Recombinant Mycobacterium ulcerans Elongation factor Ts (tsf)

What is the role of Elongation Factor Ts in M. ulcerans protein synthesis?

Elongation Factor Ts (EF-Ts) in M. ulcerans functions as a nucleotide exchange factor that directly facilitates both the formation and disassociation of the EF-Tu·GTP·aa-tRNA ternary complex, which is essential for protein synthesis. This process involves EF-Ts catalyzing the release of GDP from EF-Tu after GTP hydrolysis and facilitating the binding of a new GTP molecule to regenerate active EF-Tu, enabling it to participate in subsequent rounds of translation elongation. The efficiency of this process is particularly significant in M. ulcerans due to its exceptionally slow growth rate (doubling time of 3.5 days), where optimal protein synthesis machinery is critical for bacterial viability .

Unlike most bacterial species, Mycobacterium EF-Ts likely plays a specialized role in adapting protein synthesis rates to changing environmental conditions, potentially including host environments, which could influence virulence factor production including mycolactone toxin synthesis regulation .

How is the tsf gene organized in the M. ulcerans genome?

The tsf gene in M. ulcerans is structured similarly to other mycobacterial species, though with specific adaptations reflecting its evolutionary trajectory from M. marinum. Typically, in mycobacteria, the tsf gene is located in a conserved genomic region that contains other translation-related genes.

The genomic context of tsf in M. ulcerans demonstrates the following characteristics:

The conservation of this genomic organization across mycobacterial species suggests functional importance, while any M. ulcerans-specific variations might contribute to its unique growth properties and environmental adaptations .

What expression systems are optimal for recombinant M. ulcerans EF-Ts production?

When expressing recombinant M. ulcerans EF-Ts, researchers should consider the following expression systems, each with distinct advantages for mycobacterial protein production:

Most successful expressions utilize pET vectors with N-terminal His6-tags in E. coli systems, as demonstrated with similar mycobacterial elongation factors. When expressing M. ulcerans proteins specifically, researchers should account for the bacterium's adaptation to specific carbohydrates by supplementing expression media with glucose polymers, which have been shown to influence M. ulcerans metabolism .

What purification challenges are specific to M. ulcerans EF-Ts and how can they be addressed?

Purifying recombinant M. ulcerans EF-Ts presents several challenges due to mycobacterial protein characteristics. A systematic approach to addressing these challenges includes:

• Solubility enhancement: M. ulcerans proteins often have hydrophobic regions that can cause aggregation. Using solubility-enhancing fusion partners (SUMO, MBP, TrxA) significantly improves yields of soluble protein.

• Optimized lysis buffers: Incorporate components that maintain protein stability:

  • 50 mM Tris-HCl (pH 8.0)

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

  • 0.5% Triton X-100 (to help solubilize membrane-associated fractions)

• Multi-step purification strategy:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged proteins

  • Ion exchange chromatography to remove nucleic acid contamination

  • Size exclusion chromatography for final polishing and buffer exchange

• Stability assessment: Use differential scanning fluorimetry to identify buffer conditions that maximize protein stability during storage. Many mycobacterial proteins benefit from the presence of glycerol (10-20%) and reducing agents during storage .

What assays can accurately measure M. ulcerans EF-Ts activity?

Multiple complementary approaches can be employed to characterize M. ulcerans EF-Ts activity comprehensively:

• Nucleotide exchange assays:

  • Measure the rate of mantGDP dissociation from EF-Tu in the presence and absence of EF-Ts using fluorescence spectroscopy

  • Quantify the acceleration of GDP/GTP exchange rates on EF-Tu

  • Typical values for mycobacterial EF-Ts show 10-50 fold acceleration of exchange rates

• Ternary complex formation kinetics:

  • Monitor the formation of EF-Tu·GTP·aa-tRNA using fluorescence anisotropy

  • Compare rates with and without EF-Ts to determine catalytic efficiency

  • Stopped-flow techniques provide high temporal resolution for fast kinetics

• In vitro translation assays:

