Recombinant Chlamydophila caviae Triosephosphate isomerase (tpiA)

What is triosephosphate isomerase (TPI) and what is its functional significance in C. caviae?

Triosephosphate isomerase (TPI) is a highly evolved enzymatic catalyst that interconverts dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (G3P) in both glycolysis and the Calvin-Benson cycle . In most organisms, including C. caviae, TPI functions as an obligate dimer that folds into the characteristic (β-α)8 barrel known as the TIM barrel . The enzyme catalyzes this isomerization reaction near diffusion rate-limiting speed, without requiring any cofactors or metal ions .

In the context of Chlamydophila caviae, TPI plays a crucial role in central carbon metabolism. Like other bacteria, C. caviae relies on this enzyme for energy production through glycolysis. The enzyme's high catalytic efficiency is particularly important in intracellular pathogens like C. caviae, which must compete with host cells for nutrients during their developmental cycle.

What are the general structural characteristics of bacterial triosephosphate isomerases?

Bacterial triosephosphate isomerases, including those from Chlamydial species, typically exhibit the following structural characteristics:

• Quaternary structure: TPI forms a homodimer, with each monomer folding into a (β-α)8 barrel .

• Active site location: The catalytic site often forms at the dimer interface, but the four key catalytic residues (typically Asn11, Lys13, His95, and Glu167) come from the same subunit .

• Catalytic mechanism: Glu167 generally serves as the catalytic base. Upon substrate binding, conformational changes occur, particularly the closure of loop-6 and loop-7, which shield the active site from bulk solvent and stabilize the enediolate intermediate .

• Dynamic elements: The enzyme features flexible loops that undergo substantial conformational changes during catalysis. For instance, loop-6 closes upon substrate binding, moving the Glu167 carboxylate moiety approximately 2 Å toward the substrate .

These structural features are highly conserved across different species, though specific amino acid variations may affect properties such as redox sensitivity.

How can Chlamydophila caviae be successfully transformed for recombinant protein expression?

Successfully transforming C. caviae for recombinant protein expression can be achieved using Protocol B as described in recent research. This protocol involves:

• Preparation of the bacteria in 50 mM CaCl2 for 30 minutes at room temperature.

• Co-incubation with freshly trypsinized cells for 20 minutes.

• Selection using appropriate antibiotics (e.g., 5 μg/ml ampicillin for C. caviae) .

Importantly, research has shown that Protocol B is effective for C. caviae transformation, while Protocol A (100 mM CaCl2 for 1 hour) and alternative protocols were unsuccessful . The table below summarizes transformation protocol effectiveness for different Chlamydia species:

Successful transformation has been demonstrated using the shuttle vector pUC-Ccavpl-GFP, which contains the whole plasmid of C. caviae strain GPIC along with a beta-lactamase gene for selection and a GFP marker for visualization .

What factors influence the redox sensitivity of triosephosphate isomerases and how might this apply to C. caviae TPI?

The redox sensitivity of triosephosphate isomerases varies significantly between species and appears to be related to the presence and accessibility of reactive cysteine residues. Research comparing TPIs from different organisms provides insights that may be relevant to C. caviae TPI:

• Accessibility of cysteine residues: In SyTPI (from Synechocystis), only one of three cysteines (C176) is solvent-exposed, yet it remains relatively insensitive to redox modification in vitro . This contrasts with plant TPIs, which contain redox-sensitive cysteines that are accessible and reactive.

• Response to oxidizing agents: SyTPI demonstrates resistance to inhibition by oxidizing agents like diamide (DA) and H2O2, as well as thiol-conjugating agents such as oxidized glutathione (GSSG) and methyl methanethiosulfonate (MMTS) at concentrations that inactivate plant TPIs .

• Evolutionary considerations: It has been proposed that the replacement of cyanobacterial TPI by eukaryotic TPI in chloroplasts may be related to the latter's redox-sensitive cysteines, which allow post-translational modifications to modulate enzymatic activity .

For C. caviae TPI specifically, a comprehensive analysis of its cysteine content and positioning would be necessary to predict its redox sensitivity. If the pattern follows that of other bacterial TPIs, it may have fewer accessible reactive cysteines and therefore demonstrate greater resistance to oxidative stress conditions compared to eukaryotic counterparts.

