Recombinant Mycobacterium smegmatis Elongation factor Tu (tuf)

What is the function of Elongation Factor Tu in Mycobacterium smegmatis?

Elongation Factor Tu (EF-Tu) in M. smegmatis, like in other bacteria, plays an essential role in protein biosynthesis by promoting the binding of aminoacyl-tRNAs to ribosomes during the elongation phase of translation . This GTP-binding protein forms a ternary complex with GTP and aminoacyl-tRNA, which then interacts with the ribosome to ensure accurate codon recognition. EF-Tu undergoes conformational changes during this process, cycling between GTP-bound (active) and GDP-bound (inactive) states. The protein's activity is tightly regulated to control the rate of protein synthesis, which directly impacts bacterial growth and survival.

How does the structure of M. smegmatis EF-Tu compare to other bacterial EF-Tu proteins?

M. smegmatis EF-Tu shares significant structural homology with other bacterial elongation factors. Comparison studies reveal strong sequence similarity with EF-Tu from other mycobacterial species, particularly M. tuberculosis . Sequence analysis has shown that mycobacterial EF-Tu also exhibits considerable homology with elongation factors from diverse organisms, including chloroplasts of Arabidopsis thaliana (approximately 65% identity) and mitochondrial EF-Tu from Saccharomyces cerevisiae . The conserved regions primarily include the GTP-binding domain and the domains involved in aminoacyl-tRNA recognition, reflecting the evolutionary conservation of this essential translational component.

What is known about the tuf gene encoding EF-Tu in mycobacteria?

The tuf gene encoding EF-Tu has been fully sequenced in several mycobacterial species, including M. tuberculosis . The gene has been characterized through screening of genomic libraries using specific DNA probes and monoclonal antibodies. In M. tuberculosis, the complete tuf gene has been isolated from a λgt11 expression library, enabling the expression of the native protein in E. coli as a lysogen . Restriction mapping and sequence analysis have provided detailed information about the gene structure, including important regulatory elements. The gene contains sequences typical of bacterial translational machinery components, with conserved motifs for GTP binding and interaction with ribosomes and aminoacyl-tRNAs.

What are the most effective systems for expressing recombinant M. smegmatis EF-Tu?

For recombinant expression of M. smegmatis EF-Tu, several systems have proven effective, with M. smegmatis itself being an excellent host for expressing mycobacterial proteins. This homologous expression system provides the proper cellular environment with appropriate chaperones and post-translational modification machinery . For M. smegmatis EF-Tu expression, the T7 promoter-based expression system has been successfully employed, similar to methods used for other mycobacterial proteins . The pYUB1062 shuttle vector, which has been adapted with a TEV cleavable C-terminal GFP tag, allows for monitoring of protein expression via fluorescence . This system offers advantages over E. coli-based expression, particularly for mycobacterial proteins that may require specific folding conditions or post-translational modifications.

How can researchers optimize purification protocols for recombinant M. smegmatis EF-Tu?

Optimizing purification of recombinant M. smegmatis EF-Tu requires careful consideration of protein properties and expression methods. A successful approach involves:

• Affinity tag selection: The addition of a poly-histidine tag enables efficient initial purification using immobilized metal affinity chromatography (IMAC) .

• Fusion protein strategies: Expression as a GFP-fusion protein offers dual benefits - monitoring expression levels via fluorescence and enhancing solubility .

• Cleavage options: Incorporating a TEV protease cleavage site between the target protein and tags allows for removal of tags post-purification, yielding native EF-Tu .

• Buffer optimization: Since EF-Tu binds nucleotides, purification buffers must be carefully formulated to maintain protein stability while controlling nucleotide binding state.

• Additional purification steps: Following IMAC, size exclusion chromatography effectively removes aggregates and provides a homogeneous preparation suitable for structural and functional studies .

For phosphorylated EF-Tu studies, researchers should consider including phosphatase inhibitors throughout the purification process to preserve the modification state.

What are the critical factors that influence the solubility and stability of recombinant M. smegmatis EF-Tu?

