Recombinant Francisella philomiragia subsp. philomiragia Serine hydroxymethyltransferase (glyA)

What are the recommended approaches for cloning the glyA gene from Francisella philomiragia?

Based on successful approaches used for other Francisella species, the following methodological workflow is recommended:

• Primer Design and Amplification Strategy:

  • Design gene-specific primers that target the glyA coding sequence, including appropriate start (ATG) and stop codons.

  • Incorporate partial recombinational cloning sites (e.g., attB sites) in the primers to facilitate downstream cloning.

  • For genes below 2,000 bp (glyA typically falls in this range), use a two-step PCR approach: first amplify with gene-specific primers containing partial attB sites, then perform a second PCR with universal primers to complete the attB recombinational cloning sites .

• PCR Optimization:

  • Use high-fidelity DNA polymerase to minimize mutation rates (improves from 1/608 to 1/3,939 base pairs with higher fidelity polymerases) .

  • Optimize PCR conditions including annealing temperature, extension time, and MgCl₂ concentration.

  • Verify amplification success using agarose gel electrophoresis.

• Cloning Strategy:

  • Utilize recombinational cloning systems (e.g., Gateway) for efficient transfer between vectors.

  • Capture the PCR product in an entry vector such as pDONR221 using BP clonase reaction .

  • Use E. coli strain DH5αT1 for propagation of the clones .

• Clone Verification:

  • Perform colony PCR to identify positive transformants.

  • Conduct comprehensive sequence verification to ensure the absence of PCR-induced mutations.

  • Store verified clones as both plasmid DNA and bacterial glycerol stocks (15% v/v glycerol) .

This approach has been successfully used to create a full-genomic sequence-verified protein-coding gene collection for Francisella tularensis with a success rate of over 96% , suggesting it would be effective for F. philomiragia glyA as well.

How can recombinant F. philomiragia glyA be expressed and purified effectively?

The following methodological protocol is recommended for effective expression and purification of recombinant F. philomiragia glyA:

• Expression Vector Selection:

  • Use pDEST17 or similar vector that provides an N-terminal 6xHIS tag for purification purposes .

  • The vector should contain a strong, inducible promoter (e.g., T7) for controlled expression.

• Host Strain Selection:

  • BL21 star (DE3) pLysS is recommended as it has been successfully used for other Francisella proteins .

  • This strain provides tight regulation of the T7 promoter and contains the pLysS plasmid to reduce basal expression.

• Expression Conditions:

  • Grow transformants at 37°C in appropriate media to an OD₆₀₀ of approximately 0.7.

  • Induce protein expression with 1 mM IPTG .

  • Consider testing different temperatures for induction (16°C, 25°C, 37°C) to optimize soluble protein yield.

• Cell Harvest and Lysis:

  • Harvest cells by centrifugation and resuspend in appropriate lysis buffer.

  • Lyse cells using sonication, French press, or chemical methods.

  • Include protease inhibitors to prevent degradation of the target protein.

• Purification Strategy:

  • Perform immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to capture the His-tagged glyA.

  • Elute with an imidazole gradient (50-300 mM).

  • Consider additional purification steps such as size exclusion chromatography to enhance purity.

• Quality Control:

  • Verify purity using SDS-PAGE.

  • Confirm protein identity using Western blotting or mass spectrometry.

  • Assess enzymatic activity using appropriate assays (see section 1.4).

This expression and purification protocol has been effective for producing proteins from the related organism F. tularensis with success rates over 72% , suggesting it would be suitable for F. philomiragia glyA as well.

How can the enzymatic activity of purified recombinant glyA be verified?

Verification of glyA enzymatic activity requires carefully designed assays that monitor the reversible conversion of serine to glycine. The following methodological approaches are recommended:

• Spectrophotometric Assays:

  • Forward Reaction (Serine to Glycine): Monitor the formation of 5,10-methylene-THF by coupling to NADPH oxidation via methylenetetrahydrofolate reductase, measuring absorbance decrease at 340 nm.

  • Reverse Reaction (Glycine to Serine): Measure the consumption of 5,10-methylene-THF and formation of THF by monitoring changes in absorbance at specific wavelengths.

• Radiometric Assays:

  • Use ¹⁴C-labeled serine as substrate and measure the formation of ¹⁴C-labeled glycine.

  • Separate reaction products using thin-layer chromatography and quantify radioactivity.

• HPLC-Based Assays:

  • Derivatize amino acids pre- or post-column for detection.

  • Quantify the conversion of serine to glycine or vice versa over time.

  • Compare reaction rates with established enzyme standards.

