Recombinant Staphylococcus aureus Elongation factor Tu (tuf)

What is Elongation factor Tu (EF-Tu) and what is its primary function in S. aureus?

Elongation factor Tu (EF-Tu) is a highly abundant G protein that plays an essential role in protein biosynthesis. Its primary canonical function is to catalyze the binding of aminoacyl-tRNA to the A-site of the ribosome during translation elongation. In this process, EF-Tu ensures translational accuracy by facilitating the correct amino acid addition to the growing polypeptide chain. After the aminoacyl-tRNA docks with the mRNA, GTPase activity induces a conformational change that releases EF-Tu from the ribosome, allowing the translation process to continue . This function is conserved across bacterial species, including Staphylococcus aureus, where EF-Tu can comprise a significant percentage of the total cellular protein.

What are the structural characteristics of S. aureus EF-Tu?

S. aureus EF-Tu has an approximate molecular mass of 41,000 Da . Like other bacterial EF-Tu proteins, it consists of three functional domains:

• Domain I (amino acids 1-200): Forms a helix structure with Rossmann fold topology, a structural motif found in proteins that bind nucleotides. This domain houses the GTP/GDP binding sites and is often referred to as the Ras-like domain due to its similarity to the eukaryotic G protein Ras .

• Domain II (amino acids 209-299): Predominantly composed of beta sheets .

• Domain III (amino acids 301-393): Also largely comprised of beta sheets .

Domains II and III form anti-parallel beta-barrels that allosterically regulate Domain I activity, including enhancing affinity for GDP over GTP . The full-length protein sequence is available in recombinant protein data sheets .

How does S. aureus EF-Tu differ from EF-Tu in other bacterial species?

S. aureus EF-Tu exhibits notable differences from its counterparts in other bacteria, particularly Escherichia coli:

• Nucleotide binding: S. aureus EF-Tu binds negligible amounts of [³H]GDP compared to E. coli EF-Tu .

• Antibiotic sensitivity: S. aureus EF-Tu is significantly less sensitive to certain antibiotics:

  • Pulvomycin: 50% inhibitory concentration is approximately 100 μM for S. aureus vs. 2 μM for E. coli .

  • Aurodox: 50% inhibitory concentration is approximately 1,000 μM for S. aureus vs. 0.2 μM for E. coli .

• Complex formation: S. aureus EF-Tu forms a more stable complex with EF-Ts (molecular mass 34,000 Da), which affects its interaction with guanine nucleotides and inhibitors .

These differences have significant implications for antimicrobial development and understanding species-specific translation mechanisms.

What are the optimal storage conditions for maintaining recombinant S. aureus EF-Tu stability?

For optimal stability of recombinant S. aureus EF-Tu, follow these evidence-based storage recommendations:

• Temperature: Store at -20°C for standard storage, or -80°C for extended storage periods .

• Reconstitution: Briefly centrifuge vials before opening to bring contents to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Cryoprotection: Add 5-50% glycerol (final concentration) before aliquoting for long-term storage at -20°C/-80°C. The default final concentration of glycerol is typically 50% .

• Aliquoting: Prepare working aliquots to avoid repeated freeze-thaw cycles. Working aliquots can be stored at 4°C for up to one week .

• Shelf life: Liquid form generally maintains stability for 6 months at -20°C/-80°C, while lyophilized form can maintain stability for 12 months at -20°C/-80°C .

These storage protocols help preserve protein activity and prevent degradation, which is crucial for experimental reproducibility.

How can researchers verify the purity and activity of recombinant S. aureus EF-Tu preparations?

To ensure experimental validity, researchers should verify both the purity and functional activity of recombinant S. aureus EF-Tu:

Purity Assessment:

• SDS-PAGE analysis: Commercially available recombinant proteins typically have >85% purity as determined by SDS-PAGE . Researchers should confirm this upon receipt.

• Western blotting: Use specific antibodies against S. aureus EF-Tu to confirm identity and assess potential degradation products.

Activity Assessment:

• GTPase activity assay: Measure the GTP hydrolysis rate using colorimetric or fluorometric methods. Note that S. aureus EF-Tu forms a stable complex with EF-Ts that affects nucleotide binding .

