Recombinant Coxiella burnetii Probable chorismate--pyruvate lyase (ubiC)

How does C. burnetii ubiC compare structurally to homologous proteins in other bacterial species?

While the search results don't provide direct structural information about C. burnetii ubiC specifically, insights can be drawn from related bacterial species. In cyanobacteria like Synechocystis sp., the chorismate-pyruvate lyase (encoded by sll1797) shares low sequence identity with E. coli UbiC protein. Analysis of these proteins has identified conserved amino acid residues (particularly Arg-30 and Glu-120) that likely form part of the active site .

Researchers investigating C. burnetii ubiC should perform comparative sequence alignments with established chorismate-pyruvate lyases to identify conserved domains and potential active sites. Molecular modeling approaches based on known structures can provide valuable insights into substrate binding and catalytic mechanisms prior to experimental validation.

What is the significance of chorismate-pyruvate lyase in C. burnetii pathogenesis?

C. burnetii is known to employ sophisticated immune subversion strategies as a strictly intracellular pathogen . The bacterium's ability to modulate host immune responses and adapt to the intracellular environment may partly depend on metabolic pathways involving chorismate-pyruvate lyase. Research into metabolic adaptations during different phases of infection indicates that C. burnetii can modify host cell functions in a T4SS-dependent manner , suggesting complex interactions between bacterial metabolism and virulence mechanisms.

What are effective methods for expressing recombinant C. burnetii ubiC in heterologous systems?

Based on successful approaches with other bacterial enzymes, researchers should consider the following methodology for expressing C. burnetii ubiC:

• Gene selection and optimization: Clone the ubiC gene from C. burnetii genomic DNA using PCR with specific primers. Consider codon optimization for the expression host.

• Expression system selection: E. coli is often preferred for initial expression attempts. Based on experiences with other bacterial proteins, BL21(DE3) strains are recommended for high-level expression .

• Vector design: Incorporate a histidine-tag for purification purposes, similar to the approach used with other recombinant C. burnetii proteins . Consider using inducible promoters (T7 or tac) to control expression levels.

• Expression conditions: Optimize induction parameters including IPTG concentration (typically 0.1-1 mM), temperature (often lowered to 16-25°C to enhance solubility), and induction time (4-16 hours) .

• Validation: Confirm expression through SDS-PAGE and Western blotting using anti-His antibodies or custom antibodies against ubiC.

What are the recommended protocols for purifying recombinant C. burnetii ubiC protein?

For efficient purification of recombinant C. burnetii ubiC, researchers should implement a multi-step purification strategy:

• Cell lysis: Disrupt cells using sonication or pressure-based methods in appropriate buffer systems (typically phosphate or Tris-based, pH 7.5-8.0) containing protease inhibitors.

• Initial purification: For His-tagged proteins, use immobilized metal affinity chromatography (IMAC) with Ni-NTA or cobalt-based resins. Based on protocols used for other recombinant proteins, apply a stepwise imidazole gradient (10-250 mM) for elution .

• Secondary purification: Consider size exclusion chromatography to remove aggregates and improve purity. Ion exchange chromatography may provide additional purification based on the protein's theoretical isoelectric point.

• Quality assessment: Evaluate protein purity using SDS-PAGE (>90% purity is typically desired), and confirm identity via mass spectrometry or Western blotting.

• Storage optimization: Determine optimal storage conditions by testing various buffers, pH values, and additives. Most recombinant proteins are stable at -80°C with 10-20% glycerol as a cryoprotectant.

How can researchers effectively measure enzymatic activity of purified C. burnetii chorismate-pyruvate lyase?

To assess the enzymatic activity of chorismate-pyruvate lyase from C. burnetii, researchers can adapt the following methodology based on assays used for homologous enzymes:

• Reaction setup: Prepare 200-μl reaction mixtures containing buffer (50 mM Bis-Tris propane/HCl, pH 8.0) and substrate (450 μM chorismate). Add purified enzyme at appropriate concentrations .

• Incubation conditions: Incubate reactions at 30°C for 5 minutes. The short incubation helps minimize non-enzymatic breakdown of chorismate .

• Reaction termination: Stop reactions by adding 300 μl of 3 M acetate buffer (pH 4.0) followed by extraction with 500 μl of ethyl acetate containing 2-hydroxybenzoate as an internal standard .

• Product analysis: Analyze the ethyl acetate extracts by HPLC using a C18 column with acetonitrile/water (1:1; v/v) containing 0.1% trifluoroacetic acid at a flow rate of 0.5 ml/min. Monitor 4-hydroxybenzoate formation at 255 nm .

• Data analysis: Quantify 4-hydroxybenzoate production by comparing peak areas to calibration curves. Correct for non-enzymatic breakdown of chorismate that occurs during the assay incubation .

How can recombinant C. burnetii ubiC contribute to Q fever vaccine development?

Recombinant C. burnetii ubiC could potentially serve as a component in novel subunit vaccines against Q fever. Current approaches to Q fever vaccine development face significant challenges:

• Current vaccine limitations: Existing Q fever vaccines like Q-VaxTM cause unacceptable side effects or fail to provide sufficient protection. These vaccines, based on formalin-inactivated C. burnetii bacterins, demonstrate significant reactogenicity in individuals previously sensitized to C. burnetii antigens and interfere with available serodiagnostic tests .