• Enzyme Kinetics Analysis:

  Parameter	Forward Reaction	Reverse Reaction
  Substrate	L-Serine, THF	Glycine, 5,10-methylene-THF
  K<sub>m</sub> range	0.5-2.0 mM (Serine)	0.1-0.5 mM (Glycine)
  pH optimum	7.5-8.5	7.0-8.0
  Temperature optimum	35-40°C	35-40°C
  Cofactor requirement	Pyridoxal 5'-phosphate	Pyridoxal 5'-phosphate

• Controls and Validation:

  • Include positive controls (commercial SHMT or recombinant glyA from related species).

  • Include negative controls (heat-inactivated enzyme, reaction without substrate).

  • Verify the dependence on pyridoxal 5'-phosphate by testing activity with and without this cofactor.

These assays should be performed under optimized conditions, considering factors such as pH, temperature, buffer composition, and cofactor concentration that might affect the activity of F. philomiragia glyA.

What role might glyA play in F. philomiragia virulence and pathogenicity?

While direct evidence for glyA's contribution to F. philomiragia virulence is limited, its central metabolic function suggests potential roles in pathogenesis:

• Metabolic Support for Intracellular Replication:

  • F. philomiragia can infect and proliferate in various mammalian cells, including murine macrophages J774A.1, human lung epithelial A549 cells, and human hepatocytes HepG2 cells .

  • glyA's role in one-carbon metabolism likely supports the biosynthetic demands of intracellular replication, providing essential building blocks for nucleic acids and proteins.

• Adaptation to Host Microenvironments:

  • Different host cell types present distinct metabolic conditions to which F. philomiragia must adapt.

  • glyA might be differentially regulated in response to these varying conditions, potentially explaining the observed differences in cytotoxicity between cell types (cytotoxic in U937 and J774A.1 cells but non-cytotoxic in HepG2 and A549 cells) .

• Potential Contribution to Virulence in Animal Models:

  • F. philomiragia demonstrates lethal infections in both Galleria mellonella (LD₅₀ of 1.8 × 10³) and BALB/c mice via intranasal infection (LD₅₀ of 3.45 × 10³) .

  • The metabolic functions of glyA may be critical for establishing and maintaining these infections, particularly in the respiratory tract where one-carbon metabolism could support adaptation to pulmonary environments.

• Experimental Approaches to Investigate glyA's Role in Virulence:

  Approach	Methodology	Expected Outcome
  Gene knockout/knockdown	CRISPR-Cas or transposon mutagenesis to disrupt glyA	Assess changes in growth rate, intracellular survival, and virulence in models
  Conditional expression	Inducible promoter systems to control glyA expression	Determine minimum expression levels required for virulence
  Metabolic bypass	Provide metabolites downstream of glyA function	Identify which aspects of glyA function are most critical for virulence
  Host response analysis	Transcriptomics/proteomics of host cells infected with wild-type vs. glyA-deficient bacteria	Understand how glyA affects host-pathogen interactions

These investigations would help establish whether glyA represents a potential therapeutic target for treating F. philomiragia infections, which though rare, can be serious in near-drowning victims or immunocompromised individuals.

Which animal and cell culture models are most appropriate for studying F. philomiragia glyA function in pathogenesis?

Based on established research, the following models are recommended for investigating F. philomiragia glyA function in pathogenesis:

• Cell Culture Models:

  Cell Type	Advantages	Key Findings with F. philomiragia	Application for glyA Studies
  J774A.1 murine macrophages	Well-characterized, easy to culture, phagocytic	F. philomiragia replicates to higher levels than F. tularensis subspecies at 24h	Ideal for studying intracellular replication and the role of glyA in supporting bacterial metabolism within macrophages
  A549 human lung epithelial cells	Models lung infection relevant to near-drowning cases	F. philomiragia can infect but is non-cytotoxic	Useful for investigating glyA's role in adaptation to pulmonary environments
  HepG2 human hepatocellular cells	Models liver infection	F. philomiragia can infect but is non-cytotoxic	Appropriate for studying metabolic adaptations during hepatic infection
  U937 human macrophage-like cells	Human-derived, allows investigation of species-specific interactions	F. philomiragia infection is cytotoxic after 24h	Valuable for comparing glyA function between different host species

• In Vivo Models:

  Model	Advantages	Established Parameters	Methodology for glyA Studies
  Galleria mellonella	Ethical advantages, cost-effective, has innate immune system	LD₅₀ of 1.8 × 10³ for F. philomiragia	Inject larvae with wild-type vs. glyA-modified bacteria; monitor survival and bacterial loads
  BALB/c mice (intranasal infection)	Mammalian model, allows study of complex host-pathogen interactions	LD₅₀ of 3.45 × 10³ for F. philomiragia	Administer bacteria intranasally; assess bacterial burden in lungs and other organs; evaluate histopathology

This comprehensive approach would provide insights into glyA's role across different stages of F. philomiragia pathogenesis and in different host environments, enabling the identification of potential therapeutic intervention points.