Several factors significantly impact the solubility and stability of recombinant M. smegmatis EF-Tu:

• Expression temperature: Lower induction temperatures (16-25°C) often improve proper folding and solubility compared to standard 37°C expression.

• Nucleotide binding: The presence of GTP or GDP stabilizes EF-Tu structure; therefore, including low concentrations (0.1-0.5 mM) of these nucleotides in purification buffers enhances stability .

• Fusion partners: Expression as a GFP fusion has been shown to enhance solubility of mycobacterial proteins, including EF-Tu .

• Buffer composition: Optimized buffer systems containing 50-100 mM salt, glycerol (5-10%), and reducing agents like DTT or β-mercaptoethanol prevent aggregation and oxidation.

• Post-translational modifications: Phosphorylation status affects EF-Tu stability and activity; unphosphorylated and phosphorylated forms may require different stabilization strategies .

• Storage conditions: Flash-freezing purified EF-Tu in liquid nitrogen with glycerol as a cryoprotectant helps maintain activity during long-term storage at -80°C.

What methods are most effective for assessing the GTP-binding activity of recombinant M. smegmatis EF-Tu?

Several robust methods have been established to assess the GTP-binding activity of recombinant M. smegmatis EF-Tu:

• Filter binding assays: This classic approach uses radiolabeled GTP ([γ-32P]GTP or [α-32P]GTP) to quantify nucleotide binding to EF-Tu. The protein-nucleotide complex is captured on nitrocellulose filters, and bound radioactivity is measured by scintillation counting .

• Fluorescence-based assays: Non-radioactive alternatives include using fluorescent GTP analogs like BODIPY-GTP or mant-GTP, which exhibit changes in fluorescence intensity or anisotropy upon binding to EF-Tu.

• Surface Plasmon Resonance (SPR): This technique enables real-time analysis of EF-Tu-GTP binding kinetics, providing association and dissociation rate constants.

• Isothermal Titration Calorimetry (ITC): ITC provides a comprehensive thermodynamic profile of EF-Tu-GTP interactions, measuring binding affinity, stoichiometry, and enthalpy changes.

• Differential Scanning Fluorimetry (DSF): This method measures thermal stability shifts upon GTP binding, offering a rapid assessment of functional binding.

Research has demonstrated that phosphorylation of M. tuberculosis EF-Tu by PknB reduces its interaction with GTP, which can be detected using these assays . Similar approaches can be applied to M. smegmatis EF-Tu to evaluate the impact of post-translational modifications or mutations on nucleotide binding function.

How can researchers effectively study the interaction between EF-Tu and aminoacyl-tRNAs in mycobacterial systems?

Studying EF-Tu and aminoacyl-tRNA interactions in mycobacterial systems requires specialized approaches:

• Purification of components: Both recombinant EF-Tu and mycobacterial tRNAs must be prepared. tRNAs can be enzymatically aminoacylated in vitro using purified aminoacyl-tRNA synthetases.

• Gel mobility shift assays: Native PAGE can detect the formation of EF-Tu:GTP:aminoacyl-tRNA ternary complexes by their reduced electrophoretic mobility.

• Filter binding assays: Similar to GTP binding studies, radiolabeled aminoacyl-tRNAs can be used to quantify ternary complex formation.

• Fluorescence anisotropy: Fluorescently labeled tRNAs provide a sensitive readout of binding to EF-Tu:GTP.

• Cryo-EM studies: Advanced structural analysis of the ternary complex reveals detailed interaction interfaces.

• Ribosome binding assays: To assess functional relevance, researchers can measure delivery of aminoacyl-tRNAs to mycobacterial ribosomes using purified translation components.

• Cross-linking approaches: Chemical cross-linking followed by mass spectrometry identifies specific residues involved in the interaction.

It's important to note that phosphorylation of EF-Tu may alter its interaction with aminoacyl-tRNAs, similar to how it affects GTP binding . Comparing wild-type and phosphorylated forms provides insights into regulatory mechanisms specific to mycobacteria.

