Recombinant Coxiella burnetii Phosphoglycerate kinase (pgk)

What is Coxiella burnetii and why is its phosphoglycerate kinase of research interest?

Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for Q fever, a disease that typically presents as a severe flu-like illness. The bacterium has been classified as a risk group 3 organism due to its high infectivity and disease severity . Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate, generating ATP in the process. In C. burnetii, PGK is of particular interest because:

• It represents a potential drug target given its essential metabolic role

• Its study provides insights into the pathogen's unique metabolic adaptations for intracellular survival

• As a conserved protein, it allows for comparative analyses between different bacterial species

• Recombinant PGK can be used to develop detection methods and diagnostic tools for Q fever

What expression systems are suitable for producing recombinant C. burnetii PGK?

Recombinant C. burnetii PGK can be successfully expressed using several systems, with E. coli being the most commonly employed. Based on protocols similar to those used for other C. burnetii proteins:

• E. coli expression systems: Most researchers use BL21(DE3) or similar strains with pET-based vectors for high-yield expression. The choice of fusion tags (His, GST, etc.) depends on downstream applications and purification strategies.

• Vector selection: Vectors allowing insertion of the pgk gene with appropriate restriction enzymes (such as BamHI and EcoRI) as described for other C. burnetii genes are typically used .

• Induction conditions: IPTG induction at concentrations of 0.5-1.0 mM when cultures reach OD600 of 0.6-0.8, followed by expression at lower temperatures (16-25°C) often yields better results for soluble protein production.

• Codon optimization: Given the difference in codon usage between C. burnetii and E. coli, codon optimization of the pgk gene sequence is recommended for improved expression.

What are the optimal conditions for purifying recombinant C. burnetii PGK?

Purification of recombinant C. burnetii PGK typically involves:

• Cell lysis: Sonication or pressure-based disruption in buffer systems containing protease inhibitors. Standard lysis buffers include 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and 1 mM DTT.

• Affinity chromatography: For His-tagged PGK, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with imidazole gradients (20-250 mM) effectively separates the target protein.

• Size exclusion chromatography: Further purification via gel filtration to remove aggregates and ensure monomeric protein preparation.

• Buffer optimization: Final protein storage typically requires 20-50 mM Tris-HCl or phosphate buffer (pH 7.4-8.0) with 100-150 mM NaCl and 5-10% glycerol to maintain stability.

• Storage conditions: Purified protein is generally stable at -80°C for extended periods, with flash freezing in liquid nitrogen recommended to prevent activity loss.

How can I confirm the identity and purity of recombinant C. burnetii PGK?

Multiple analytical techniques should be employed to validate the identity and purity of recombinant C. burnetii PGK:

• SDS-PAGE analysis: Should show a single band at the expected molecular weight (approximately 43-45 kDa).

• Western blotting: Using anti-His antibodies (for His-tagged protein) or antibodies specific to C. burnetii PGK.

• Mass spectrometry: MALDI-TOF or LC-MS/MS analysis to confirm protein identity through peptide mass fingerprinting.

• Enzymatic activity assay: Spectrophotometric assays measuring ATP production from 1,3-bisphosphoglycerate and ADP, or the reverse reaction.

• Circular dichroism (CD) spectroscopy: To verify proper protein folding and secondary structure composition.

How does the enzymatic activity of C. burnetii PGK compare to PGK from other bacterial species?

The enzymatic properties of C. burnetii PGK can be characterized and compared to other bacterial PGKs through:

• Kinetic parameter determination:

  • Km values for substrates (1,3-bisphosphoglycerate and ADP)

  • kcat and catalytic efficiency (kcat/Km)

  • pH optimum and pH stability profiles

  • Temperature optimum and thermal stability

• Structural comparisons:

  • X-ray crystallography to determine three-dimensional structure

  • Comparative modeling with PGKs from other pathogens to identify unique features

• Inhibition studies:

  • Sensitivity to known PGK inhibitors

  • Identification of C. burnetii-specific inhibitors through screening approaches

Due to C. burnetii's adaptation to the acidic environment of the parasitophorous vacuole (PV), its PGK may exhibit unique properties compared to other bacterial PGKs, particularly regarding pH optimum and stability .

What role might PGK play in C. burnetii's intracellular life cycle and virulence?

