Recombinant Bartonella quintana Holo-[acyl-carrier-protein] synthase (acpS)

What is the primary function of Holo-[acyl-carrier-protein] synthase (acpS) in Bartonella quintana?

AcpS in B. quintana catalyzes the essential post-translational modification of apo-acyl carrier protein (apo-ACP) to its holo form by transferring the 4'-phosphopantetheine moiety from coenzyme A (CoA) onto a conserved serine residue of apo-ACP . This reaction converts the inactive apo-ACP to the functionally active holo-ACP, which plays a central role in fatty acid biosynthesis by mediating the transfer of acyl intermediates via the thiol group of the 4'-phosphopantetheine . In B. quintana and other bacteria, this conversion is critical because holo-ACP serves as the central coenzyme in fatty acid and phospholipid biosynthesis, making AcpS an essential enzyme for bacterial survival and growth . The functional significance of AcpS is further demonstrated by studies showing that conditional acpS mutants accumulate apo-ACP under nonpermissive conditions, highlighting the enzyme's indispensable role in bacterial metabolism .

How does B. quintana AcpS compare structurally and functionally to AcpS from other bacterial species?

While specific structural data for B. quintana AcpS is limited in the provided search results, comparative analysis can be inferred from studies of related AcpS enzymes. Research has demonstrated that AcpS enzymes from different bacterial species, including gram-negative, gram-positive, and mycoplasma bacteria, share fundamental catalytic mechanisms but exhibit distinct biochemical properties . For example, AcpS enzymes from E. coli, M. pneumoniae, and S. pneumoniae have been purified and characterized with significant variations in substrate specificity and catalytic efficiency .

Homologs of AcpS have been identified across bacterial species, suggesting evolutionary conservation of this essential enzyme family . Based on the close phylogenetic relationship between B. quintana and B. henselae (both alphaproteobacteria in the Bartonellaceae family), we can infer that B. quintana AcpS likely shares structural similarities with B. henselae AcpS, which has been characterized as a recombinant protein . Functional conservation is expected in the core catalytic domain, while species-specific differences may exist in substrate recognition regions that define interaction specificity with their cognate apo-ACP targets.

What experimental approaches are recommended for initial expression and purification of recombinant B. quintana AcpS?

Based on established protocols for related bacterial AcpS proteins, a recommended experimental approach for B. quintana AcpS would include:

• Gene Cloning Strategy: PCR amplification of the acpS gene from B. quintana genomic DNA using primers designed based on the published genome sequence. The amplified gene should be cloned into an expression vector like pET-11a using appropriate restriction sites (NdeI and BamHI) as demonstrated for other bacterial acpS genes .

• Expression System: Transform the expression construct into E. coli BL21(DE3) pLysS or similar strains optimized for recombinant protein expression . Culture cells in LB medium supplemented with appropriate antibiotics at 35°C until mid-log phase (OD590 0.5-0.6), followed by induction with 1 mM IPTG for 3 hours .

• Cell Lysis and Initial Fractionation: Harvest cells by centrifugation, wash with phosphate-buffered saline, and disrupt using a French press in buffer containing 50 mM Tris-HCl (pH 7.0) and 100 mM KCl. Clear lysate by ultracentrifugation at approximately 160,000 × g for 40 minutes at 4°C .

• Chromatographic Purification: Apply the supernatant to a Source S cation exchange column equilibrated with the lysis buffer. Elute the protein using a linear KCl gradient (0.1-1.0 M) in the same buffer . Monitor fractions by SDS-PAGE (16% Tricine gels) to identify those containing AcpS.

• Storage Conditions: Adjust purified protein fractions to contain 1 mM DTT and 15% glycerol, then store in small aliquots at -80°C to prevent repeated freeze-thaw cycles .

This protocol has been effective for related AcpS enzymes and should serve as a starting point for B. quintana AcpS purification, with potential modifications based on protein-specific properties.

What methods can be used to assess the enzymatic activity of purified recombinant B. quintana AcpS?

