Recombinant Francisella tularensis subsp. tularensis Elongation factor G (fusA), partial

What is the significance of studying Elongation Factor G in Francisella tularensis subsp. tularensis?

Elongation Factor G (fusA) plays an essential role in protein synthesis, making it central to F. tularensis survival and virulence. As F. tularensis subsp. tularensis is one of the most infectious human pathogens with an infectious dose of <10 CFU in humans , understanding the function of its essential proteins provides insights into its exceptional virulence. Since protein synthesis is a primary target for antibiotics, studying fusA may reveal novel therapeutic approaches against this potential biothreat agent. Additionally, comparative analysis of fusA between different subspecies could help explain virulence differences between the highly pathogenic subsp. tularensis and the less virulent subsp. holarctica.

How does fusA differ between Francisella tularensis subspecies?

While the search results don't specifically address fusA sequence differences, they highlight that F. tularensis subspecies (tularensis and holarctica) share approximately 97-99% nucleotide identity despite significant differences in virulence . Genomic comparisons have revealed numerous DNA rearrangements between subspecies, particularly between type A (tularensis) and type B (holarctica) strains . These rearrangements are often flanked by repeated DNA insertion sequence elements, suggesting evolution through homologous recombination events. Given these broader genomic differences, fusA might exhibit subtle but functionally significant variations between subspecies that could contribute to differences in translational efficiency, stress responses, or antibiotic susceptibility.

What are the general structural and functional properties of bacterial Elongation Factor G?

Bacterial Elongation Factor G is a GTPase that catalyzes the translocation step during protein synthesis, moving the tRNA-mRNA complex through the ribosome. In bacterial pathogens like F. tularensis, EF-G typically consists of several domains that work together for proper ribosomal interaction and translocation function. As F. tularensis undergoes significant stress during infection and host-pathogen interactions, EF-G may play roles beyond canonical translation, potentially including stress adaptation or virulence regulation. Understanding these properties in the context of F. tularensis's unusual pathogenicity is essential for developing targeted antimicrobials.

What expression systems are most effective for producing recombinant F. tularensis fusA?

Producing recombinant F. tularensis proteins presents unique challenges due to the organism's pathogenicity and specialized growth requirements. For fusA expression, E. coli-based systems remain the most accessible option, though several considerations must be addressed:

• Codon optimization: F. tularensis has a low G+C content of approximately 32% , which differs from E. coli, potentially necessitating codon optimization.

• Toxicity management: Overexpression of translation factors can be toxic to host cells; therefore, tight regulation using systems like pET with T7 lysozyme co-expression may be necessary.

• Solubility enhancement: Fusion partners such as MBP (maltose-binding protein) or SUMO may improve solubility of recombinant fusA.

• Expression temperature: Lower temperatures (16-25°C) often improve proper folding of complex proteins like EF-G.

• Selection of E. coli strain: BL21(DE3) derivatives optimized for expression of proteins from low G+C organisms may provide better yields.

Table: Recommended Expression Systems for Recombinant F. tularensis Proteins

How can I optimize purification of recombinant F. tularensis fusA?

Purifying recombinant fusA requires a strategic approach to maintain protein function and stability:

• Affinity chromatography: His-tagged fusA can be purified using Ni-NTA resins, but buffer optimization is crucial as EF-G typically requires specific ion concentrations for stability.

• Buffer considerations: Inclusion of glycerol (10-20%), reducing agents (DTT or β-mercaptoethanol), and appropriate salt concentrations (typically 100-300 mM NaCl) helps maintain fusA stability.

• Sequential chromatography: After initial affinity purification, ion exchange chromatography followed by size exclusion chromatography can significantly improve purity for functional studies.

• Activity preservation: GTP or non-hydrolyzable GTP analogs in purification buffers may stabilize the protein's native conformation.

• Contaminant removal: F. tularensis contains numerous immunologically active components including lipopolysaccharides . Include steps to remove these potential contaminants, such as Triton X-114 phase separation or polymyxin B treatment.

What are the challenges in verifying the functionality of recombinant fusA?