Understanding PGK's potential roles in C. burnetii pathogenesis requires consideration of:

• Metabolic adaptations:

  • C. burnetii survives in acidic phagolysosome-like compartments where glycolytic enzymes like PGK may be crucial for energy production

  • The bacterium has a prolonged growth cycle, making efficient energy metabolism essential for survival

• Potential moonlighting functions:

  • Beyond its glycolytic role, PGK may serve additional functions in bacterial pathogens

  • These could include surface localization, interaction with host factors, or modulation of host signaling

• Regulation during infection:

  • Expression patterns of pgk during different stages of infection

  • Potential post-translational modifications affecting PGK activity in response to environmental cues

• Experimental approaches:

  • Targeted gene disruption using newly developed genetic tools for C. burnetii

  • Protein-protein interaction studies to identify potential host targets

  • Localization studies using immunofluorescence microscopy during infection

Can C. burnetii PGK be used as a diagnostic marker for Q fever?

The potential of C. burnetii PGK as a diagnostic tool warrants investigation:

• Serological detection:

  • Recombinant PGK could serve as an antigen in ELISA or other immunoassays

  • Need to evaluate sensitivity and specificity compared to current diagnostic antigens

• Nucleic acid-based detection:

  • PCR targeting the pgk gene could complement existing molecular diagnostics

  • The RPA-LF (recombinase polymerase amplification with lateral flow) method described for 23S rRNA detection could be adapted for pgk

• Comparative analysis with current diagnostic methods:

  Detection Method	Target	Time Required	Sensitivity	Specificity	Equipment Needs
  RPA-LF (potential for pgk)	Gene-specific	~30 min	7-10 copies/reaction	High	Minimal
  RT-qPCR	23S rRNA, IS1111, com1	1-2 hours	1-10 copies/reaction	High	Specialized
  IFA	Whole cell antigens	3-4 hours	Lower in early infection	Variable	Fluorescence microscope

• Distinguishing acute vs. chronic infections:

  • Potential for PGK-specific antibody profiles to differentiate disease stages

  • Need for longitudinal serological studies using recombinant PGK

How might post-translational modifications affect C. burnetii PGK function during infection?

Investigation of post-translational modifications (PTMs) of C. burnetii PGK requires:

• Identification of potential modifications:

  • Phosphorylation sites (C. burnetii activates host kinases during infection)

  • Acetylation, methylation, or other modifications affecting enzyme activity

  • Potential host-mediated modifications during intracellular growth

• Functional impact assessment:

  • Site-directed mutagenesis of identified or predicted PTM sites

  • In vitro modification systems to recapitulate and study specific PTMs

  • Activity assays comparing modified vs. unmodified PGK forms

• Temporal dynamics:

  • Changes in modification patterns throughout the bacterial life cycle

  • Correlation with phases of infection and PV biogenesis

C. burnetii is known to modulate host signaling pathways, including PKC, PKA, p38, and other kinases during infection , which might affect bacterial proteins through host-mediated phosphorylation.

What structural features distinguish C. burnetii PGK from human PGK, and how can these differences be exploited for drug development?

Structure-based drug design targeting C. burnetii PGK requires:

• Structural characterization:

  • X-ray crystallography or cryo-EM analysis of C. burnetii PGK

  • Molecular dynamics simulations to identify flexible regions and binding pockets

  • Comparison with human PGK crystal structure to identify unique features

• Identification of targetable differences:

  • Catalytic site variations affecting substrate binding

  • Allosteric sites unique to bacterial PGKs

  • Surface loops or domains absent in human counterparts

• Virtual screening approaches:

  • In silico docking of compound libraries against identified unique sites

  • Fragment-based screening for lead compound identification

  • Pharmacophore modeling based on structural differences

• Validation of inhibitor candidates:

  • Biochemical assays to confirm target engagement and inhibition

  • Selectivity testing against human PGK

  • Cellular models to assess bacterial growth inhibition

  • Testing in C. burnetii infection models

What controls are essential when working with recombinant C. burnetii PGK?

Proper experimental design for studies involving recombinant C. burnetii PGK should include:

• Protein quality controls:

  • Heat-inactivated PGK to establish baseline in activity assays

  • Known concentrations of commercial PGK from other species as reference standards

  • Buffer-only controls for background signal determination

• Specificity controls:

  • Related kinases from C. burnetii or other bacteria for comparative analyses

  • Human PGK when assessing inhibitor specificity

  • Site-directed mutants affecting catalytic residues as negative controls

• Expression system controls:

  • Empty vector transformants processed identically to PGK-expressing constructs

  • Unrelated recombinant proteins expressed under identical conditions

• Biological relevance controls:

  • Wild-type vs. pgk-mutant C. burnetii strains (if available)

  • Complementation experiments to verify phenotypic effects

How can I develop a high-throughput screening system to identify inhibitors of C. burnetii PGK?