  • Reconstituted translation systems supplemented with varying concentrations of EF-Ts

  • Measure the rate of polypeptide synthesis using labeled amino acids

  • Assess the impact of environmental factors (pH, temperature, ion concentrations) that may be relevant to M. ulcerans environmental persistence

• Protein-protein interaction analysis:

  • Surface plasmon resonance to determine binding kinetics between EF-Ts and EF-Tu

  • Isothermal titration calorimetry for thermodynamic parameters of the interaction

  • Pull-down assays to identify potential additional interaction partners in M. ulcerans lysates

These assays should be performed under conditions that mimic the natural growth environment of M. ulcerans, potentially including carbohydrate supplements known to influence its metabolism .

How does M. ulcerans EF-Ts function relate to mycolactone production?

While direct evidence linking EF-Ts to mycolactone production is limited, several important connections can be hypothesized and experimentally investigated:

• Metabolic coordination: Under specific carbohydrate-rich conditions that downregulate mycolactone production, protein synthesis pathways including EF-Ts-mediated translation may be redirected toward alternative metabolic activities such as siderophore production. This metabolic shift suggests a potential regulatory network where translation factors like EF-Ts contribute to resource allocation between virulence factor production and other cellular processes .

• Translational regulation: Mycolactone synthesis involves giant polyketide synthases encoded on a virulence plasmid. While research indicates that mycolactone regulation is not primarily at the transcriptional or translational level, the efficiency of translation (mediated by EF-Ts and other factors) could influence the cellular capacity for producing these complex enzymes .

• Environmental adaptation: M. ulcerans adjusts mycolactone production based on environmental conditions, including its transition to intramacrophage growth where toxin production appears to be downregulated. This adaptation likely involves coordinated changes in protein synthesis patterns that would involve EF-Ts activity .

Research approaches to investigate these connections could include comparative proteomics of M. ulcerans under conditions that alter mycolactone production, coupled with targeted studies of translation efficiency .

How might targeting M. ulcerans EF-Ts contribute to Buruli ulcer treatment strategies?

Targeting M. ulcerans EF-Ts represents a promising avenue for developing novel therapeutic approaches for Buruli ulcer, which currently lacks effective medical treatments beyond surgery. Strategic approaches include:

• Selective inhibition: The structural differences between bacterial and eukaryotic elongation factors provide potential for designing selective inhibitors that target M. ulcerans EF-Ts without affecting host translation. Structure-based drug design approaches could identify compounds that specifically disrupt the EF-Ts:EF-Tu interaction surface unique to mycobacteria .

• Growth inhibition: M. ulcerans' extremely slow growth rate (3.5 days doubling time) makes it particularly vulnerable to translation inhibition. Even moderate reduction in EF-Ts activity could significantly impair bacterial replication, potentially allowing host immune mechanisms to more effectively clear the infection .

• Virulence attenuation: By targeting EF-Ts, it may be possible to alter the balance of protein synthesis in M. ulcerans, potentially reducing mycolactone production. Since this toxin is responsible for the extensive tissue necrosis and local immunosuppression characteristic of Buruli ulcer, reducing its production could mitigate disease severity even if bacterial clearance is not complete .

• Combination therapy approaches: EF-Ts inhibitors could potentially enhance the effectiveness of existing antibiotics by compromising the bacterium's ability to synthesize proteins necessary for antibiotic resistance or stress responses. This approach might be particularly effective during the intramacrophage growth phase of M. ulcerans .

What role might M. ulcerans EF-Ts play in the bacterium's immune evasion strategies?