• Ribosome binding assay: Assess the ability of EF-Tu to deliver aminoacyl-tRNA to ribosomes in a reconstituted translation system.

• For moonlighting function studies: Conduct binding assays with host molecules such as plasminogen, fibronectin, or other extracellular matrix components .

When interpreting activity results, researchers should be aware that S. aureus EF-Tu behaves differently from the well-characterized E. coli EF-Tu, particularly regarding nucleotide binding and antibiotic sensitivity profiles .

What are the methodological considerations for studying S. aureus EF-Tu interactions with antibiotics?

When investigating S. aureus EF-Tu interactions with antibiotics, particularly elfamycins, researchers should consider these methodological approaches:

• Differential sensitivity analysis:

  • Compare inhibitory concentrations between S. aureus and E. coli EF-Tu. For example, S. aureus EF-Tu requires significantly higher concentrations of pulvomycin (50x) and aurodox (5000x) for inhibition compared to E. coli EF-Tu .

  • Establish clear IC₅₀ values using purified components in translation assays.

• Complex stability considerations:

  • Account for the stable EF-Tu.EF-Ts complex formation in S. aureus, which affects interactions with nucleotides and inhibitors .

  • Design experiments to determine whether this complex formation is responsible for differential antibiotic sensitivity.

• Mechanism of action studies:

  • Consider the two distinct mechanisms of elfamycin antibiotics:
    a) Kirromycin/enacyloxin IIa: Prevention of EF-Tu:GDP dissociation from the ribosome
    b) Pulvomycin/GE2270 A: Inhibition of EF-Tu:GTP and aminoacyl-tRNA ternary complex formation

  • Design assays that can distinguish between these mechanisms.

• Structural binding studies:

  • Employ techniques like X-ray crystallography or cryo-EM to determine binding sites and conformational changes.

  • Identify structural differences in S. aureus EF-Tu that might explain differential antibiotic sensitivity.

These methodological considerations are essential for accurately characterizing antibiotic interactions and developing potential novel therapeutics targeting S. aureus EF-Tu.

How does S. aureus EF-Tu function as a moonlighting protein on the bacterial cell surface?

S. aureus EF-Tu exhibits significant moonlighting functions beyond its canonical role in translation. As a cell surface-associated protein, it:

• Traffics to and is retained on the bacterial cell surface despite lacking classical secretion signals .

• Mediates adhesion to host extracellular matrix components, facilitating bacterial colonization and invasion .

• Interacts with diverse host molecules, including plasminogen, which can be converted to plasmin in the presence of plasminogen activators, potentially enhancing tissue invasion .

• Undergoes multiple processing events on the cell surface, generating fragments that retain binding capabilities to host proteins .

These moonlighting functions contribute to S. aureus virulence and pathogenesis. Research methodologies to study these functions typically include surface protein extraction techniques, binding assays with purified host components, and surface plasmon resonance to quantify binding affinities. Researchers investigating these aspects must carefully design experiments that distinguish between canonical and moonlighting functions.

What structural features of S. aureus EF-Tu enable its moonlighting functions?

The moonlighting capabilities of S. aureus EF-Tu are facilitated by specific structural features:

• Short Linear Motifs (SLiMs):

  • Surface-exposed, non-conserved regions containing SLiMs play a key role in moonlighting functions .

  • These motifs enable interaction with various host molecules without compromising the structural constraints needed for canonical function.

• Positively Charged Amino Acid Accumulation:

  • Bioinformatics and structural modeling studies indicate that the accumulation of positively charged amino acids in SLiMs promotes multifunctional behavior .

  • In Staphylococcus aureus and other pathogens, these charged residues facilitate binding to negatively charged host molecules.

• Protein Processing:

  • EF-Tu undergoes multiple processing events on the cell surface, characterized through N-terminomics pipelines .

  • These processing events generate fragments that retain binding capabilities to host proteins, potentially expanding the functional repertoire.