Multiple complementary approaches can be employed to rigorously assess B. quintana AcpS enzymatic activity:

• ESMS Analysis: Electrospray mass spectrometry provides definitive evidence of AcpS-catalyzed phosphopantetheinylation by detecting the mass increase (~340 Da) corresponding to the addition of the 4'-phosphopantetheine moiety to apo-ACP . A standard reaction mixture should contain 50 mM Tris-HCl buffer (pH 7.0), 10 mM MgCl₂, 1 mM DTT, 300 μM CoA, 5 μM purified AcpS, and ~50-120 μM of substrate apo-ACP (either native B. quintana apo-ACP or a validated surrogate). Incubate at room temperature for 1 hour before ESMS analysis .

• HPLC-Based Assay: High-pressure liquid chromatography offers quantitative assessment of the conversion of apo-ACP to holo-ACP. Reaction mixtures (100 μl) should be injected onto a reverse-phase column (Vydac Selectapore 300M or equivalent) and separated using a 25-100% acetonitrile gradient containing 0.1% trifluoroacetic acid . The substrate (apo-ACP) and product (holo-ACP) can be detected by absorption at 220 nm, with activity quantified by comparing peak areas .

• Kinetic Parameter Determination: For comprehensive enzyme characterization, determine kinetic parameters (Km and kcat) by varying one substrate while keeping the other fixed. For CoA kinetics, maintain apo-ACP at 1 μM while varying CoA concentration (0.5-100 μM). For apo-ACP kinetics, maintain CoA at a saturating concentration (determined experimentally, typically 20-100 μM) while varying apo-ACP concentration .

• Gel-Shift Assay: Conformational differences between apo-ACP and holo-ACP can be visualized using non-denaturing PAGE, as the addition of the phosphopantetheine group often alters migration properties. This provides a simple qualitative assessment of enzymatic activity.

These methods provide complementary data on enzyme activity, substrate specificity, and reaction kinetics, offering a comprehensive characterization of recombinant B. quintana AcpS.

How can researchers establish an effective in vitro system to study interactions between B. quintana AcpS and potential inhibitors?

Establishing an effective in vitro system for inhibitor studies requires:

• High-Throughput Screening Assay Development: Modify the HPLC-based assay described above for a microplate format using fluorescent or colorimetric detection methods. This could involve developing a coupled enzymatic assay that monitors CoA release or using fluorescently labeled CoA analogs.

• Inhibitor Binding Studies:

  • Thermal Shift Assays: Use differential scanning fluorimetry to detect compound binding by monitoring changes in protein thermal stability upon inhibitor binding.

  • Isothermal Titration Calorimetry (ITC): Determine binding constants and thermodynamic parameters of inhibitor interactions.

  • Surface Plasmon Resonance (SPR): Measure real-time binding kinetics of potential inhibitors to immobilized AcpS.

• Structural Biology Approaches:

  • X-ray Crystallography: Determine the three-dimensional structure of B. quintana AcpS alone and in complex with inhibitors to identify binding sites and inform structure-based drug design.

  • Molecular Docking: Use computational methods to predict inhibitor binding modes and optimize lead compounds.

• Control Experiments:

  • Include E. coli AcpS as a reference enzyme since it has been well-characterized .

  • Test inhibitor specificity against human phosphopantetheinyl transferases to assess selectivity.

  • Validate hits with orthogonal assays to confirm mechanism of action.

• Data Analysis Framework:

  • Determine IC50 values for inhibitors using dose-response curves.

  • Calculate Ki values to understand inhibition mechanism (competitive, noncompetitive, uncompetitive).

  • Correlate inhibition constants with structural features of inhibitors to establish structure-activity relationships.

This systematic approach enables rigorous evaluation of potential inhibitors and provides a foundation for therapeutic development targeting B. quintana infections.

How does B. quintana AcpS substrate specificity compare with other bacterial AcpS enzymes?

While specific data on B. quintana AcpS substrate specificity is not directly available in the search results, comparative analysis based on related bacterial AcpS enzymes provides valuable insights:

• CoA Substrate Utilization:
  Bacterial AcpS enzymes typically show high specificity for CoA as the phosphopantetheine donor. Kinetic studies with purified AcpS enzymes from E. coli, M. pneumoniae, and S. pneumoniae have demonstrated varying Km values for CoA, indicating species-specific differences in CoA binding affinity . B. quintana AcpS would likely exhibit similar specificity for CoA, potentially with unique kinetic parameters reflecting its evolutionary adaptation.