What experimental approaches can be used to study the dynamics of flexible loops in C. caviae TPI during catalysis?

Studying the dynamics of flexible loops in C. caviae TPI during catalysis requires sophisticated experimental approaches that capture both structural and temporal aspects of enzyme function:

• X-ray crystallography with substrate analogs: By co-crystallizing the enzyme with transition-state analogs like 2-phosphoglycolate, researchers can capture different conformational states of the flexible loops . For C. caviae TPI, this would involve comparing structures with and without bound substrates or analogs to visualize loop movements.

• Molecular dynamics simulations: Based on crystal structures, computational simulations can reveal the dynamic behavior of loops over time, particularly the movements of loop-6 and loop-7, which are critical for catalysis .

• NMR spectroscopy: Solution NMR can provide insights into loop dynamics in real-time, revealing information about both the rate and extent of conformational changes during catalysis.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can identify regions of high flexibility and conformational changes upon substrate binding.

• Site-directed mutagenesis of loop residues: By systematically mutating residues in loops 6 and 7 of C. caviae TPI and measuring the effects on catalysis, researchers can identify key residues involved in loop dynamics and substrate binding.

• FRET-based approaches: By strategically placing fluorophores near the flexible loops, researchers can monitor conformational changes in real-time during catalysis.

These approaches, used in combination, would provide a comprehensive understanding of how loop dynamics contribute to the catalytic efficiency of C. caviae TPI.

How can GFP-tagged C. caviae TPI be used for in vivo studies of enzyme localization and infection dynamics?

GFP-tagged C. caviae TPI offers valuable opportunities for in vivo visualization of both enzyme localization and infection progression:

• Infection dissemination tracking: Successfully transformed C. caviae expressing GFP can be used in guinea pig infection models to better understand the dissemination of chlamydial infections . This approach allows real-time visualization of infected cells and tissues without the need for fixation and immunostaining.

• Subcellular enzyme localization: By tagging TPI specifically with GFP, researchers can track the localization of this enzyme within C. caviae cells during different stages of the developmental cycle. This may reveal dynamic reorganization of metabolic enzymes during the transition between elementary bodies and reticulate bodies.

• Host-pathogen interface visualization: GFP-tagged TPI could potentially be used to study interactions between bacterial metabolic enzymes and host cell components, especially if TPI has moonlighting functions beyond its catalytic role.

• Quantitative assessment of expression levels: Flow cytometry or fluorescence microscopy can be used to quantify GFP expression levels, providing insights into the regulation of TPI expression under different conditions.

For these applications, the shuttle vector pUC-Ccavpl-GFP has been successfully used to transform C. caviae strain GPIC . The stability of the transformed strain should be assessed through multiple passages with and without selection pressure to ensure reliable expression during in vivo experiments.

What are the key considerations for designing site-directed mutagenesis experiments to study catalytic mechanisms of C. caviae TPI?

When designing site-directed mutagenesis experiments to investigate the catalytic mechanism of C. caviae TPI, researchers should consider the following key aspects:

By systematically applying these approaches, researchers can build a comprehensive model of the catalytic mechanism specific to C. caviae TPI and identify any unique features compared to other bacterial and eukaryotic TPIs.

What are the optimal conditions for expression and purification of recombinant C. caviae TPI?

Based on general practices for bacterial TPI expression and the specific characteristics of C. caviae, the following protocol represents an optimized approach for expression and purification of recombinant C. caviae TPI:

Expression System Selection:

• E. coli BL21(DE3) or Rosetta(DE3) strains are recommended for high-level expression of potentially difficult-to-express chlamydial proteins.

• Expression vectors containing T7 promoter (pET series) with either N-terminal or C-terminal His-tag are suitable for subsequent purification.

Culture Conditions:

• LB or 2xYT medium supplemented with appropriate antibiotics.

• Induction at OD600 of 0.6-0.8 with 0.5-1.0 mM IPTG.

• Post-induction temperature of 25-30°C for 4-16 hours to enhance soluble protein expression.

Cell Lysis:

• Resuspension in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitor cocktail.

• Lysis via sonication or French press, followed by centrifugation at 20,000 × g for 30 minutes at 4°C.

Purification Strategy:

• Immobilized Metal Affinity Chromatography (IMAC)

  • Ni-NTA or Co-NTA resin with binding buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol.