Establishing a robust high-throughput screening (HTS) platform requires:

• Assay development and optimization:

  • Miniaturized spectrophotometric assays coupling PGK activity to NAD+/NADH conversion

  • Fluorescence-based assays for enhanced sensitivity

  • Determination of optimal reaction conditions (pH, temperature, ionic strength)

• Assay validation metrics:

  Parameter	Target Value	Importance
  Z'-factor	>0.7	Statistical validity of assay
  Signal-to-background ratio	>3	Assay window
  Coefficient of variation	<10%	Assay reproducibility
  DMSO tolerance	≥1%	Compatibility with compound libraries
  Stability over time	<10% drift	Assay robustness

• Screening cascade:

  • Primary screen at single concentration (10-20 μM typical)

  • Dose-response confirmation of hits

  • Counter-screening against human PGK

  • Orthogonal assays to confirm mechanism of action

  • Cell-based secondary screens using C. burnetii-infected cells

• Data analysis and hit selection:

  • Statistical methods for hit identification (typically >50% inhibition)

  • Structure-activity relationship analysis of confirmed hits

  • Clustering of chemical scaffolds for prioritization

How do I establish a cell-based assay to study the role of PGK in C. burnetii infection?

Developing cellular models to investigate PGK function requires:

• Cell line selection:

  • Human THP-1 macrophage-like cells and HeLa cells are established models for C. burnetii infection

  • Primary human alveolar macrophages for physiologically relevant studies

• Infection protocols:

  • Standardized MOI (multiplicity of infection) determination

  • Synchronized infection procedures

  • Assessment of PV formation kinetics

• Gene manipulation approaches:

  • CRISPR/Cas9 editing of pgk in C. burnetii

  • Conditional expression systems

  • Complementation with wild-type or mutant variants

• Readout methods:

  • Immunofluorescence microscopy to assess PV formation

  • qPCR to quantify bacterial replication

  • Live-cell imaging to track infection dynamics

  • Fluorescent reporter systems to monitor PGK expression or activity

C. burnetii exhibits a unique dependence on host kinase activity for establishing its parasitophorous vacuole , suggesting potential interplay between bacterial metabolic enzymes like PGK and host signaling pathways.

What approaches can be used to investigate potential non-canonical functions of C. burnetii PGK?

To explore potential moonlighting functions of PGK beyond its glycolytic role:

• Protein-protein interaction studies:

  • Pull-down assays using recombinant PGK to identify bacterial or host binding partners

  • Yeast two-hybrid screening

  • Proximity labeling methods (BioID, APEX) in infected cells

  • Co-immunoprecipitation from infected cell lysates

• Localization analyses:

  • Immunofluorescence microscopy to determine PGK distribution during infection

  • Cell fractionation followed by Western blotting

  • Surface biotinylation to assess potential membrane association

• Functional assessments:

  • Domain mapping to identify regions responsible for non-canonical functions

  • Site-directed mutagenesis to separate glycolytic and potential moonlighting activities

  • Competition assays with recombinant PGK added extracellularly during infection

• Comparative genomics and transcriptomics:

  • Analysis of pgk expression patterns during different phases of infection

  • Correlation with expression of virulence factors

  • Comparison with PGK function in related bacterial pathogens

What strategies can resolve issues with low solubility of recombinant C. burnetii PGK?

When encountering solubility problems with recombinant PGK:

• Expression optimization:

  • Lower induction temperature (16-20°C)

  • Reduced IPTG concentration (0.1-0.5 mM)

  • Co-expression with chaperones (GroEL/GroES, DnaK/DnaJ)

  • Use of solubility-enhancing tags (MBP, SUMO, TRX)

• Buffer optimization:

  • Screen various pH conditions (typically pH 6.5-8.5)

  • Test different salt concentrations (100-500 mM NaCl)

  • Addition of stabilizing agents (glycerol, arginine, trehalose)

  • Inclusion of reducing agents (1-5 mM DTT or β-mercaptoethanol)

• Refolding approaches (if inclusion bodies form):

  • Denaturation with 6-8 M urea or guanidine hydrochloride

  • Stepwise dialysis for gradual refolding

  • On-column refolding during affinity purification

  • Pulse refolding with dilution methods

• Alternative expression systems:

  • Cell-free protein synthesis

  • Eukaryotic expression systems (yeast, insect cells)

  • Psychrophilic bacterial hosts for cold-adapted expression

How can I address inconsistent enzymatic activity in purified recombinant C. burnetii PGK preparations?