• apo-ACP Recognition:
  AcpS enzymes demonstrate remarkable specificity for their cognate apo-ACP substrates. Cross-reactivity studies have shown that while some AcpS enzymes can recognize heterologous apo-ACPs, the catalytic efficiency is often significantly reduced . This substrate discrimination occurs through specific protein-protein interactions that position the conserved serine residue of apo-ACP correctly in the AcpS active site.

• Species-Specific Variations:
  The biochemical characterization of AcpS enzymes from diverse bacterial species has revealed significant differences in their kinetic parameters with respect to both CoA and apo-ACP substrates . These variations reflect evolutionary adaptations to the specific metabolic requirements and environmental niches of different bacteria. Given B. quintana's adaptation to human hosts and its intracellular lifestyle, its AcpS may have evolved distinct substrate recognition properties optimized for its unique ecological niche.

• Potential for Engineered Specificity:
  Understanding the molecular basis of substrate specificity could enable the engineering of B. quintana AcpS variants with altered or expanded substrate recognition, potentially useful for biochemical applications or pathway engineering.

To experimentally determine B. quintana AcpS substrate specificity, researchers should systematically test the enzyme with various apo-ACP substrates from related and divergent bacterial species, as well as with CoA analogs to establish a comprehensive substrate recognition profile.

What are the critical residues in the catalytic site of B. quintana AcpS and how do they contribute to enzyme function?

While the specific catalytic residues of B. quintana AcpS have not been directly identified in the provided search results, we can infer likely critical residues based on published structures and mutagenesis studies of homologous AcpS enzymes:

• Active Site Architecture:
  Bacterial AcpS enzymes typically function as trimers with three active sites formed at the interface between adjacent subunits. Each active site contains:

  • A magnesium binding site for coordination of the phosphate groups of CoA

  • Positively charged residues that interact with the phosphate groups of CoA

  • Hydrophobic residues that position the pantetheine moiety

  • Residues that recognize and position the conserved serine of apo-ACP

• Key Catalytic Residues:
  Based on E. coli AcpS studies, critical catalytic residues likely include:

  • Conserved acidic residues (Asp or Glu) that coordinate the essential Mg²⁺ cofactor

  • Basic residues (Arg or Lys) that stabilize the developing negative charge during phosphoryl transfer

  • Hydrophobic residues forming a binding pocket for the pantetheine moiety

• Functional Impact of Mutations:
  The E. coli MP4 strain with the G4D mutation in AcpS (AcpS1) exhibited approximately 5-fold reduction in catalytic efficiency compared to wild-type AcpS, demonstrating that even subtle changes in the protein sequence can significantly impact enzyme function . This suggests that the N-terminal region of AcpS plays an important role in catalysis or substrate binding.

• Structure-Function Relationships:
  To definitively identify critical residues in B. quintana AcpS, researchers should:

  • Generate a homology model based on crystallized AcpS structures

  • Perform site-directed mutagenesis of conserved residues

  • Assess the impact of mutations on kinetic parameters (kcat and Km)

  • Use computational approaches such as molecular dynamics simulations to predict the effects of mutations on enzyme function

Understanding these critical residues would facilitate the rational design of selective inhibitors targeting B. quintana AcpS while minimizing off-target effects on human phosphopantetheinyl transferases.

What are the primary technical challenges in expressing and purifying functional B. quintana AcpS, and how can they be addressed?

Researchers working with B. quintana AcpS may encounter several technical challenges:

• Low Solubility and Aggregation:

  • Challenge: Bacterial proteins often form inclusion bodies when overexpressed in E. coli.

  • Solutions:
    a) Optimize expression conditions by reducing induction temperature (16-25°C), lowering IPTG concentration (0.1-0.5 mM), and using slower growth media.
    b) Express protein with solubility-enhancing tags (MBP, SUMO, TrxA) that can be later removed by specific proteases.
    c) Co-express with bacterial chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) to assist proper folding.
    d) If inclusion bodies persist, develop a refolding protocol using stepwise dialysis from denaturing conditions.

• Instability of Purified Enzyme:

  • Challenge: Purified AcpS may exhibit limited stability or lose activity during storage.