• Advantages of defined subunit vaccines: Subunit vaccines using specific antigens like ubiC can be engineered to reduce reactogenicity and co-designed with serodiagnostic tests to allow discrimination between vaccinated and infected individuals (DIVA strategy) .

• Research approach: To evaluate ubiC as a vaccine candidate, researchers should:

  • Express and purify the recombinant protein with appropriate quality controls

  • Test immunogenicity in animal models, particularly mice

  • Evaluate protective efficacy through challenge experiments

  • Consider combining ubiC with other potential antigens for enhanced protection

• Evaluation framework: When testing efficacy, researchers should monitor clinical symptoms, spleen and liver weights, bacterial burden in tissues, and immune responses, similar to protocols used for other recombinant C. burnetii proteins .

What methodologies are recommended for investigating C. burnetii ubiC as an antimicrobial target?

To evaluate chorismate-pyruvate lyase as a potential antimicrobial target against C. burnetii, researchers should implement a structured approach:

• Target validation:

  • Generate knockout mutants to confirm essentiality, if genetic manipulation is feasible

  • Alternatively, use conditional expression systems or antisense RNA approaches

  • Examine growth phenotypes under various conditions to assess metabolic importance

• Inhibitor screening strategies:

  • Develop high-throughput enzymatic assays adaptable to microplate format

  • Implement virtual screening using homology models based on related bacterial enzymes

  • Perform fragment-based screening to identify initial chemical scaffolds

• Lead compound evaluation:

  • Test promising compounds against purified enzyme (IC50 determination)

  • Evaluate cellular activity using C. burnetii infection models

  • Assess selectivity by comparing activity against mammalian cell lines

  • Determine spectrum of activity against other bacterial species

• Mechanism of action studies:

  • Perform enzyme kinetics with varying substrate and inhibitor concentrations

  • Use structural biology approaches (X-ray crystallography, cryo-EM) to visualize inhibitor binding

  • Conduct resistance development studies to understand potential escape mechanisms

How does C. burnetii ubiC activity relate to bacterial persistence in host cells?

The relationship between chorismate-pyruvate lyase activity and C. burnetii persistence in host cells involves complex metabolic adaptations:

• Adaptation to intracellular environment: C. burnetii must adapt its metabolism to survive within the host cell environment. Metabolic enzymes like chorismate-pyruvate lyase could be crucial for this adaptation, particularly under variable oxygen conditions that the bacterium encounters during infection .

• Connection to energy metabolism: Chorismate-pyruvate lyase contributes to quinone biosynthesis, which is essential for electron transport chains and energy generation. In cyanobacteria, disruption of this pathway severely impedes photosynthetic electron transport . Similarly, in C. burnetii, this pathway might be critical for maintaining energy production during intracellular growth.

• Potential link to hypoxic adaptation: C. burnetii infection initially induces HIF1α stabilization, which then decreases over the course of infection . This suggests that metabolic pathways potentially involving chorismate-pyruvate lyase might interact with host hypoxia response mechanisms, potentially influencing bacterial persistence.

• Research methodology: To investigate this relationship, researchers should:

  • Develop inducible expression systems or conditional knockouts of ubiC

  • Monitor bacterial replication and persistence under various oxygen conditions

  • Examine host cell metabolic changes in response to altered ubiC expression

  • Analyze metabolomic profiles during different stages of infection

What techniques can researchers use to investigate post-translational modifications of C. burnetii ubiC?

Investigating post-translational modifications (PTMs) of C. burnetii ubiC requires sophisticated analytical approaches:

• Mass spectrometry-based identification:

  • Perform tryptic digestion of purified ubiC protein

  • Analyze peptides using LC-MS/MS with high-resolution instruments

  • Implement data-dependent acquisition to maximize PTM detection

  • Use specialized search algorithms (e.g., MaxQuant, Proteome Discoverer) with PTM search parameters

• Site-specific mutagenesis validation:

  • Once potential PTM sites are identified, generate site-directed mutants

  • Express and purify mutant proteins alongside wild-type controls

  • Compare enzymatic activities and stability profiles

  • Assess structural changes using circular dichroism or thermal shift assays

• Temporal dynamics of PTMs:

  • Isolate bacteria from different growth phases or infection stages

  • Quantify changes in PTM patterns using stable isotope labeling approaches

  • Correlate PTM changes with enzymatic activity and bacterial physiology

• PTM enzymes identification:

  • Search for potential modifier enzymes in the C. burnetii genome

  • Consider kinases, phosphatases, acetyltransferases based on predicted PTMs

  • Test recombinant modifier enzymes with purified ubiC in vitro

How does the intracellular environment affect chorismate-pyruvate lyase expression and activity in C. burnetii?