• Genome-Influenced Features:

  • In some bacteria like Mycoplasma, codon bias resulting from A+T rich genomes may influence how positively charged residues accumulate in SLiMs, affecting moonlighting capabilities .

Researchers investigating these structural features should consider employing techniques such as site-directed mutagenesis to modify charged residues, truncation studies to examine fragment functionality, and structural analyses to map interaction domains.

What experimental approaches are most effective for distinguishing between canonical and moonlighting functions of S. aureus EF-Tu?

To effectively differentiate between canonical translation roles and moonlighting functions of S. aureus EF-Tu, researchers should consider these methodological approaches:

• Subcellular Localization Studies:

  • Cell fractionation to separate cytoplasmic from membrane/surface fractions

  • Immunofluorescence microscopy with anti-EF-Tu antibodies to visualize surface expression

  • Surface biotinylation followed by pull-down assays to confirm surface exposure

• Domain-Specific Mutational Analysis:

  • Generate point mutations or domain deletions that affect moonlighting functions while preserving canonical activity

  • Create recombinant fragments corresponding to processed forms identified in N-terminomics studies

  • Test mutants in both translation assays and host-binding assays to determine functional separation

• Binding Studies with Host Components:

  • Surface plasmon resonance (SPR) or bio-layer interferometry to quantify binding kinetics

  • Solid-phase binding assays with purified host components (plasminogen, fibronectin, etc.)

  • Competition assays between full-length EF-Tu and specific fragments

• In vivo Functional Separation:

  • Develop S. aureus strains expressing EF-Tu variants with mutations affecting surface localization but not translation

  • Assess virulence phenotypes in infection models compared to translation efficiency

• Comparative Analysis:

  • Compare the moonlighting capabilities of EF-Tu from S. aureus with those from other species to identify species-specific functions

  • Correlate structural features with functional differences

These approaches collectively provide robust evidence for distinguishing between canonical and moonlighting functions, essential for understanding the full biological significance of S. aureus EF-Tu.

How can researchers exploit S. aureus EF-Tu's unique properties for antimicrobial development?

The unique properties of S. aureus EF-Tu present several strategic approaches for antimicrobial development:

• Targeting Elfamycin Resistance Mechanisms:

  • S. aureus EF-Tu shows reduced sensitivity to elfamycin antibiotics like pulvomycin and aurodox compared to E. coli .

  • Researchers can:
    a) Identify structural differences responsible for reduced sensitivity
    b) Design modified elfamycins that overcome these barriers
    c) Develop combination therapies that destabilize the EF-Tu.EF-Ts complex to enhance elfamycin effectiveness

• Disrupting Moonlighting Functions:

  • Target the SLiMs and positively charged regions involved in host interactions .

  • Design peptide mimetics or small molecules that:
    a) Block EF-Tu surface exposure
    b) Interfere with host molecule binding
    c) Inhibit the processing events that generate functional fragments

• Exploiting Structural Vulnerabilities:

  • Target regions unique to S. aureus EF-Tu that differ from human elongation factors.

  • Focus on domains involved in:
    a) EF-Tu.EF-Ts complex formation
    b) Surface localization
    c) Species-specific structural elements

• Immunotherapeutic Approaches:

  • Develop antibodies or vaccines targeting surface-exposed EF-Tu.

  • Design strategies that:
    a) Block moonlighting functions without affecting commensals
    b) Enhance opsonization of S. aureus through EF-Tu recognition
    c) Neutralize virulence-associated functions of EF-Tu

These approaches require integrated structural biology, medicinal chemistry, and microbiology methodologies to develop effective antimicrobials that overcome the limitations of current elfamycins, which have poor pharmacokinetics and solubility issues .

What are the challenges in analyzing post-translational modifications and processing events of S. aureus EF-Tu?

Investigating post-translational modifications (PTMs) and processing events of S. aureus EF-Tu presents several methodological challenges:

• Surface vs. Cytoplasmic Population Discrimination:

  • Challenge: Distinguishing modifications specific to surface-exposed EF-Tu from the cytoplasmic pool.

  • Approach: Implement careful cell fractionation protocols followed by comparative proteomics between fractions.

