Recombinant Francisella philomiragia subsp. philomiragia Triosephosphate isomerase (tpiA)

What is the genomic context of the tpiA gene in Francisella philomiragia?

The tpiA gene in Francisella philomiragia is part of the complete genome that was sequenced and deposited in GenBank under accession number NC_010336.1. The genome of F. philomiragia subsp. philomiragia ATCC 25017 consists of a 2,045,775 bp chromosome with 32.0% G+C content and a small 3,936-bp cryptic plasmid designated pFPHIo1 with 28.4% G+C content . Unlike many metabolic genes that might be duplicated in bacterial genomes, the tpiA gene is typically present as a single copy in the F. philomiragia genome, reflecting its conserved role in central carbon metabolism. The gene organization surrounding tpiA is important for understanding potential co-regulation with other glycolytic enzymes, though specific operon structures would require experimental confirmation.

How does F. philomiragia tpiA differ structurally from homologs in other species?

While the precise structural differences of tpiA aren't explicitly detailed in the provided search results, comparative analysis can be inferred from genomic differences between Francisella species. F. philomiragia has a larger genome (2.045 Mb) compared to F. tularensis (1.9 Mb) , suggesting potential differences in metabolic enzyme configurations.

The triosephosphate isomerase enzyme generally maintains a highly conserved TIM barrel fold across species, but species-specific variations in substrate binding pockets and catalytic residues may exist. These differences could be particularly relevant in the context of F. philomiragia's adaptation to environmental niches versus the host-adapted lifestyle of pathogenic Francisella species. Detailed structural studies would be required to elucidate these specific differences.

What expression systems are most effective for producing recombinant F. philomiragia tpiA?

Based on research with other Francisella proteins, several expression systems can be considered for recombinant F. philomiragia tpiA production:

• Shuttle vector systems: The development of pF242- and pF243-based shuttle vectors has enabled transformation of Francisella strains with high efficiency through electroporation . These vectors, derived from plasmids isolated from F. philomiragia strains ATCC 25016 and ATCC 25017, provide excellent platforms for homologous expression.

• E. coli-based systems: Standard E. coli expression systems with T7 promoters are likely effective for heterologous expression, though codon optimization may be necessary due to the lower G+C content (32.0%) of F. philomiragia compared to E. coli.

For optimal expression, construct design should include:

• A strong, inducible promoter (like T7 or tac)

• Appropriate tags for purification (His6 or GST)

• Consideration of the protein's native folding requirements

Successful expression has been shown for other Francisella proteins using electroporation, which is significantly more efficient than cryotransformation for introducing recombinant vectors into Francisella strains .

How does tpiA contribute to F. philomiragia's metabolic adaptation to aquatic environments?

F. philomiragia demonstrates an apparent connection to diverse saline, brackish, and sea-water environments, showing greater resistance than F. tularensis to grazing by protists . This environmental adaptation suggests that metabolic enzymes like tpiA may play specialized roles in F. philomiragia's survival strategy.

The triosephosphate isomerase enzyme catalyzes a critical step in glycolysis, enabling flexible carbon metabolism that may contribute to F. philomiragia's ability to persist in aquatic habitats. Several adaptations may be relevant:

• Temperature stability: The enzyme likely maintains activity across the wider temperature ranges encountered in aquatic environments

• Osmotic adaptation: TpiA may function optimally under varying salt concentrations characteristic of different water bodies

• Energy efficiency: The enzyme may show kinetic properties optimized for energy conservation in nutrient-limited aquatic settings

Research comparing enzymatic properties of tpiA between F. philomiragia and strictly host-associated Francisella species would provide insights into metabolic specialization for environmental persistence versus host invasion.

What role might tpiA play in the virulence mechanisms of F. philomiragia in immunocompromised hosts?

F. philomiragia infections occur predominantly in immunosuppressed individuals, suggesting opportunistic rather than primary pathogen behavior . While tpiA is primarily a metabolic enzyme, it may contribute to virulence through several mechanisms:

• Metabolic adaptation: During infection, tpiA could help redirect carbon flux to support survival within host cells

• Moonlighting functions: Beyond its canonical role in glycolysis, tpiA might have secondary functions in host-pathogen interactions

• Stress response: The enzyme may support bacterial survival under oxidative stress conditions encountered within phagocytes

The genome of F. philomiragia harbors substantial differences in virulence factors compared to F. tularensis Schu S4, including variations in the intracellular growth locus proteins and phospholipase C . These variations likely influence the pathogenic potential and host range, potentially interacting with metabolic systems in which tpiA participates.