Variability in enzymatic activity may result from multiple factors:

• Protein quality issues:

  • Verify complete removal of imidazole or other purification reagents

  • Check for protein aggregation by dynamic light scattering

  • Analyze for potential proteolytic degradation by SDS-PAGE and mass spectrometry

  • Verify correct disulfide bond formation if applicable

• Assay optimization:

  • Standardize substrate quality and preparation

  • Confirm linear range of the assay for accurate kinetic measurements

  • Control temperature precisely during measurements

  • Establish consistent enzyme handling protocols

• Storage and stability:

  • Determine optimal storage conditions (buffer composition, pH, additives)

  • Test stability at different temperatures

  • Evaluate freeze-thaw sensitivity

  • Consider flash-freezing aliquots to maintain consistent quality

• Validation approaches:

  • Circular dichroism to confirm consistent secondary structure

  • Thermal shift assays to assess stability across preparations

  • Size exclusion chromatography to verify oligomeric state

What are potential pitfalls when comparing recombinant C. burnetii PGK activity with native enzyme activity?

Several factors can complicate direct comparisons between recombinant and native PGK:

• Technical considerations:

  • Differing purification methods affecting activity

  • Tag presence/absence on recombinant protein

  • Buffer composition differences

  • Contaminating activities in partially purified native preparations

• Biological factors:

  • Post-translational modifications present only in native enzyme

  • Potential binding partners or cofactors in cellular context

  • Allosteric regulators present in vivo but absent in vitro

  • Differences in oligomeric state between recombinant and native forms

• Experimental design approaches:

  • Use of cell lysates from C. burnetii-infected cells vs. purified recombinant enzyme

  • In situ activity measurements when possible

  • Reconstitution experiments with potential cofactors

  • Side-by-side comparisons under identical conditions

• Data interpretation caveats:

  • Careful normalization based on active site titration rather than protein concentration

  • Consideration of microenvironment effects on activity parameters

  • Accounting for temperature and pH differences between in vitro and in vivo conditions

How might systems biology approaches enhance our understanding of C. burnetii PGK in the context of metabolic networks?

Integrative systems biology can provide deeper insights into PGK function:

• Metabolic modeling:

  • Flux balance analysis incorporating PGK in the context of C. burnetii metabolism

  • Identification of synthetic lethal interactions with other metabolic enzymes

  • Prediction of growth phenotypes under various perturbations

• Multi-omics integration:

  • Correlation of pgk expression with metabolomic profiles during infection

  • Proteome-wide interaction networks centered on PGK

  • Identification of regulatory mechanisms controlling pgk expression

• Comparative systems analyses:

  • Cross-species comparison of metabolic networks involving PGK

  • Evolutionary analysis of PGK conservation and adaptation

  • Identification of C. burnetii-specific metabolic features

• Computational predictions for validation:

  • In silico prediction of PGK inhibitor effects on bacterial growth

  • Modeling of metabolic rewiring upon PGK perturbation

  • Simulation of host-pathogen metabolic interactions

What novel applications might emerge from structural and functional studies of C. burnetii PGK?

Beyond basic understanding, C. burnetii PGK research may lead to:

• Therapeutic applications:

  • Structure-based design of specific inhibitors targeting C. burnetii PGK

  • Development of PGK-based subunit vaccines

  • Identification of allosteric modulators with antimicrobial potential

• Diagnostic innovations:

  • PGK-based detection systems for environmental monitoring

  • Novel serological assays for different stages of Q fever

  • Point-of-care diagnostic tools suitable for resource-limited settings

• Biotechnological applications:

  • Engineered PGK variants with enhanced thermostability or catalytic efficiency

  • Biosensors utilizing PGK for metabolite detection

  • Biocatalytic applications in synthesis of high-energy compounds

• Research tools:

  • PGK-based reporter systems for tracking C. burnetii in vivo

  • Affinity-tagged PGK for studying protein-protein interactions

  • Modified PGK variants as probes for metabolic pathway analysis