M. ulcerans EF-Ts likely contributes to immune evasion mechanisms through its support of the translation machinery required for bacterial adaptation within host environments:

• Supporting mycolactone synthesis: While mycolactone regulation occurs primarily at post-translational levels, efficient translation machinery is necessary for producing the enzymatic machinery that synthesizes this immunosuppressive toxin. Mycolactone has been shown to inhibit multiple immune functions, including:

  • 95% inhibition of LPS-induced TNF and IL-10 release from human monocytes

  • Blocked production of IL-2 from activated T lymphocytes

  • Abrogation of TNF-induced NF-κB activation

  • Causing loss of monocyte adherence without cell death

• Adaptation during host transitional phases: During the transition from extracellular to intramacrophage growth, M. ulcerans appears to modulate mycolactone production. This adaptation requires rapid reprogramming of protein synthesis, which would necessarily involve translation factors like EF-Ts .

• Stress response during immune challenge: When faced with host immune pressures, bacteria typically upregulate stress response proteins. The efficiency of this response depends on translation factors including EF-Ts, especially in slow-growing organisms like M. ulcerans where rapid protein synthesis may be required despite generally slow metabolism .

Experimental approaches to investigate these connections could include creating conditional EF-Ts mutants and assessing their ability to produce mycolactone and survive within macrophages or animal models .

How does M. ulcerans EF-Ts function in the context of bacterial translocation?

Recent experimental models have revealed that M. ulcerans can translocate through the digestive tract in mammals, challenging previous understanding of its transmission and infection mechanisms. This finding has important implications for EF-Ts function:

• Adaptation to digestive conditions: M. ulcerans must adapt to the harsh environments of the digestive tract, including acid exposure, bile salts, and nutrient fluctuations. Translation factors like EF-Ts likely play crucial roles in reprogramming protein synthesis during this stress response .

• Systemic dissemination pathway: Experimental evidence shows M. ulcerans can be detected in the lymphatic system including cervical and axillary lymph nodes and the spleen following digestive tract exposure. This suggests that EF-Ts supports protein synthesis during a complex life cycle involving multiple host environments .

• Mycolactone regulation during translocation: The absence of systemic mycolactone detection despite bacterial dissemination suggests that toxin production may be locally regulated. This points to sophisticated translational control mechanisms potentially involving EF-Ts that allow M. ulcerans to modulate virulence factor production based on its anatomical location .

These findings suggest that M. ulcerans EF-Ts may support distinct translational programs during different phases of infection and dissemination, representing a key area for future research .

What experimental approaches can identify novel interactions between M. ulcerans EF-Ts and other cellular components?

Advanced experimental strategies to uncover the extended interaction network of M. ulcerans EF-Ts include:

• Proximity-dependent biotinylation (BioID): By fusing EF-Ts to a biotin ligase, researchers can identify proteins that come into close proximity with EF-Ts in living mycobacteria. This approach could reveal unexpected interactions beyond the canonical EF-Tu binding partner, potentially uncovering M. ulcerans-specific regulatory mechanisms.

• Quantitative interactomics: Combining pull-down assays with mass spectrometry under various growth conditions (standard media, carbohydrate supplementation, iron limitation) could reveal condition-specific interaction partners that might link translation to mycolactone regulation .

• Cryo-electron microscopy: Structural determination of M. ulcerans EF-Ts in complex with ribosomes and other translation factors could reveal unique features that contribute to this slow-growing pathogen's protein synthesis regulation.

• Conditional depletion studies: Creating strains with regulatable EF-Ts expression would allow researchers to observe the consequences of reduced EF-Ts activity on various cellular processes, including:

  • Growth rate in different media compositions

  • Mycolactone production profiles

  • Siderophore synthesis

  • Response to environmental stresses

• Comparative systems biology: Integrating transcriptomic, proteomic, and metabolomic data from M. ulcerans under different growth conditions, with focus on translation efficiency metrics, could reveal how EF-Ts activity coordinates with broader cellular responses to environmental changes .

These approaches would significantly advance understanding of how M. ulcerans adapts its protein synthesis machinery to support its unique lifestyle as a slow-growing environmental pathogen capable of producing a potent immunosuppressive toxin.