The ability of F. philomiragia to escape phagolysosomal degradation and multiply in the cytosol, similar to other Francisella species , suggests coordination between virulence factors and metabolic systems, possibly involving tpiA in energy provision for these processes.

How can site-directed mutagenesis of tpiA elucidate its role in F. philomiragia physiology?

Site-directed mutagenesis represents a powerful approach to investigate tpiA function in F. philomiragia. Key targets and methodological considerations include:

Mutation Targets:

• Catalytic residues (typically Glu and His in the active site)

• Substrate binding residues

• Interface residues if the enzyme functions as a dimer

• Potential regulatory sites

Experimental Approach:

• Generate mutations using the shuttle vectors based on pF242 and pF243

• Introduce constructs via electroporation, which has demonstrated higher transformation efficiency than cryotransformation

• Assess phenotypes under various growth conditions (different carbon sources, stress conditions)

• Measure enzymatic activities in vitro using purified wild-type and mutant proteins

This approach would reveal residues critical for catalysis and potentially identify unexpected functions. Comparing the effects of equivalent mutations in tpiA from F. tularensis would further illuminate species-specific adaptations.

What purification strategies yield highest activity of recombinant F. philomiragia tpiA?

Optimal purification of recombinant F. philomiragia tpiA should consider the enzyme's biochemical properties, with the following recommended strategy:

Purification Protocol:

• Expression: Use pF242- or pF243-based vectors with a strong promoter and affinity tag

• Lysis: Gentle lysis methods (e.g., osmotic shock or mild detergents) to preserve protein folding

• Initial Capture: Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

• Intermediate Purification: Ion exchange chromatography based on the protein's predicted pI

• Polishing: Size exclusion chromatography to ensure removal of aggregates

Activity Preservation:

• Include glycerol (10-20%) in storage buffers

• Maintain reducing conditions with DTT or β-mercaptoethanol

• Determine optimal pH and temperature stability profiles

• Test activity with and without potential cofactors

The purification should be monitored at each step using SDS-PAGE and activity assays measuring the reversible interconversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Circular dichroism spectroscopy can confirm proper folding of the purified enzyme.

How can researchers develop specific assays to distinguish F. philomiragia tpiA activity from host enzymes?

Developing assays that distinguish F. philomiragia tpiA from host homologs is critical for infection studies. Several approaches can be employed:

• Antibody-based detection:

  • Generate antibodies against unique epitopes in F. philomiragia tpiA

  • Employ immunoprecipitation followed by activity assays

  • Develop ELISA-based quantification methods

• Kinetic differentiation:

  • Identify substrate analogs or conditions where bacterial and host enzymes show different kinetic parameters

  • Design assays exploiting differences in temperature optima, pH dependence, or inhibitor sensitivity

• Genetic approaches:

  • Create tagged versions of tpiA that can be specifically tracked

  • Use the second generation of shuttle vectors containing GFP for fluorescent tracking

The development of specific assays would enable monitoring of bacterial enzyme activity during infection processes, providing insights into metabolic adaptation during host interaction.

What are the best approaches for studying protein-protein interactions involving F. philomiragia tpiA?

Understanding tpiA's protein interaction network could reveal unexpected functions beyond glycolysis. Recommended approaches include:

In vitro methods:

• Pull-down assays: Using recombinant tagged tpiA as bait to capture interacting partners from F. philomiragia lysates

• Surface Plasmon Resonance: For quantitative measurement of binding kinetics with candidate interactors

• Isothermal Titration Calorimetry: To determine thermodynamic parameters of interactions

In vivo methods:

• Bacterial two-hybrid systems: Adapted for use in Francisella or surrogate hosts

• Proximity labeling: Utilizing BioID or APEX2 fused to tpiA to identify proximal proteins in living bacteria

• Fluorescence techniques: Using FRET or BiFC with the second generation of shuttle vectors containing GFP

Computational approaches:

• Prediction of interaction partners based on genomic context

• Structural modeling of potential protein complexes

• Cross-species comparison of known glycolytic enzyme interactions

This multi-faceted strategy would reveal both conserved interactions within the glycolytic pathway and potentially novel interactions specific to F. philomiragia's lifestyle.

How does the kinetic profile of F. philomiragia tpiA compare to orthologs from pathogenic Francisella species?

A comparative kinetic analysis between F. philomiragia tpiA and orthologs from pathogenic Francisella species would likely reveal adaptations related to their different ecological niches. Expected differences include:

F. philomiragia appears better adapted to survive in aquatic environments compared to F. tularensis , suggesting its metabolic enzymes might show broader tolerance to environmental variations. Conversely, F. tularensis enzymes may be optimized for conditions encountered specifically within mammalian hosts.