  • Washing with increasing imidazole concentrations (10-40 mM).

  • Elution with 250-300 mM imidazole.

• Size Exclusion Chromatography

  • Superdex 75 or 200 column equilibrated with 25 mM Tris-HCl pH 7.5, 150 mM NaCl.

  • Collection of dimeric fraction (expected MW ~53 kDa) .

• Optional Ion Exchange Chromatography

  • Q-Sepharose column for additional purification if needed.

Quality Control:

• SDS-PAGE for purity assessment (>95% purity expected).

• Western blotting with anti-His and anti-TPI antibodies.

• Enzymatic activity assay using the coupled assay with α-glycerophosphate dehydrogenase.

• Dynamic light scattering to confirm dimeric state and homogeneity.

Storage Conditions:

• For short-term: 4°C in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT.

• For long-term: -80°C in the same buffer supplemented with 20% glycerol.

This protocol may require optimization based on the specific properties of C. caviae TPI, particularly if it shows unexpected solubility or stability characteristics.

What approaches can be used to evaluate the catalytic mechanism of recombinant C. caviae TPI?

Evaluating the catalytic mechanism of recombinant C. caviae TPI requires a multi-faceted approach combining kinetic, structural, and computational methods:

1. Steady-State Kinetics:

• Determination of kcat and Km using a coupled assay with α-glycerophosphate dehydrogenase.

• pH-rate profiles to identify the pKa values of catalytic residues.

• Temperature dependence studies to calculate activation energies and thermodynamic parameters.

• Solvent isotope effects using D2O to probe proton transfer steps.

2. Pre-Steady-State Kinetics:

• Stopped-flow spectroscopy to measure the rates of individual steps in the catalytic cycle.

• Rapid chemical quench experiments to trap reaction intermediates.

• Single-turnover kinetics to isolate the chemical step from product release.

3. Structural Analysis:

• X-ray crystallography of enzyme-substrate complexes using substrate analogs or cryocrystallography.

• Analysis of loop conformations in "open" and "closed" states to understand the role of dynamics in catalysis .

• Hydrogen-deuterium exchange mass spectrometry to identify regions with altered dynamics during catalysis.

4. Inhibition Studies:

• Testing with transition state analogs like 2-phosphoglycolate .

• Analysis of inhibition patterns (competitive, noncompetitive, uncompetitive) to gain insights into binding mechanisms.

5. Computational Methods:

• Molecular dynamics simulations to visualize loop movements and water molecule positions.

• Quantum mechanics/molecular mechanics (QM/MM) calculations to model the reaction coordinate.

• Computational mutagenesis to predict the effects of specific amino acid substitutions.

6. Comparative Analysis:

• Comparison with well-characterized TPIs from other species, particularly focusing on the four key catalytic residues (equivalents of Asn11, Lys13, His95, and Glu167) .

• Analysis of species-specific variations that might influence catalytic efficiency or substrate specificity.

By integrating data from these complementary approaches, researchers can develop a comprehensive model of the C. caviae TPI catalytic mechanism, including the roles of specific residues, loop dynamics, and any unique features compared to other TPIs.

How can recombinant C. caviae TPI contribute to understanding chlamydial metabolism during infection?

Recombinant C. caviae TPI serves as a valuable tool for investigating chlamydial metabolism during the infection process through several research approaches:

• Metabolic flux analysis: By monitoring the activity of recombinant TPI under various conditions that mimic the intracellular environment, researchers can gain insights into how glycolytic flux is regulated during different stages of the chlamydial developmental cycle. This is particularly relevant for understanding energy production in these obligate intracellular parasites.

• Host-induced modifications: Expression of recombinant C. caviae TPI in eukaryotic cells can reveal whether host factors induce post-translational modifications that might regulate enzymatic activity during infection.

• Nutrient competition studies: Comparing the kinetic parameters of C. caviae TPI with human TPI can provide insights into potential competition for metabolic intermediates between the pathogen and host cells.

• Drug target validation: As a central metabolic enzyme, TPI represents a potential therapeutic target. Recombinant enzyme enables high-throughput screening for specific inhibitors that could selectively target chlamydial TPI without affecting the human ortholog.