What impact does EF-Tu have on antibiotic resistance in M. smegmatis?

EF-Tu influences antibiotic resistance in M. smegmatis through several mechanisms:

• Direct interaction with antibiotics: EF-Tu is the target of kirromycin, an antibiotic that inhibits protein synthesis by preventing conformational changes in EF-Tu . Research has shown that phosphorylation of EF-Tu affects its sensitivity to kirromycin – phosphorylated Mtb EF-Tu shows reduced sensitivity compared to the unphosphorylated form .

• Regulatory effects on growth rate: By controlling protein synthesis rates, EF-Tu levels and activity influence bacterial growth speed, which can indirectly affect susceptibility to antibiotics that target actively dividing cells.

• Relationship with other resistance mechanisms: While not directly implicated with rifampicin resistance like the marRAB operon in M. smegmatis , altered EF-Tu function may contribute to multi-drug resistance phenotypes through growth rate modulation.

• Stress response connection: Under antibiotic stress, bacteria often modify translation machinery components, including EF-Tu, as part of adaptive responses.

Understanding these mechanisms provides potential strategies for enhancing antimicrobial efficacy or designing new drugs that target protein synthesis in mycobacteria. The observation that post-translational modifications alter drug sensitivity highlights the importance of considering protein modification states when developing new antibiotics .

How does phosphorylation regulate the activity of EF-Tu in mycobacteria?

Phosphorylation serves as a critical regulatory mechanism for EF-Tu activity in mycobacteria. Research has demonstrated that M. tuberculosis EF-Tu is phosphorylated by the serine/threonine protein kinase PknB on multiple sites, including Thr118, which is required for optimal activity of the protein . This phosphorylation significantly impacts EF-Tu function through several mechanisms:

• Reduced GTP binding: Phosphorylation by PknB reduces EF-Tu's interaction with GTP, which is essential for its function in protein synthesis .

• Decreased protein synthesis: Consistent with reduced GTP binding, overexpression of PknB in M. smegmatis results in decreased protein synthesis levels .

• Altered antibiotic sensitivity: Phosphorylated EF-Tu exhibits different sensitivity to antibiotics compared to the unphosphorylated form. For example, kirromycin (an EF-Tu-specific antibiotic) significantly affects the nucleotide binding of unphosphorylated EF-Tu but has minimal effect on the phosphorylated protein .

• Modulation of growth rate: By regulating protein synthesis efficiency, phosphorylation of EF-Tu contributes to control of bacterial growth rate, potentially promoting persistence under stress conditions.

This phosphorylation-dependent regulation represents a unique mechanism by which mycobacteria can rapidly adjust protein synthesis in response to environmental stresses and growth conditions.

What other post-translational modifications have been identified in mycobacterial EF-Tu?

Beyond phosphorylation, mycobacterial EF-Tu undergoes several other post-translational modifications that influence its function and interactions:

• Acetylation: While not explicitly documented in the provided search results for mycobacterial EF-Tu, acetylation has been observed in EF-Tu from other bacterial species, particularly E. coli . Based on the evolutionary conservation of this protein, similar acetylation patterns may exist in mycobacterial EF-Tu.

• Methylation: Methylation has been reported as a regulatory modification of EF-Tu in various bacterial species . This modification can affect protein-protein interactions and stability.

• Association with the cell wall: M. tuberculosis EF-Tu has been found to be associated with the cell wall , suggesting potential lipid modifications or interactions that facilitate membrane localization.

• Oxidative modifications: Under stress conditions, EF-Tu may undergo oxidative modifications that alter its activity and stability.

The interplay between these various modifications likely creates a complex regulatory network that fine-tunes EF-Tu function according to cellular needs. Understanding the complete modification profile of mycobacterial EF-Tu remains an active area of research, with significant implications for bacterial physiology and potential drug development.

What experimental approaches are most effective for studying the phosphorylation state of M. smegmatis EF-Tu?