  • Validation: Use site-specific antibodies that recognize modified forms in different cellular compartments.

• Processing Event Characterization:

  • Challenge: Identifying physiologically relevant cleavage products versus experimental artifacts.

  • Approach: Apply N-terminomics pipelines as described in the literature for EF-Tu characterization .

  • Validation: Compare processed forms detected in vitro with those present in vivo during infection.

• PTM Identification:

  • Challenge: Low abundance of specific modifications and potential heterogeneity.

  • Approach: Employ enrichment strategies combined with high-resolution mass spectrometry.

  • Analysis: Develop specialized search algorithms that account for S. aureus-specific modification patterns.

• Functional Correlation:

  • Challenge: Connecting specific modifications to functional outcomes.

  • Approach: Generate site-directed mutants that mimic or prevent specific modifications.

  • Assessment: Evaluate effects on both canonical translation activity and moonlighting functions.

• Species-Specific Differences:

  • Challenge: Extrapolating findings from model organisms to S. aureus.

  • Approach: Conduct comparative studies between S. aureus EF-Tu and other bacterial species.

  • Interpretation: Account for differences in EF-Tu sequence, processing machinery, and cellular context.

• Technical Limitations:

  • Challenge: Maintaining native modification patterns during protein purification.

  • Approach: Develop gentle extraction protocols that preserve labile modifications.

  • Control: Include appropriate inhibitors of proteases and enzymes that remove modifications.

Addressing these challenges requires integrated approaches combining advanced proteomics, molecular biology, and functional assays to generate a comprehensive understanding of EF-Tu processing and modification in S. aureus.

What experimental design considerations are important when studying interactions between S. aureus EF-Tu and host immune system components?

When investigating interactions between S. aureus EF-Tu and host immune components, researchers should implement these experimental design considerations:

• Physiologically Relevant Protein Preparations:

  • Use recombinant proteins with proper folding and post-translational modifications.

  • Consider comparing laboratory-produced recombinant EF-Tu with native protein isolated from S. aureus.

  • Verify activity through functional assays before immune interaction studies.

• Host System Selection:

  • Choose appropriate host cell types relevant to S. aureus infection sites.

  • Consider species-specific differences when using animal models versus human cells.

  • Include primary cells alongside cell lines to capture physiological responses.

• Experimental Conditions:

  • Match protein concentrations to those encountered during infection:

    • Surface EF-Tu concentration

    • Soluble EF-Tu released during bacterial lysis

  • Include appropriate controls:

    • Other surface proteins from S. aureus

    • EF-Tu from non-pathogenic bacteria

    • Heat-inactivated or denatured EF-Tu

• Comprehensive Immune Response Assessment:

  • Evaluate multiple immune pathways:

    • Pattern recognition receptor activation (TLRs, NODs)

    • Complement system interactions

    • Antibody recognition and epitope mapping

    • Cellular immune responses (neutrophils, macrophages)

  • Measure both pro-inflammatory and anti-inflammatory outcomes.

• Fragment and Domain Analysis:

  • Test both full-length EF-Tu and naturally occurring fragments .

  • Use domain-specific constructs to map immunogenic regions.

  • Investigate whether processing affects immunogenicity.

• In vivo Validation:

  • Confirm in vitro findings in appropriate animal models.

  • Assess immune responses during actual infection versus purified protein exposure.

  • Consider host genetic background effects on immune recognition.

• Temporal Considerations:

  • Evaluate both immediate and delayed immune responses.

  • Track dynamic changes in immune recognition during different infection phases.

These considerations help ensure robust, physiologically relevant results that accurately characterize the immunological significance of S. aureus EF-Tu during host-pathogen interactions.

How do the structural and functional properties of S. aureus EF-Tu compare with EF-Tu from other pathogenic bacteria?

A systematic comparison of EF-Tu across pathogenic bacteria reveals both conserved elements and species-specific adaptations:

This comparative analysis reveals that while the canonical translation function of EF-Tu is conserved across species, pathogenic bacteria have evolved species-specific adaptations in:

• Nucleotide binding properties

• Antibiotic sensitivity profiles

• Protein-protein interactions

• Surface exposure mechanisms

• Host molecule binding preferences

These differences likely reflect evolutionary adaptations to specific host environments and selective pressures, including antibiotic exposure. Researchers investigating S. aureus EF-Tu should consider these comparative aspects when designing experiments and interpreting results, particularly when extrapolating findings from model organisms.