  • Solutions:
    a) Include stabilizing additives in purification buffers (glycerol 10-15%, reducing agents like DTT or TCEP).
    b) Identify optimal pH and ionic strength conditions through stability screening.
    c) Add protease inhibitors during purification to prevent degradation.
    d) Store enzyme in small aliquots at -80°C to avoid repeated freeze-thaw cycles .

• Co-purification of Endogenous E. coli AcpS:

  • Challenge: Contamination with host E. coli AcpS can confound activity assays.

  • Solutions:
    a) Use histidine-tagged constructs with stringent IMAC purification conditions.
    b) Employ additional purification steps (ion exchange, size exclusion chromatography).
    c) Verify purity by mass spectrometry analysis.
    d) Express in E. coli strains with reduced or conditional expression of endogenous acpS.

• Production of Cognate B. quintana apo-ACP Substrate:

  • Challenge: Authentic activity assessment requires the native substrate.

  • Solutions:
    a) Clone and express B. quintana ACP using similar strategies to those outlined for AcpS.
    b) Ensure apo-form production by expressing in E. coli in the presence of elevated pantothenic acid (25 mM) to inhibit conversion to holo-form .
    c) Purify apo-ACP using anion exchange chromatography (Source Q) .
    d) Confirm apo-state by mass spectrometry before use in activity assays.

These methodological approaches provide a systematic framework for addressing common challenges in recombinant B. quintana AcpS production, enabling researchers to obtain sufficient quantities of active enzyme for structural and functional studies.

How can researchers troubleshoot activity loss during purification and storage of B. quintana AcpS?

Activity loss is a common challenge when working with enzymes like B. quintana AcpS. A systematic troubleshooting approach includes:

• Diagnostic Testing Protocol:

  • Establish a baseline activity measurement immediately after cell lysis.

  • Track activity after each purification step to identify where losses occur.

  • Perform parallel purifications with variations in buffer conditions to identify optimal parameters.

  • Use E. coli AcpS as a positive control, as it has established purification protocols and known stability characteristics .

• Buffer Optimization Matrix:

  Parameter	Test Range	Monitoring Method
  pH	6.5-8.5 in 0.5 increments	Activity assay, thermal shift
  Salt concentration	50-500 mM KCl or NaCl	Activity assay, aggregation monitoring
  Reducing agents	0.5-5 mM DTT, TCEP, or β-ME	Activity assay, oxidation status
  Divalent cations	1-10 mM MgCl₂, MnCl₂	Activity assay, thermal shift
  Stabilizing additives	5-20% glycerol, 0.1-1% CHAPS	Activity assay, aggregation monitoring
  Metal chelators	0.1-1 mM EDTA	Activity assay with Mg²⁺ supplementation

• Storage Condition Optimization:

  • Compare activity retention at different storage temperatures (-80°C, -20°C, 4°C).

  • Evaluate freeze-thaw stability by subjecting aliquots to multiple freeze-thaw cycles.

  • Test lyophilization with cryoprotectants as an alternative storage method.

  • Compare storage of the enzyme at different concentrations (dilute vs. concentrated).

• Structural Integrity Assessment:

  • Monitor protein folding using circular dichroism spectroscopy.

  • Assess quaternary structure using size exclusion chromatography or analytical ultracentrifugation.

  • Evaluate thermal stability using differential scanning fluorimetry.

  • Check for proteolysis using SDS-PAGE and mass spectrometry.

• Recovery Strategies for Inactive Enzyme:

  • Attempt refolding by dialysis against optimal buffer conditions.

  • Try adding fresh cofactors (Mg²⁺) and reducing agents.

  • Explore chemical chaperones (arginine, proline) to promote proper folding.

  • Consider fusion to stability-enhancing protein tags.

By systematically applying these approaches, researchers can identify and address the specific factors contributing to activity loss, significantly improving the yield of functional B. quintana AcpS for downstream applications.

What is the relationship between AcpS function and B. quintana pathogenesis?