Several complementary experimental approaches can effectively characterize the phosphorylation state of M. smegmatis EF-Tu:

• Mass spectrometry-based phosphoproteomics:

  • LC-MS/MS analysis of tryptic digests of purified EF-Tu can identify specific phosphorylation sites

  • Quantitative approaches using SILAC or TMT labeling enable comparison of phosphorylation levels under different conditions

  • Targeted MS methods (PRM/MRM) allow precise quantification of specific phosphopeptides

• Phospho-specific antibodies:

  • Generation of antibodies that specifically recognize phosphorylated forms of EF-Tu

  • Western blotting with these antibodies provides a straightforward method to detect phosphorylation

  • Immunoprecipitation can be used to isolate phosphorylated EF-Tu from cell lysates

• In vitro kinase assays:

  • Recombinant PknB can be used to phosphorylate purified EF-Tu in vitro

  • Radioactive [γ-32P]ATP incorporation provides a sensitive measure of phosphorylation

  • Mutational analysis of potential phosphorylation sites (e.g., Thr118) confirms specific targets

• Functional assays to assess phosphorylation impact:

  • GTP binding assays comparing wild-type and phosphorylated EF-Tu

  • Antibiotic sensitivity tests (e.g., kirromycin) that show differential effects based on phosphorylation status

  • Protein synthesis assays in the presence of varying levels of kinase activity

• Phosphomimetic and phosphoablative mutations:

  • Creation of EF-Tu variants where phosphorylation sites are mutated to aspartate/glutamate (phosphomimetic) or alanine (phosphoablative)

  • These mutants can be used to study the functional consequences of permanent phosphorylation or its absence

These approaches have revealed that phosphorylation of EF-Tu by PknB reduces its interaction with GTP and affects protein synthesis levels in mycobacteria .

How does M. smegmatis EF-Tu differ from M. tuberculosis EF-Tu in structure and function?

M. smegmatis and M. tuberculosis EF-Tu share significant similarities but also exhibit notable differences:

Structural Similarities:

• Both proteins maintain the three-domain architecture characteristic of bacterial EF-Tu proteins

• High sequence homology reflects the essential nature of this protein in translation

• Conservation of key functional regions, including the GTP-binding pocket and aminoacyl-tRNA interaction surfaces

Functional Differences:

• Phosphorylation patterns may differ, though both can be phosphorylated by mycobacterial protein kinases including PknB

• M. tuberculosis EF-Tu has been found associated with the cell wall , a localization that may be different in M. smegmatis

• The two proteins may show different sensitivities to antibiotics based on subtle structural variations

• Expression levels and regulation might differ based on the different growth rates of these species (M. smegmatis being faster-growing than M. tuberculosis)

Experimental Considerations:

• When using M. smegmatis as a model for studying M. tuberculosis EF-Tu, researchers should be aware that while core functions are conserved, regulatory mechanisms may vary

• Studies have successfully used M. smegmatis for heterologous expression of M. tuberculosis proteins, suggesting the cellular machinery for proper folding and modification is compatible

Understanding these similarities and differences is crucial when using M. smegmatis as a model organism for studying the more pathogenic M. tuberculosis.

What insights can comparative studies of EF-Tu across mycobacterial species provide for tuberculosis research?

Comparative studies of EF-Tu across mycobacterial species offer valuable insights for tuberculosis research:

• Evolutionary conservation and specialization:

  • Identifying conserved regions across mycobacterial EF-Tu proteins highlights functionally critical domains

  • Species-specific variations may reveal adaptations to different environmental niches and host interactions

• Drug target potential:

  • Regions that are highly conserved in mycobacteria but divergent from human elongation factors represent potential selective drug targets

  • Comparing how different mycobacterial EF-Tu proteins interact with antibiotics like kirromycin provides insights for drug development

• Regulatory mechanisms:

  • Differences in phosphorylation patterns and other post-translational modifications between fast-growing (M. smegmatis) and slow-growing (M. tuberculosis) species may explain growth rate variations

  • The phosphorylation of EF-Tu by PknB and its effect on protein synthesis suggests a conserved regulatory mechanism that may be exploited therapeutically