What research approaches are recommended for investigating the role of S. aureus EF-Tu in biofilm formation and persistence?

To effectively investigate S. aureus EF-Tu's role in biofilm formation and persistence, researchers should implement these methodological approaches:

• Gene Expression and Localization Analysis:

  • Quantify EF-Tu expression levels during different biofilm development stages using RT-qPCR.

  • Employ immunofluorescence microscopy with anti-EF-Tu antibodies to track protein localization within biofilm structures.

  • Use reporter gene fusions to monitor dynamic expression patterns in real-time.

• Genetic Manipulation Strategies:

  • Generate conditional EF-Tu mutants (since complete deletion would affect viability) using:
    a) Inducible promoter systems to control expression levels
    b) Domain-specific mutations that affect moonlighting but not translational functions
    c) Surface-display mutations that prevent externalization while maintaining cytoplasmic activity

  • Complement mutants with wild-type and modified EF-Tu variants to confirm phenotypes.

• Biofilm Quantification Methods:

  • Implement crystal violet staining for biomass quantification under different conditions.

  • Use confocal laser scanning microscopy to assess three-dimensional biofilm architecture.

  • Apply computational image analysis to quantify structural parameters (thickness, roughness, etc.).

  • Perform viable count assessments to determine cell numbers within biofilms.

• Molecular Interaction Studies:

  • Identify binding partners of EF-Tu within the biofilm matrix using pull-down assays coupled with mass spectrometry.

  • Investigate interactions with extracellular DNA, polysaccharides, and other matrix components.

  • Determine if EF-Tu processing differs between planktonic and biofilm growth states.

• Stress Response and Persistence Analysis:

  • Evaluate how EF-Tu contributes to biofilm resistance against:
    a) Antimicrobial agents
    b) Host immune factors
    c) Environmental stressors

  • Monitor persister cell formation in EF-Tu modified strains.

• Multi-species Biofilm Considerations:

  • Assess EF-Tu's role in mixed-species biofilms relevant to clinical scenarios.

  • Investigate interspecies interactions mediated by EF-Tu.

These approaches should be implemented in both laboratory models and clinically relevant systems to ensure translational relevance. Researchers should also consider the dual role of EF-Tu in both normal cellular function and biofilm-specific activities when designing experiments and interpreting results.

How can researchers effectively study the immunogenicity of S. aureus EF-Tu and its potential as a vaccine candidate?

To comprehensively evaluate S. aureus EF-Tu's immunogenicity and vaccine potential, researchers should implement these strategic approaches:

• Epitope Mapping and Immunodominant Region Identification:

  • Employ peptide arrays covering the entire EF-Tu sequence to identify B-cell epitopes.

  • Use bioinformatic predictions combined with experimental validation to identify T-cell epitopes.

  • Distinguish between epitopes in surface-exposed versus hidden regions of the protein.

  • Map epitopes to the three-dimensional structure to understand accessibility.

• Cross-reactivity Assessment:

  • Evaluate antibody cross-reactivity with:
    a) Human mitochondrial EF-Tu homologs
    b) EF-Tu from commensal bacteria
    c) EF-Tu from other pathogens

  • Identify S. aureus-specific epitopes to minimize off-target effects.

• Immune Response Characterization:

  • Analyze both humoral and cellular responses:
    a) Antibody isotype profiling (IgG, IgA, IgM)
    b) T-cell subset activation (Th1, Th2, Th17)
    c) Cytokine production patterns
    d) Memory response development

  • Evaluate responses in multiple animal models with appropriate controls.