What methodologies can be employed to assess the impact of glyA inhibition on F. philomiragia metabolism?

Comprehensive assessment of glyA inhibition effects requires multi-level analysis of bacterial metabolism. The following methodological approaches are recommended:

• Chemical Inhibition Studies:

  Inhibitor Type	Examples	Methodological Considerations
  Competitive inhibitors	Glycine analogues, serine analogues	Test at various concentrations; determine IC₅₀ values
  Mechanism-based inhibitors	Compounds targeting pyridoxal 5'-phosphate	Assess reversibility with excess cofactor
  Allosteric inhibitors	Novel compounds identified through screening	Evaluate effects on enzyme kinetics

• Metabolic Flux Analysis:

  • Use ¹³C-labeled substrates (e.g., [¹³C]serine, [¹³C]glycine) to trace metabolic pathways.

  • Quantify flux changes through one-carbon metabolism pathways upon glyA inhibition.

  • Identify compensatory metabolic routes activated during glyA inhibition.

• Comprehensive Metabolomic Profiling:

  Technique	Application	Expected Outcomes
  LC-MS/MS	Quantify amino acids, nucleotides, one-carbon metabolites	Identify accumulation or depletion of key metabolites
  GC-MS	Analyze TCA cycle intermediates, fatty acids	Assess broader metabolic impact
  NMR spectroscopy	Real-time metabolic changes	Monitor dynamic responses to inhibition

• Growth and Survival Assays:

  • Determine effects of glyA inhibition on growth rates in different media compositions.

  • Assess survival under various stress conditions (oxidative stress, nutrient limitation, antimicrobial exposure).

  • Evaluate biofilm formation capacity when glyA is inhibited.

• Cellular Infection Models with glyA Inhibition:

  • Infect J774A.1 macrophages with F. philomiragia in the presence of glyA inhibitors.

  • Quantify bacterial replication, host cell viability, and inflammatory responses.

  • Compare effects across different cell types (macrophages vs. epithelial cells).

• In Vivo Metabolic Imaging:

  • Use PET imaging with radiolabeled metabolites in animal models.

  • Track metabolic adaptations during infection with and without glyA inhibition.

These methodologies provide a comprehensive framework for understanding how glyA inhibition affects F. philomiragia metabolism, potentially revealing new therapeutic strategies and improving our understanding of this pathogen's metabolic vulnerabilities.

How can structural biology approaches advance our understanding of F. philomiragia glyA?

Structural biology offers powerful tools to elucidate the molecular basis of glyA function in F. philomiragia. The following methodological approaches are recommended:

• Structure-Function Analysis:

  Structural Feature	Functional Significance	Experimental Approach
  Active site architecture	Substrate binding and catalysis	Site-directed mutagenesis of key residues
  Protein oligomerization	Enzyme stability and regulation	Size exclusion chromatography, analytical ultracentrifugation
  Conformational dynamics	Catalytic cycle progression	Hydrogen-deuterium exchange mass spectrometry
  Cofactor binding pocket	Pyridoxal 5'-phosphate interaction	Co-crystallization with cofactors and substrates

• Comparative Structural Analysis:

  • Generate homology models of F. philomiragia glyA based on known structures if crystallization proves challenging.

  • Compare structural features with glyA from other Francisella species and human SHMT.

  • Identify unique structural elements that could explain functional differences or serve as specific drug targets.

• Structural Basis for Inhibition:

  • Co-crystallize glyA with known inhibitors to understand binding modes.

  • Perform molecular docking studies to identify potential binding sites for novel inhibitors.

  • Use structure-based drug design to develop specific inhibitors of F. philomiragia glyA.

• Cryo-Electron Microscopy:

  • For challenging crystallization targets or to capture conformational heterogeneity.

  • Study glyA in complex with partner proteins or larger assemblies.

  • Visualize different conformational states during the catalytic cycle.

• Biophysical Characterization:

  Technique	Application	Expected Insights
  Circular dichroism	Secondary structure assessment	Stability under various conditions
  Differential scanning fluorimetry	Thermal stability	Effects of ligands and buffer conditions
  Isothermal titration calorimetry	Binding thermodynamics	Affinity for substrates and inhibitors
  Surface plasmon resonance	Binding kinetics	Association/dissociation rates

• Molecular Dynamics Simulations:

  • Simulate glyA behavior in different environments.

  • Investigate conformational changes during catalysis.

  • Predict effects of mutations or inhibitor binding.

These approaches would provide unprecedented insights into the molecular basis of glyA function in F. philomiragia, facilitating both fundamental understanding of one-carbon metabolism in this organism and the development of potential therapeutic strategies targeting this enzyme.