• Pathogenesis connections:

  • M. tuberculosis EF-Tu has been implicated in binding to human plasminogen , suggesting roles beyond translation

  • Comparing this function across pathogenic and non-pathogenic mycobacteria could reveal virulence mechanisms

• Model system validation:

  • Understanding similarities and differences justifies when M. smegmatis can serve as an appropriate model for M. tuberculosis studies

  • This is particularly important for heterologous protein expression and drug screening approaches

These comparative insights can accelerate tuberculosis research by identifying both universal mycobacterial targets and pathogen-specific mechanisms.

How do expression systems for EF-Tu differ between M. smegmatis and M. tuberculosis?

Expression systems for EF-Tu differ between M. smegmatis and M. tuberculosis in several important aspects:

Natural Expression Patterns:

• Growth rate influence: M. smegmatis grows significantly faster than M. tuberculosis, which affects the baseline expression levels of translation machinery components including EF-Tu

• Regulatory responses: The tuf gene regulation may respond differently to stress conditions between species

• Association patterns: M. tuberculosis EF-Tu has been found associated with the cell wall and is induced under anaerobic conditions , associations that may differ in M. smegmatis

Heterologous Expression Systems:

• Vector compatibility: The T7 promoter-based pYUB1062 shuttle vector system works effectively in M. smegmatis for expressing mycobacterial proteins

• Expression efficiency: M. smegmatis generally provides higher protein yields due to its faster growth rate

• Post-translational modifications: While M. smegmatis can perform many of the same modifications as M. tuberculosis (including phosphorylation by PknB) , subtle differences in modification patterns may exist

• Monitoring strategies: GFP-fusion systems have been effectively used in M. smegmatis to monitor protein expression levels , providing a valuable tool for optimization

Practical Considerations:

• Safety advantages: Working with M. smegmatis requires lower biosafety levels than M. tuberculosis

• Experimental timeline: Experiments with M. smegmatis can be completed more rapidly due to faster growth

• Protein folding: M. smegmatis provides a more authentic environment for mycobacterial protein folding than E. coli systems

These differences highlight the importance of choosing the appropriate expression system based on specific research goals, whether focused on basic characterization, drug development, or understanding species-specific regulation.

What strategies can be employed to study the interaction of M. smegmatis EF-Tu with the ribosome?

Studying the interaction between M. smegmatis EF-Tu and ribosomes requires specialized techniques:

• Ribosome isolation and reconstitution:

  • Purification of intact M. smegmatis ribosomes using sucrose gradient ultracentrifugation

  • Reconstitution of translation systems with purified components (ribosomes, EF-Tu, aminoacyl-tRNAs)

  • In vitro translation assays to measure functional interactions

• Cryo-electron microscopy (Cryo-EM):

  • Visualization of EF-Tu-ribosome complexes at near-atomic resolution

  • Capture of different states during the translation cycle

  • Comparison with structures from other bacterial species to identify mycobacteria-specific features

• Cross-linking mass spectrometry (XL-MS):

  • Chemical cross-linking of EF-Tu to ribosomal proteins during active translation

  • Mass spectrometry identification of cross-linked peptides

  • Mapping of interaction interfaces between EF-Tu and the ribosome

• Fluorescence-based approaches:

  • Fluorescently labeled EF-Tu to track binding kinetics to ribosomes

  • FRET pairs between EF-Tu and ribosomal proteins to measure conformational changes

  • Single-molecule techniques to observe individual binding events

• Effects of phosphorylation:

  • Comparison of phosphorylated versus unphosphorylated EF-Tu interactions with ribosomes

  • Investigation of how PknB-mediated phosphorylation influences EF-Tu release from ribosomes

  • Analysis of how phosphorylation-induced changes in GTP binding affect ribosome interactions

• Antibiotic probes:

  • Use of kirromycin, which stabilizes EF-Tu on the ribosome, as a tool to trap specific conformational states

  • Comparison of antibiotic effects on different phosphorylation states of EF-Tu

These approaches can reveal how M. smegmatis EF-Tu's interaction with ribosomes may differ from other bacterial species and how regulatory mechanisms like phosphorylation specifically impact the mycobacterial translation process.