Confirming that recombinant fusA maintains native functionality presents several challenges:

• GTPase activity assays: Measuring GTP hydrolysis using colorimetric phosphate detection or coupled enzyme assays can confirm basic enzymatic function.

• Ribosome binding assays: Surface plasmon resonance or filter binding assays using purified ribosomes can verify interaction with the translational machinery.

• In vitro translation systems: Reconstituted translation systems can test fusA's ability to support protein synthesis, with comparison to commercially available EF-G from model organisms.

• Structural validation: Circular dichroism spectroscopy can verify proper secondary structure formation as compared to predicted models.

• Thermostability assessment: Differential scanning fluorimetry can evaluate protein stability and compare variants from different F. tularensis subspecies.

How does the genomic context of fusA differ between F. tularensis subspecies?

While specific information about the fusA genomic context isn't provided in the search results, broader genomic comparisons offer relevant insights:

F. tularensis subspecies show significant genomic reorganization despite high sequence identity (97-99%) . The more virulent subspecies, F. tularensis subsp. tularensis (type A), displays more genomic rearrangements compared to F. tularensis subsp. holarctica (type B) . These rearrangements are often flanked by insertion sequence elements, suggesting homologous recombination as the mechanism . Such genomic plasticity may affect the expression regulation of essential genes like fusA through changes in promoter contexts or operon structures.

Additionally, pseudogenization is a notable feature in F. tularensis evolution, with the more virulent subspecies containing approximately 200-300 pseudogenes compared to only 14 in the less pathogenic F. novicida . This differential gene inactivation pattern may influence interaction networks involving translation factors.

What can comparative analysis of fusA tell us about Francisella evolution?

Analyzing fusA across Francisella species and subspecies could provide several evolutionary insights:

• Selection pressure: As an essential gene, fusA likely experiences purifying selection, but specific domains might show signatures of positive selection, particularly at host-pathogen interfaces.

• Adaptation signatures: Comparison of fusA sequences between environmental and highly pathogenic strains may reveal adaptations associated with virulence evolution.

• Evolutionary lineage markers: Given the high conservation of translation factors, subtle variations in fusA might help resolve phylogenetic relationships between closely related Francisella isolates.

• Host adaptation: F. tularensis infects over 250 animal species , and fusA variations might reflect adaptation to diverse host environments and immune pressures.

How might fusA function differ between virulent F. tularensis and the Live Vaccine Strain (LVS)?

The Live Vaccine Strain (LVS) is an attenuated derivative of F. tularensis subsp. holarctica that has been used experimentally as a vaccine . While specific fusA differences between virulent strains and LVS aren't detailed in the search results, several inferences can be made:

• Functional constraints: As an essential protein, major changes in fusA function would likely be deleterious. Therefore, any differences would likely be subtle rather than dramatic functional alterations.

• Translation efficiency: Minor sequence variations in fusA might affect translation rates under stress conditions, potentially contributing to LVS attenuation.

• Protein interaction networks: LVS contains 448 polymorphisms in non-transposase coding sequences compared to the OSU18 strain , which might include changes affecting fusA interactions with other cellular components.

• Post-translational modifications: Differences in post-translational modification patterns of fusA between virulent strains and LVS could affect protein function without sequence changes.

How can fusA be leveraged for developing diagnostic tools for F. tularensis?

Though highly conserved, fusA may offer opportunities for diagnostic development:

• Subspecies differentiation: Minor sequence variations in fusA between subspecies tularensis and holarctica might be detectable through high-resolution melting analysis or subspecies-specific PCR.

• Recombinant antigen applications: Purified recombinant fusA could serve as a capture antigen in immunoassays, though its intracellular location may limit antibody production during natural infection.

• CRISPR-Cas diagnostics: Cas13-based nucleic acid detection systems targeting subspecies-specific sequences within fusA could enable rapid, field-deployable diagnostics.

• Mass spectrometry identification: fusA peptides could serve as marker ions for mass spectrometry-based identification and typing of F. tularensis isolates.

• Aptamer development: DNA or RNA aptamers against unique epitopes in F. tularensis fusA could enable sensitive detection systems.