• Protective Efficacy Determination:

  • Challenge immunized animals with:
    a) Different S. aureus strains (MRSA, MSSA)
    b) Various infection models (skin, systemic, biofilm)
    c) Multiple infectious doses

  • Measure specific protection parameters:
    a) Bacterial burden reduction
    b) Clinical symptom amelioration
    c) Survival rate improvement
    d) Biofilm prevention capabilities

• Vaccine Formulation Optimization:

  • Test different antigen formats:
    a) Full-length recombinant EF-Tu
    b) Selected domain constructs
    c) Multi-epitope peptide vaccines
    d) DNA vaccines encoding EF-Tu

  • Evaluate adjuvant combinations to enhance immunogenicity.

  • Determine optimal dose, route, and schedule through systematic testing.

• Mechanism of Protection Studies:

  • Investigate whether protection works through:
    a) Neutralization of EF-Tu moonlighting functions
    b) Opsonization of surface-exposed EF-Tu
    c) Cell-mediated immunity against EF-Tu-presenting bacteria
    d) Inhibition of biofilm formation

• Population Variability Considerations:

  • Analyze pre-existing immunity to EF-Tu in human populations.

  • Evaluate genetic polymorphisms in EF-Tu across S. aureus clinical isolates.

  • Test vaccine efficacy in immunocompromised models.

These methodologies provide a comprehensive framework for evaluating EF-Tu as a vaccine candidate, addressing both basic immunological questions and translational vaccine development challenges.

What are the most promising research directions for understanding S. aureus EF-Tu's role in antimicrobial resistance?

The intersection of S. aureus EF-Tu with antimicrobial resistance presents several high-priority research directions:

• Elfamycin Resistance Mechanisms:

  • Investigate the structural basis for S. aureus EF-Tu's reduced sensitivity to elfamycin antibiotics .

  • Determine how the stable EF-Tu.EF-Ts complex contributes to resistance.

  • Explore whether acquired mutations in tuf genes correlate with changes in elfamycin susceptibility.

  • Develop methodologies to screen for novel compounds that overcome resistance mechanisms.

• Translation Modulation During Stress:

  • Examine how S. aureus EF-Tu activity changes under antibiotic pressure.

  • Investigate whether EF-Tu contributes to the formation of persister cells through translation regulation.

  • Study potential interactions between EF-Tu and other translation factors during stress responses.

  • Develop assays to monitor EF-Tu activity in real-time during antibiotic exposure.

• Moonlighting Functions and Resistance:

  • Determine whether surface-exposed EF-Tu contributes to biofilm-associated resistance.

  • Investigate potential roles in sequestering antimicrobials away from their targets.

  • Explore interactions between EF-Tu and host immune components that might affect antibiotic efficacy.

  • Develop strategies targeting moonlighting functions to enhance antibiotic effectiveness.

• Combination Therapy Approaches:

  • Test whether agents targeting EF-Tu can sensitize resistant S. aureus to existing antibiotics.

  • Evaluate synergistic effects between elfamycins and other translation inhibitors.

  • Develop methodologies for high-throughput screening of combination therapies.

  • Investigate whether targeting EF-Tu can prevent resistance development to other antibiotics.

These research directions require integrated approaches combining structural biology, molecular genetics, biochemistry, and clinical microbiology to develop a comprehensive understanding of EF-Tu's role in antimicrobial resistance and to identify novel therapeutic strategies.

How can researchers address the challenges of developing EF-Tu-targeted therapeutics for S. aureus infections?

Developing EF-Tu-targeted therapeutics for S. aureus presents specific challenges requiring methodological innovations:

• Target Selectivity Challenges:

  • Challenge: Distinguishing between bacterial EF-Tu and human mitochondrial EF-Tu (TUFM) .

  • Approach: Conduct comprehensive structural comparisons to identify S. aureus-specific regions.

  • Methodology: Employ computational modeling to design selective inhibitors that exploit structural differences.

  • Validation: Develop cellular assays that assess both antimicrobial activity and host cell toxicity.

• Pharmacokinetic/Pharmacodynamic Optimization:

  • Challenge: Poor pharmacokinetics and solubility of existing elfamycins .

  • Approach: Apply medicinal chemistry to modify elfamycin scaffolds for improved properties.

  • Methodology: Implement systematic structure-activity relationship studies.

