Recombinant Bartonella quintana Phosphatidylserine decarboxylase proenzyme (psd)

What are the optimal expression systems for recombinant B. quintana psd?

For recombinant expression of B. quintana psd, E. coli-based systems typically yield the best results, particularly BL21(DE3) strains containing the pET vector system. Temperature optimization is crucial, as B. quintana proteins show temperature-specific characteristics, with optimal expression often occurring at lower temperatures (16-20°C) to enhance protein solubility . The following expression parameters have been shown to improve yield:

Including a histidine or GST tag is recommended to facilitate purification, with consideration that N-terminal tags may affect proenzyme processing.

How can I confirm the structural integrity of purified recombinant psd?

Confirming structural integrity requires multiple analytical approaches:

• SDS-PAGE analysis to verify molecular weight (typically ~30-35 kDa for the proenzyme)

• Western blotting using anti-His or specific anti-psd antibodies

• Circular dichroism (CD) spectroscopy to assess secondary structure composition

• Thermal shift assays to evaluate protein stability under various buffer conditions

• Size exclusion chromatography to confirm monomeric/oligomeric state

Properly folded psd typically exhibits characteristic secondary structure elements including both α-helices and β-sheets. Any significant deviations in the CD spectrum compared to native enzyme may indicate structural issues .

What are the optimal assay conditions for measuring B. quintana psd enzymatic activity?

B. quintana psd activity can be measured using the following protocol:

• Buffer composition: 50 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM DTT

• Substrate preparation: Phosphatidylserine liposomes (0.1-0.5 mM)

• Reaction temperature: 37°C (for host conditions) or 28°C (for vector conditions)

• Detection methods:

  • Direct measurement of phosphatidylethanolamine formation by TLC or HPLC

  • Release of CO₂ using radiolabeled substrates

  • Coupled enzyme assays measuring released serine

Activity is often temperature-dependent, mirroring B. quintana's adaptation to different host environments, with distinct activity profiles observed at 37°C versus 28°C .

How does B. quintana psd compare to psd enzymes from other bacterial species?

B. quintana psd shares structural homology with psd enzymes from other alpha-proteobacteria but has several distinctive features:

• Sequence identity: Approximately 60-70% with other Bartonella species (similar to the 59.7% sequence identity observed between B. quintana and B. henselae BafA proteins)

• Temperature sensitivity: More pronounced temperature-dependent activity shifts compared to other bacterial psds, reflecting B. quintana's adaptation to both human host (37°C) and body louse vector (28°C)

• Catalytic efficiency: Generally lower K​m values for phosphatidylserine compared to E. coli psd

• Autoprocessing: Similar self-cleavage mechanism but potentially different rates of conversion from proenzyme to active enzyme

These differences likely reflect B. quintana's unique lifecycle and membrane adaptation requirements in distinct host environments.

What mechanisms regulate the autoprocessing of B. quintana psd proenzyme into its active form?

B. quintana psd proenzyme undergoes self-catalyzed cleavage at a conserved (G/S)S(T/S)K motif, generating α and β subunits. This process is regulated by:

• Local pH conditions: Optimal autoprocessing occurs at pH 6.5-7.2

• Membrane association: Lipid composition influences cleavage rates

• Redox state: Oxidizing conditions typically inhibit processing

• Temperature-dependent conformational changes: Processing efficiency differs at 28°C versus 37°C, reflecting the bacterium's temperature-specific gene expression patterns

Experimental approaches to study this process include:

• Site-directed mutagenesis of the cleavage site residues

• Time-course analysis of processing using SDS-PAGE and western blotting

• Mass spectrometry to identify precise cleavage sites

• Fluorescence resonance energy transfer (FRET) assays with labeled proenzyme

These studies are particularly important given B. quintana's temperature-specific transcriptome deployment, which may extend to post-translational regulatory mechanisms .

How does B. quintana psd contribute to the pathogen's immune evasion strategies?