What insights can crystallographic studies of F. philomiragia tpiA provide for understanding species-specific adaptations?

Crystal structures of F. philomiragia tpiA would provide valuable insights into species-specific adaptations through:

• Active site architecture: Revealing subtle differences in substrate binding that might reflect metabolic specialization for different ecological niches

• Surface properties: Identifying unique surface features that could mediate species-specific protein-protein interactions

• Conformational flexibility: Determining regions of structural plasticity that might contribute to environmental adaptation

Comparative structural analysis with other Francisella species would be particularly informative for:

• Identifying structural determinants of thermal stability differences

• Pinpointing species-specific surface patches that might interact with other proteins

• Revealing potential allosteric sites unique to environmental versus host-adapted species

How does the regulation of tpiA expression differ between F. philomiragia and other Francisella species?

Regulation of tpiA expression likely differs between F. philomiragia and other Francisella species, reflecting their distinct ecological adaptations. Several regulatory mechanisms warrant investigation:

• Transcriptional regulation:

  • Promoter architecture differences affecting binding of transcription factors

  • Potential integration with stress response systems relevant to aquatic environments

  • Co-regulation with other metabolic genes

• Post-transcriptional control:

  • mRNA stability differences

  • Small RNA regulation specific to environmental versus pathogenic species

  • Ribosome binding site accessibility

• Environmental responsiveness:

  • Carbon source-dependent expression patterns

  • Temperature-responsive regulation reflecting environmental versus host ranges

  • Response to oxidative stress conditions

Experimental approaches could utilize the second generation of shuttle vectors containing the Francisella groES promoter to compare expression patterns across species under different conditions. The development of Francisella-specific genetic tools has significantly advanced the ability to investigate such regulatory differences.

How might F. philomiragia tpiA be utilized as a target for developing species-specific detection methods?

The development of species-specific detection methods based on tpiA would be valuable for environmental monitoring and clinical diagnostics. Promising approaches include:

• PCR-based detection:

  • Design of primers targeting unique regions of the F. philomiragia tpiA sequence

  • Development of real-time PCR assays with species-specific probes

  • Multiplex PCR systems to differentiate various Francisella species

• Immunological methods:

  • Production of antibodies against unique epitopes in F. philomiragia tpiA

  • Development of lateral flow assays for rapid detection

  • ELISA systems optimized for environmental and clinical samples

• Aptamer development:

  • Selection of DNA or RNA aptamers with high specificity for F. philomiragia tpiA

  • Integration with biosensor platforms for real-time detection

These approaches would build upon existing detection methods for Francisella species, such as the Real-time PCR assay Fc50 that specifically detects F. noatunensis , by adding another target for enhanced specificity.

What insights might metabolic flux analysis provide about the role of tpiA in F. philomiragia's adaptation to different environments?

Metabolic flux analysis (MFA) would provide dynamic insights into how carbon flows through central metabolism in F. philomiragia under different conditions, highlighting the specific role of tpiA:

• Comparative flux patterns:

  • Measure glycolytic versus pentose phosphate pathway flux in different environments

  • Quantify the balance between catabolic and anabolic processing of triose phosphates

  • Track carbon allocation to biomass versus energy production

• Adaptation mechanisms:

  • Identify metabolic rewiring in response to nutrient limitation

  • Characterize flux changes during transition from environmental to host-mimicking conditions

  • Determine the impact of oxidative stress on central carbon metabolism

• Methodological approach:

  • Utilize 13C-labeled substrates to track carbon flow

  • Implement computational models integrating genomic and experimental data

  • Compare results with those from pathogenic Francisella species

How can computational modeling advance our understanding of tpiA's role in F. philomiragia metabolism?

Computational modeling offers powerful approaches to understand tpiA's role in the broader context of F. philomiragia metabolism:

• Genome-scale metabolic reconstruction:

  • Development of a comprehensive metabolic model for F. philomiragia

  • In silico gene knockout studies to predict the systemic effects of tpiA disruption

  • Comparison with models of F. tularensis to identify niche-specific metabolic adaptations

• Enzyme network analysis:

  • Identification of synthetic lethal interactions involving tpiA

  • Prediction of alternative metabolic routes when tpiA function is compromised

  • Assessment of metabolic robustness under different environmental conditions

• Evolutionary modeling:

  • Reconstruction of the evolutionary history of tpiA across Francisella species

  • Identification of signatures of selection in environmental versus host-adapted lineages

  • Prediction of functional consequences of observed sequence variations

Such computational approaches would complement experimental studies and could guide the design of targeted experiments to validate predictions about tpiA's role in F. philomiragia's distinct ecological lifestyle compared to pathogenic Francisella species.