What role might fusA play in F. tularensis stress response and virulence?

Beyond its canonical role in translation, Elongation Factor G may contribute to stress adaptation and virulence:

• Stress response integration: In other bacteria, translation factors participate in various stress responses. F. tularensis faces extreme stress within phagocytes, and fusA may help coordinate adaptive responses.

• Interaction with virulence factors: The proteome of F. tularensis contains numerous virulence-associated proteins . fusA might interact with these factors, particularly during stress conditions.

• Moonlighting functions: Some translation factors perform secondary "moonlighting" functions beyond protein synthesis. Given that F. tularensis lacks many classical virulence factors , essential proteins like fusA might have evolved additional functions.

• Role in persistence: F. tularensis can persist within macrophages and other host cells. Regulated translation through fusA activity may contribute to this persistence capability.

How might recombinant fusA contribute to vaccine development efforts?

Vaccine development against F. tularensis remains challenging, with no licensed vaccine currently available . Recombinant fusA could contribute to these efforts:

• Subunit vaccine component: Though intracellular, portions of fusA might be processed and presented to immune cells, potentially eliciting protective responses.

• Attenuated strain development: Strategic mutations in fusA could potentially create attenuated strains with reduced translation efficiency under in vivo conditions while maintaining immunogenicity.

• Adjuvant effects: Some bacterial proteins exhibit intrinsic adjuvant properties. If fusA contains immunostimulatory domains, it might enhance responses to other antigens.

• Correlates of protection studies: Analysis of immune responses to fusA following vaccination with LVS or other candidates may identify correlates of protection .

What controls should be included when studying recombinant F. tularensis fusA?

Robust experimental design for fusA studies should include:

• Species/subspecies comparisons: Include fusA from multiple F. tularensis subspecies (tularensis, holarctica) and F. novicida to identify subspecies-specific properties .

• Structural and functional controls: Compare with well-characterized E. coli EF-G to benchmark functional assays and structural properties.

• Mutation controls: Generate known GTPase-deficient mutants (e.g., mutations in the G domain) as negative controls for enzymatic assays.

• Expression system controls: Include empty vector controls and unrelated protein expressions to distinguish fusA-specific effects from expression system artifacts.

• Endotoxin considerations: F. tularensis contains immunologically active components . Include appropriate endotoxin testing and removal controls, particularly for immunological studies.

How should researchers approach studying fusA in the context of F. tularensis pathogenesis?

Given the extreme virulence of F. tularensis subsp. tularensis (requiring BSL-3 containment), researchers should consider:

• Biosafety level requirements: F. tularensis subsp. tularensis requires BSL-3 facilities, while F. tularensis subsp. holarctica LVS can be handled at BSL-2 . Consider using LVS for initial studies.

• Animal model selection: Different animal models show varying susceptibility to F. tularensis. The search results mention both mouse and vole models . Choose models appropriate for the specific aspect of fusA biology under investigation.

• In vitro infection models: Macrophage infection models can provide insights into fusA's role during intracellular growth without requiring animal studies.

• Complementation approaches: For genetic studies, complementation with fusA variants can distinguish direct effects from polar effects when creating mutants.

• Integrated multi-omics: Combine transcriptomics, proteomics, and metabolomics to place fusA function within broader pathogenesis networks.

What technical considerations are important when analyzing fusA expression during infection?

Analyzing fusA expression and function during infection presents technical challenges:

• Low bacterial numbers: F. tularensis can establish infection with <10 CFU , making detection from in vivo samples challenging. Consider amplification methods like Proximity Ligation Assay for protein detection.

• Host material interference: Abundant host proteins can mask bacterial proteins in samples. Use techniques like immunomagnetic separation to enrich bacterial cells before analysis.

• Temporal considerations: Expression may vary throughout infection stages. Design time-course experiments to capture dynamics.

• Single-cell variation: Population-level measurements may mask important cell-to-cell variation. Where possible, employ single-cell techniques like single-cell RNA-seq with pathogen enrichment.

• Translation vs. transcription: fusA mRNA levels may not correlate perfectly with protein levels. Use techniques like ribosome profiling to assess actual translation activity.