How can researchers effectively troubleshoot issues with recombinant M. smegmatis EF-Tu purification?

When encountering challenges with M. smegmatis EF-Tu purification, researchers can implement these troubleshooting strategies:

Problem: Low expression levels

• Solution: Optimize induction conditions (temperature, duration, inducer concentration)

• Solution: Test different expression constructs, including GFP fusions which enable direct monitoring of expression

• Solution: Consider codon optimization for the expression host

• Solution: Evaluate alternative promoter systems beyond the T7 promoter-based pYUB1062 system

Problem: Poor solubility

• Solution: Lower induction temperature (16-20°C) to slow protein folding

• Solution: Try different fusion tags (MBP, SUMO, GFP) that can enhance solubility

• Solution: Add low concentrations of non-ionic detergents to lysis buffers

• Solution: Include stabilizing additives like glycerol (5-10%) and nucleotides (GTP/GDP)

Problem: Co-purification of contaminants

• Solution: Include higher imidazole concentrations in wash buffers for His-tagged constructs

• Solution: Add nuclease treatment to remove nucleic acid contamination

• Solution: Implement additional purification steps (ion exchange, size exclusion chromatography)

• Solution: Consider dual affinity tags with orthogonal purification steps

Problem: Loss of activity

• Solution: Include protease inhibitors to prevent degradation

• Solution: For studying phosphorylated EF-Tu, include phosphatase inhibitors throughout purification

• Solution: Maintain reducing conditions with DTT or β-mercaptoethanol

• Solution: Include appropriate nucleotides (GTP/GDP) in buffers to stabilize protein conformation

Problem: Aggregation during concentration/storage

• Solution: Add stabilizing agents like glycerol or arginine to storage buffers

• Solution: Concentrate to lower final concentrations, avoiding protein precipitation

• Solution: Flash-freeze in liquid nitrogen with cryoprotectants rather than slow freezing

• Solution: Store multiple smaller aliquots to avoid freeze-thaw cycles

By systematically applying these strategies based on specific purification issues, researchers can significantly improve yields and quality of recombinant M. smegmatis EF-Tu preparations.

What are the emerging areas of research regarding M. smegmatis EF-Tu as a drug target?

Emerging research on M. smegmatis EF-Tu as a drug target encompasses several promising directions:

• Structure-based drug design:

  • High-resolution structural studies of mycobacterial EF-Tu to identify unique pockets for selective targeting

  • Comparison with human elongation factors to ensure antimicrobial specificity

  • Development of in silico screening approaches for virtual compound libraries

• Phosphorylation-sensitive inhibitors:

  • Design of compounds that specifically target either phosphorylated or unphosphorylated forms of EF-Tu

  • Research has shown that phosphorylation alters sensitivity to antibiotics like kirromycin

  • This approach could lead to drugs that remain effective regardless of the phosphorylation state

• Allosteric modulators:

  • Identification of allosteric sites that could modulate EF-Tu function

  • Targeting protein-protein interactions between EF-Tu and other translation components

  • Disruption of EF-Tu interactions with regulatory kinases like PknB

• Nucleotide binding pocket targeting:

  • Development of GTP analogs that compete with GTP binding

  • Compounds that lock EF-Tu in specific conformational states

  • Strategies that exploit the reduced GTP binding of phosphorylated EF-Tu

• Combination therapies:

  • Exploration of synergistic effects between EF-Tu inhibitors and existing antibiotics

  • Investigation of combinations with compounds that modulate PknB activity

  • Dual-targeting approaches addressing both EF-Tu and other components of translation machinery

• Screening platforms:

  • Development of high-throughput screening systems using recombinant M. smegmatis EF-Tu

  • GFP-fusion systems for monitoring compound effects on protein stability

  • Cell-based assays measuring translation efficiency in the presence of potential inhibitors

These approaches recognize the essential role of EF-Tu in mycobacterial protein synthesis and leverage our growing understanding of its regulation through post-translational modifications to develop novel antimicrobial strategies.