  • Assessment: Develop animal models specifically designed to evaluate pharmacokinetic parameters.

• Resistance Development Risk:

  • Challenge: Potential for resistance development through tuf gene mutations.

  • Approach: Study the fitness cost of resistance mutations in the essential tuf gene.

  • Methodology: Design combination therapy approaches targeting multiple EF-Tu functions.

  • Monitoring: Develop sensitive methods to detect emerging resistance.

• Target Accessibility Issues:

  • Challenge: Accessing cytoplasmic EF-Tu through the cell envelope.

  • Approach: Design membrane-permeable compounds or exploit moonlighting surface exposure.

  • Methodology: Develop targeted delivery systems specific to S. aureus.

  • Evaluation: Compare efficacy against planktonic bacteria versus biofilm-embedded populations.

• Functional Redundancy:

  • Challenge: Potential functional redundancy in translation factors.

  • Approach: Investigate synergistic targeting of multiple translation components.

  • Methodology: Conduct genetic interaction studies to identify synthetic lethal partners.

  • Assessment: Evaluate resistance development frequency with single versus dual targeting.

• Therapeutic Index Considerations:

  • Challenge: Achieving sufficient therapeutic index for clinical use.

  • Approach: Exploit differences in binding properties between S. aureus and human EF-Tu.

  • Methodology: Develop high-throughput screening assays specifically for S. aureus EF-Tu.

  • Validation: Implement appropriate in vivo models that recapitulate human infections.

Addressing these challenges requires collaborative efforts between structural biologists, medicinal chemists, microbiologists, and clinicians to develop effective therapeutics targeting this essential but challenging bacterial protein.

What methodological innovations are needed to better understand the structure-function relationships of S. aureus EF-Tu in both canonical and moonlighting roles?

Advancing our understanding of S. aureus EF-Tu structure-function relationships requires several methodological innovations:

• Advanced Structural Biology Approaches:

  • Implement time-resolved cryo-electron microscopy to capture dynamic conformational changes during GTP/GDP cycling.

  • Develop native mass spectrometry protocols specific for membrane-associated EF-Tu complexes.

  • Apply hydrogen-deuterium exchange mass spectrometry to map interaction interfaces with host molecules.

  • Implement single-molecule FRET techniques to monitor conformational dynamics in real-time.

• Integrated Multi-omics Strategies:

  • Combine transcriptomics, proteomics, and metabolomics to correlate EF-Tu expression with functional states.

  • Develop targeted proteomics assays that distinguish between cytoplasmic and surface-exposed EF-Tu populations.

  • Implement spatial proteomics to map EF-Tu distribution within bacterial cells and biofilms.

  • Apply systems biology approaches to model EF-Tu's dual functionality networks.

• Advanced Genetic Tools:

  • Develop CRISPR interference systems for partial knockdown of tuf gene expression.

  • Create domain-swapping methodologies to generate chimeric proteins with altered functions.

  • Implement site-specific unnatural amino acid incorporation to probe specific functions.

  • Design genetic reporter systems to monitor EF-Tu surface localization in real-time.

• Novel Protein Engineering Approaches:

  • Generate conformation-specific antibodies that recognize distinct functional states.

  • Create stabilized EF-Tu variants locked in specific conformational states.

  • Develop protein-protein interaction disruptors specific to S. aureus EF-Tu complexes.

  • Implement click chemistry approaches to track EF-Tu movement between cellular compartments.

• Advanced Imaging Methodologies:

  • Apply super-resolution microscopy to visualize EF-Tu distribution during infection.

  • Develop correlative light and electron microscopy protocols for S. aureus.

  • Implement live-cell imaging techniques to track EF-Tu dynamics during host interaction.

  • Create biosensors that report on EF-Tu conformational states in living cells.

• In Silico Prediction Tools:

  • Develop improved algorithms for predicting moonlighting functions from primary sequence.

  • Create machine learning approaches to identify SLiMs in bacterial proteins.

  • Implement molecular dynamics simulations specifically parameterized for EF-Tu.

  • Design computational screening protocols for identifying EF-Tu-targeting compounds.