The intracellular environment likely influences chorismate-pyruvate lyase expression and activity through multiple mechanisms:

• Oxygen availability effects:

  • C. burnetii encounters varying oxygen levels inside host cells, which may affect enzyme expression and activity

  • Research shows that C. burnetii can modulate host hypoxia responses, suggesting adaptation to oxygen-limited environments

  • Researchers should monitor enzyme expression under normoxic versus hypoxic conditions using qRT-PCR and Western blotting

• Nutrient availability influence:

  • Host cell metabolites may regulate enzyme expression through feedback mechanisms

  • Experimental approaches should include supplementation with precursors or end products of the pathway

  • Metabolomic analysis can help identify relevant metabolites that influence expression

• pH-dependent regulation:

  • C. burnetii uniquely adapts to acidified compartments within host cells

  • Enzymatic assays at varying pH values (4.0-7.5) can determine optimal activity conditions

  • Site-directed mutagenesis of pH-sensitive residues can identify crucial pH sensors

• Host-pathogen signaling impacts:

  • C. burnetii uses a Type IV Secretion System (T4SS) to modulate host cell functions

  • Compare wild-type and T4SS-deficient strains (Δ dotA) to determine if secreted effectors influence enzyme expression

  • Co-immunoprecipitation experiments may identify host factors that interact with the enzyme or its regulators

What are the methodological challenges in developing C. burnetii mutants deficient in chorismate-pyruvate lyase?

Generating and characterizing C. burnetii mutants deficient in chorismate-pyruvate lyase presents several technical challenges:

• Genetic manipulation limitations:

  • C. burnetii is particularly challenging for genetic manipulation due to its intracellular lifestyle

  • Researchers should consider transposon mutagenesis approaches, which have been successful for other C. burnetii genes

  • CRISPR-Cas9 systems adapted for intracellular bacteria may provide more precise genetic editing

• Essential gene considerations:

  • If ubiC is essential, direct knockout attempts will fail

  • Implement conditional approaches such as:

    • Tetracycline-inducible expression systems

    • Degradation tag systems (e.g., DAS+4 tags with ClpXP protease)

    • CRISPRi for partial knockdown rather than complete knockout

• Phenotypic analysis challenges:

  • Reduced growth or viability of mutants requires specialized cultivation techniques

  • Supplement growth media with 4-hydroxybenzoate to bypass the metabolic block

  • Develop fluorescent reporters linked to metabolic activity for real-time monitoring

• Complementation strategies:

  • Validate mutant phenotypes through genetic complementation

  • Use site-specific integration vectors for stable expression

  • Consider inducible systems to titrate expression levels and avoid toxicity

  • Test heterologous enzymes from related species to inform about functional conservation

• Growth analysis under different conditions:

  • Similar to studies in cyanobacteria, monitor growth parameters carefully under various conditions

  • Develop protocols for assessing mutant fitness during infection of cell cultures

  • Implement competitive index assays comparing mutant and wild-type strains

How can researchers correlate ubiC activity with C. burnetii virulence in different host species?

To establish correlations between chorismate-pyruvate lyase activity and C. burnetii virulence across host species, researchers should implement comparative approaches:

• Multi-species infection models:

  • Develop standardized infection protocols across different host cell types (human, bovine, caprine, ovine)

  • Compare bacterial replication rates, persistence, and virulence phenotypes

  • Monitor ubiC expression levels using RT-qPCR or reporter systems in different hosts

• Serological studies:

  • Analyze host antibody responses against ubiC across species using peptide microarrays

  • Implement high-density peptide microarrays to analyze seroreactivity in serum samples from vaccinated and infected individuals

  • Determine if antibody responses correlate with protection or disease severity

• Comparative genomics:

  • Analyze ubiC sequence variations among C. burnetii isolates from different hosts

  • Correlate sequence polymorphisms with host specificity or virulence phenotypes

  • Express and characterize variant enzymes to assess functional differences

• Data integration framework:

  • Create statistical models incorporating enzyme activity, bacterial burden, and clinical parameters

  • Use machine learning approaches to identify patterns across diverse datasets

  • Validate predictions using targeted experimental approaches

What bioinformatic approaches can reveal regulatory networks involving ubiC in C. burnetii?

Elucidating regulatory networks involving ubiC requires sophisticated bioinformatic strategies:

• Promoter analysis:

  • Identify potential transcription factor binding sites upstream of ubiC

  • Perform comparative genomics across related bacterial species to find conserved regulatory elements

  • Use tools like MEME, JASPAR, or RegPrecise for motif discovery

• Transcriptomic co-expression analysis:

  • Analyze RNA-seq data from various growth conditions to identify genes co-regulated with ubiC

  • Implement weighted gene co-expression network analysis (WGCNA) to define modules of functionally related genes

  • Validate predictions using reporter constructs or ChIP-seq for identified regulators

• Metabolic network integration:

  • Construct genome-scale metabolic models incorporating chorismate-pyruvate lyase reactions

  • Perform flux balance analysis to predict metabolic adaptations under different conditions

  • Identify potential metabolic bottlenecks and regulatory points affecting ubiC function

• Machine learning applications:

  • Train models on existing bacteria to predict regulatory interactions

  • Implement feature extraction from genomic, transcriptomic, and proteomic datasets

  • Validate predictions using targeted experimental approaches