How might research on M. smegmatis EF-Tu contribute to understanding bacterial persistence and dormancy?

Research on M. smegmatis EF-Tu offers significant potential to advance our understanding of bacterial persistence and dormancy through several key mechanisms:

• Protein synthesis regulation and growth control:

  • Phosphorylation of EF-Tu by PknB reduces GTP binding and protein synthesis rates

  • This regulatory mechanism may be central to transitioning between active growth and dormancy

  • Studies have shown that overexpression of PknB in M. smegmatis reduces protein synthesis levels , potentially mimicking aspects of dormancy

• Connection to stress response pathways:

  • EF-Tu modification states likely change under stress conditions

  • M. tuberculosis EF-Tu has been found to be induced under anaerobic conditions , suggesting a role in adaptation to hypoxia

  • The relationship between stress response signaling and translation machinery modulation can be explored using M. smegmatis models

• Drug tolerance mechanisms:

  • Phosphorylated EF-Tu shows altered sensitivity to antibiotics like kirromycin

  • This suggests post-translational modifications may contribute to drug tolerance in dormant cells

  • Understanding these mechanisms could lead to strategies for targeting persistent bacteria

• Energy conservation strategies:

  • Downregulation of protein synthesis through EF-Tu modification represents an energy conservation strategy

  • This may be crucial for long-term survival under nutrient limitation

  • M. smegmatis models allow investigation of these pathways in a more experimentally tractable system

• Integration with other persistence factors:

  • Research in M. smegmatis can explore how EF-Tu regulation integrates with other persistence mechanisms

  • Connections to toxin-antitoxin systems and stringent response pathways

  • Potential relationship with Rv2629, which delays mycobacterial entry into log-phase growth

Through these research directions, studies of M. smegmatis EF-Tu could provide crucial insights into how mycobacteria modulate protein synthesis to enter, maintain, and exit dormant states – a central challenge in tuberculosis treatment.

What technological advances are needed to better characterize the role of EF-Tu in mycobacterial pathogenesis?

Several technological advances would significantly enhance our ability to characterize EF-Tu's role in mycobacterial pathogenesis:

• Advanced in vivo imaging techniques:

  • Development of fluorescent probes to visualize EF-Tu localization and dynamics during infection

  • Super-resolution microscopy methods to study EF-Tu distribution within mycobacterial cells

  • Tools to monitor EF-Tu phosphorylation states in real-time within living bacteria

• Single-cell analysis platforms:

  • Technologies to measure protein synthesis rates in individual bacteria during infection

  • Single-cell proteomics to capture cell-to-cell variability in EF-Tu levels and modifications

  • Correlation of EF-Tu states with bacterial phenotypes in heterogeneous populations

• Synthetic biology approaches:

  • CRISPR-based systems for precise genomic manipulation of mycobacterial EF-Tu

  • Optogenetic tools to control EF-Tu activity or modification with light

  • Engineered strains with fluorescent biosensors that report on translation activity

• Improved host-pathogen models:

  • Advanced 3D cell culture systems that better mimic human tissue environments

  • Humanized mouse models for studying mycobacterial infections

  • Organoid-based infection models to study EF-Tu function during pathogenesis

• Structural biology innovations:

  • Time-resolved cryo-EM to capture dynamic states of EF-Tu during translation

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Integrative structural approaches combining multiple data types

• Chemical biology tools:

  • Activity-based probes specific for different EF-Tu conformational states

  • Selective inhibitors to distinguish between phosphorylated and unphosphorylated forms

  • Proximity labeling techniques to map the EF-Tu interactome during infection

• Advanced computational approaches:

  • Machine learning algorithms to predict EF-Tu interaction networks

  • Molecular dynamics simulations of EF-Tu under different conditions

  • Systems biology models integrating translation with other cellular processes