B. quintana psd likely plays an indirect but significant role in immune evasion through membrane phospholipid modification:

• Membrane composition affects LPS presentation: B. quintana LPS functions as a potent TLR4 antagonist, inhibiting proinflammatory cytokine production (TNF-α, IL-1β, IL-6)

• Phosphatidylethanolamine content modulates membrane fluidity and permeability, potentially affecting resistance to host antimicrobial peptides

• Altered membrane composition may influence outer membrane protein presentation, including the display of immunomodulatory proteins such as BafA homologs

Experimental approaches to investigate this relationship include:

• Comparing membrane phospholipid profiles between wild-type and psd-deficient mutants

• Assessing bacterial survival in serum bactericidal assays

• Measuring cytokine production in human monocytes exposed to bacteria with altered psd activity

• Evaluating TLR4 antagonism properties in psd-modified B. quintana

What are the technical challenges in crystallizing B. quintana psd for structural studies?

Crystallizing B. quintana psd presents several challenges:

• Proenzyme processing: Spontaneous cleavage during purification creates heterogeneous protein populations

• Membrane association: The enzyme's hydrophobic regions complicate crystallization

• Temperature sensitivity: Different conformational states at 28°C versus 37°C create structural heterogeneity

• Protein stability: The enzyme may show limited stability in typical crystallization buffers

Recommended approaches to overcome these challenges:

• Use of cleavage-site mutants to maintain proenzyme state

• Truncated constructs removing hydrophobic regions

• Fusion partners (T4 lysozyme, BRIL) to aid crystallization

• Co-crystallization with substrate analogs or inhibitors

• Lipidic cubic phase crystallization for membrane-associated regions

• Cryo-EM as an alternative approach for structural determination

How can recombinant B. quintana psd be utilized to study host-pathogen interactions?

Recombinant B. quintana psd enables several approaches to study host-pathogen interactions:

• Protein-protein interaction studies:

  • Pull-down assays to identify host cell binding partners

  • Surface plasmon resonance to quantify binding kinetics

  • Yeast two-hybrid screening with human cell libraries

• Immunological studies:

  • Assessment of psd's potential role in modulating host inflammatory responses

  • Investigation of whether psd-derived peptides trigger specific immune responses

  • Evaluation of whether antibodies against psd are present in patients with B. quintana infections

• Cellular localization:

  • Fluorescently tagged psd to track localization during infection

  • Immunohistochemistry of infected tissues using anti-psd antibodies

  • Detection of enzyme activity in different cellular compartments

• Metabolomic studies:

  • Analysis of phospholipid alterations in infected cells

  • Tracking labeled substrates to monitor psd activity during infection

  • Comparing metabolic profiles between infections with wild-type versus psd-modified bacteria

These approaches can help elucidate whether psd contributes to pathogenesis mechanisms similar to the BafA protein, which activates the VEGF pathway and contributes to angioproliferation in B. quintana infections .

How can I establish a reliable quantitative PCR-based detection system for B. quintana psd gene expression?

To establish a reliable qPCR system for B. quintana psd gene expression:

• Primer design considerations:

  • Target unique regions of the psd gene (avoid cross-reactivity with host or other bacterial psds)

  • Optimal amplicon size: 80-150 bp

  • Primer Tm around 60°C

  • GC content between 40-60%

• Reference gene selection:

  • Use multiple reference genes (rpoD, gyrB, and 16S rRNA)

  • Validate stability across experimental conditions, especially across temperature shifts

• Protocol optimization:

  • Standard curve generation using purified B. quintana genomic DNA

  • Determine PCR efficiency (should be between 90-110%)

  • Establish detection limits (typically 10-100 CFU/ml)

  • Verify specificity through melting curve analysis

• Data analysis:

  • Use the 2^(-ΔΔCt) method for relative quantification

  • Include appropriate controls (no template, no reverse transcriptase)

  • Consider temperature-specific expression patterns when comparing samples

This approach is inspired by the PCR-EIA method developed for B. henselae and B. quintana detection, which demonstrated high sensitivity (equivalent to 5 CFU in reaction mixture) and specificity .