pth as a promising target for novel antimicrobials against drug-resistant enterococcal infections.

How can genetic manipulation barriers in E. faecalis be overcome for Pth functional studies?

Conducting functional studies of Pth in E. faecalis requires overcoming significant genetic manipulation barriers. The following methodological approaches can be implemented:

• Neutralizing Restriction-Modification (RM) Systems:

  • E. faecalis contains complex type I RM systems with multiple hsdS genes and high TRD variability

  • Neutralization strategies include:
    a) Pre-methylation of plasmid DNA with appropriate methyltransferases
    b) Temporary depletion of restriction enzymes using antisense RNA
    c) Identification and targeting of phase-variable RM systems, as 24/34 E. faecalis strains analyzed contained phase-variable type I RM systems

• Optimized Electroporation Protocols:

  • Cell wall treatments: Growth in media supplemented with glycine (0.5-2%) to weaken peptidoglycan, with sucrose as an osmotic stabilizer

  • Enzymatic treatments: Application of lysozyme to log-phase cells (OD600 0.5-1.0) has improved DNA uptake in E. faecalis

  • Temperature management: Keeping all reagents (wash buffers, plasmid, cuvettes) on ice during preparation

  • Post-electroporation handling: Addition of ice-cold broth immediately after electroporation, followed by incubation on ice to delay membrane pore closure

  • DNA concentration: Using 150-1000 ng of plasmid DNA, as higher concentrations (>1 μg) can decrease transformation efficiency

• Alternative DNA Delivery Methods:

  • Conjugation: Using appropriate donor strains like E. faecalis CK111 for RepA-dependent conjugative plasmid delivery

  • Bacteriophage transduction: Utilizing temperate bacteriophages that can integrate at specific attB sites in the host genome

  • CRISPR-Cas9 delivery systems specially adapted for Enterococci

• Strain-Specific Considerations:

  • Targeting strains with reduced capsule or EPA production, as these cell wall components can reduce DNA uptake

  • Selection of laboratory-adapted strains as initial recipients before moving to clinical isolates

  • Consideration of strain-specific DNA methylation patterns determined by PacBio sequencing

• Conditional Expression/Depletion Systems for Studying Essential Genes:

  • Since Pth is essential, direct knockout studies are not feasible

  • Implementation of inducible promoter systems for controlled expression

  • Antisense RNA or CRISPRi approaches for partial depletion studies

  • Temperature-sensitive mutants for conditional functionality studies

These methodological approaches can be combined and optimized for specific E. faecalis strains to enable detailed functional studies of Pth in its native context, providing insights that may not be evident from heterologous expression systems.

What are the current challenges in developing Pth inhibitors as clinical antimicrobials?

Despite the promising potential of Pth inhibitors as novel antimicrobials, several significant challenges must be addressed in their development toward clinical applications:

• Pharmacokinetic and Pharmacodynamic Optimization:

  • While compounds like 1040-C show promising in vitro activity (IC50 of 180 nM) , optimizing drug-like properties remains challenging

  • Achieving appropriate solubility, stability, and bioavailability for systemic administration

  • Determining optimal dosing regimens based on time-kill kinetics and post-antibiotic effects

  • Addressing potential metabolism issues that could limit in vivo efficacy

• Spectrum of Activity Considerations:

  • Balancing broad-spectrum activity against selectivity for specific pathogens

  • Current Pth inhibitors like 1040-C show activity against both S. aureus and Enterococcus species (MICs 2-8 μg/mL)

  • Determining if cross-species activity is beneficial or if more targeted approaches are preferred

  • Assessing activity against diverse clinical isolates with varying resistance profiles

• Resistance Development Assessment:

  • Even though Pth is essential, bacteria might develop resistance through:
    a) Target modifications that preserve function but reduce inhibitor binding
    b) Upregulation of efflux pumps to reduce intracellular inhibitor concentrations
    c) Metabolic adaptations that compensate for partial Pth inhibition

  • Long-term studies on resistance development frequency and mechanisms are needed

• Toxicity and Safety Profiles:

  • Ensuring selectivity for bacterial Pth over human Pth-like proteins

  • Assessing potential off-target effects through comprehensive safety pharmacology

  • Evaluating effects on the human microbiome, especially for broad-spectrum inhibitors

  • Determining potential for immunomodulatory effects or hypersensitivity reactions

• Formulation and Delivery Challenges:

  • Developing appropriate formulations for different administration routes

  • Addressing challenges in penetration of biofilms, where 1040-C has shown promising activity

  • Considering combination therapy approaches to leverage synergistic effects with existing antibiotics, as demonstrated with gentamicin

  • Exploring innovative delivery systems to enhance target site concentration

• Regulatory and Clinical Development Pathways:

  • Novel mechanism antibiotics face additional regulatory scrutiny

  • Designing appropriate clinical trials for specific indications

  • Addressing the economic challenges of antibiotic development with limited market incentives

  • Navigating the complex landscape of antimicrobial stewardship considerations

Addressing these challenges requires a multidisciplinary approach combining structural biology, medicinal chemistry, microbiology, pharmacology, and clinical expertise. The promising results with compound 1040-C provide a foundation for further development, but significant optimization work remains before Pth inhibitors can advance to clinical application for treating resistant enterococcal infections.