• GFP-tracking studies: Using the successfully developed GFP-tagged C. caviae strain , researchers can track the localization and expression patterns of TPI during infection, particularly if TPI is included in the construct. This approach allows real-time visualization of metabolic enzyme deployment during the developmental cycle.

These applications collectively contribute to a more comprehensive understanding of how C. caviae adapts its central carbon metabolism during infection and may reveal novel intervention points for therapeutic development.

What comparative analyses between human and C. caviae TPI could reveal potential drug targets?

Comparative analyses between human and C. caviae triosephosphate isomerases could reveal significant differences that may be exploited for selective therapeutic targeting:

• Interface with other metabolic enzymes: Species-specific protein-protein interactions between TPI and other glycolytic enzymes could represent novel targeting opportunities that would not affect the human metabolic network.

These comparative analyses would provide a foundation for structure-based drug design efforts targeting C. caviae TPI with minimal effects on the human ortholog.

What are the emerging techniques for studying enzyme dynamics that could be applied to C. caviae TPI?

Several cutting-edge techniques are emerging for studying enzyme dynamics that could yield valuable insights when applied to C. caviae TPI:

• Cryo-electron microscopy (cryo-EM): Recent advances in resolution now enable visualization of conformational ensembles and can capture multiple states of enzymes during catalysis, potentially revealing intermediate conformations of C. caviae TPI's flexible loops that may not be captured by crystallography.

• Single-molecule FRET (smFRET): This technique could monitor the opening and closing motions of loop-6 in real-time at the single-molecule level, providing insights into the conformational dynamics and heterogeneity that may be masked in ensemble measurements.

• Time-resolved X-ray crystallography: Using X-ray free-electron lasers (XFELs) and photocaged substrates, researchers could potentially capture transient states during catalysis with unprecedented temporal resolution.

• Integrative structural biology approaches: Combining multiple experimental techniques (NMR, SAXS, cryo-EM, crystallography) with computational modeling to build a more complete picture of enzyme dynamics across different timescales.

• Nanobody-enabled stabilization: Using synthetic nanobodies to trap specific conformational states of C. caviae TPI, particularly the elusive closed-loop conformation, for structural studies.

• Deep mutational scanning: Systematic creation of all possible single amino acid variants of C. caviae TPI, coupled with high-throughput activity assays, could reveal the complete mutational landscape and identify residues critical for dynamics rather than just catalysis.

• AlphaFold2 and RoseTTAFold integration: Using these AI-powered structure prediction tools to model conformational changes and guide experimental design, particularly for regions that are difficult to resolve experimentally.

These emerging techniques, especially when used in combination, have the potential to provide unprecedented insights into the dynamic behavior of C. caviae TPI during catalysis and reveal species-specific features that could inform both basic understanding and therapeutic development.

How might genetic tools for C. caviae transformation be further developed to facilitate TPI research?

The recent success in transforming C. caviae with a shuttle vector opens up numerous possibilities for further tool development specifically aimed at TPI research:

• Inducible expression systems: Development of tetracycline or anhydrotetracycline-inducible promoters for C. caviae would allow controlled expression of modified TPI variants during infection.

• CRISPR/Cas9 adaptation: Adapting CRISPR/Cas9 technology for C. caviae would enable precise genome editing, including:

  • Introduction of point mutations in the native tpiA gene

  • Creation of conditional knockouts

  • Integration of reporter fusions at the native locus

• Split-GFP complementation systems: Engineering the TPI protein with one half of a split GFP and potential interaction partners with the complementary half would enable visualization of protein-protein interactions in living bacteria.

• Targeted protein degradation tools: Developing degron tags that function in C. caviae would allow temporal control over TPI protein levels independent of transcriptional regulation.

• Biosensor development: Creating FRET-based or other fluorescent biosensors to monitor TPI activity or substrate levels in real-time during infection.

• Optimized transformation protocols: Building on the success of Protocol B , further refinements could increase transformation efficiency, such as:

  • Testing additional CaCl2 concentrations between 50-100 mM

  • Exploring alternative divalent cations

  • Optimizing the timing of antibiotic selection

• Multiplex genetic manipulation: Developing methods for simultaneous introduction of multiple genetic modifications to facilitate complex experimental designs.

These advanced genetic tools would significantly enhance the ability to study C. caviae TPI in its native context, providing more physiologically relevant insights than those obtained from recombinant protein studies alone.