The relationship between AcpS function and B. quintana pathogenesis is multifaceted and involves several interconnected pathways:

• Essential Role in Bacterial Metabolism:
  AcpS catalyzes the conversion of apo-ACP to holo-ACP, a critical step for fatty acid biosynthesis . In bacterial pathogens, fatty acid biosynthesis is essential for:

  • Membrane phospholipid production required for bacterial growth and division

  • Lipopolysaccharide (LPS) biosynthesis in gram-negative bacteria like B. quintana

  • Production of lipid-based virulence factors

  The essential nature of AcpS is demonstrated by studies showing that conditional acpS mutants accumulate non-functional apo-ACP under nonpermissive conditions, highlighting that AcpS function is indispensable for bacterial viability .

• Connection to Virulence Factor Expression:
  While not directly mentioned for B. quintana in the search results, research on the related species B. henselae reveals important insights about pathogenesis mechanisms likely shared with B. quintana. B. quintana produces variably expressed outer membrane proteins (Vomps) that mediate host cell adhesion and invasion . The production of these and other membrane-associated virulence factors depends on proper fatty acid biosynthesis, which ultimately relies on functional AcpS.

• Role in Host Cell Interactions:
  B. quintana interacts with various host cell types, including endothelial cells, where it replicates within a Bartonella-containing vacuole . The bacterium also adheres to endothelial and epithelial cells and potentially interacts with human erythroblast cells . These host-pathogen interactions require intact bacterial membranes and surface structures that depend on fatty acid biosynthesis pathways facilitated by AcpS activity.

• Impact on Vascular Endothelial Growth Factor Secretion:
  B. quintana infections are associated with vasculoproliferative disorders, and research has shown that Vomp expression is crucial for the induction of VEGF secretion from host cells . The expression of these virulence factors is likely dependent on functional fatty acid biosynthesis pathways supported by AcpS activity.

• Therapeutic Target Potential:
  Due to its essential role in bacterial metabolism and absence of direct functional homologs in humans, AcpS represents an attractive target for therapeutic intervention against B. quintana infections . Inhibiting AcpS would disrupt fatty acid biosynthesis, impair membrane integrity, and potentially attenuate virulence factor expression.

Understanding these connections between AcpS function and B. quintana pathogenesis provides a foundation for developing novel therapeutic strategies against diseases caused by this pathogen, including trench fever, endocarditis, and vasculoproliferative disorders.

What approaches can be used to develop selective inhibitors of B. quintana AcpS as potential therapeutic agents?

Developing selective inhibitors of B. quintana AcpS requires a multidisciplinary approach combining structural biology, medicinal chemistry, and biochemical assays:

• Structure-Based Drug Design Strategy:

  • Generate a high-resolution structure of B. quintana AcpS through X-ray crystallography or cryo-EM.

  • Alternatively, create a homology model based on related bacterial AcpS structures.

  • Identify unique structural features in the active site that differ from human phosphopantetheinyl transferases.

  • Use computational docking to screen virtual compound libraries against the AcpS structure.

  • Prioritize compounds that interact with catalytic residues or that would disrupt protein-protein interactions with apo-ACP.

• Rational Inhibitor Design Approaches:

  • Develop CoA analogues that compete with the natural substrate but lack the reactive phosphopantetheine group.

  • Design transition state mimics that resemble the phosphopantetheine transfer reaction intermediate.

  • Create peptidomimetics that interfere with the AcpS-apo-ACP protein interaction surface.

  • Explore allosteric inhibitors that bind outside the active site but induce conformational changes that impair catalysis.

• High-Throughput Screening (HTS) Protocol:

  • Develop a robust, miniaturized assay suitable for screening compound libraries.

  • Primary screen options include:
    a) Fluorescence-based assays monitoring CoA consumption or holo-ACP formation
    b) FRET-based assays measuring AcpS-apo-ACP interaction
    c) Thermal shift assays to identify compounds that bind to AcpS

  • Confirm hits with orthogonal assays including the HPLC-based activity assay described earlier .

• Selectivity Profiling Framework:

  • Test lead compounds against human phosphopantetheinyl transferases to ensure selectivity.

  • Evaluate activity against AcpS enzymes from beneficial gut microbiota to minimize disruption of the microbiome.

  • Assess activity against a panel of other essential bacterial enzymes to identify potential off-target effects.

  • Monitor physiochemical properties (solubility, permeability, stability) to ensure drug-like characteristics.

• Optimization Pipeline for Lead Compounds:

  Stage	Criteria	Methods
  Initial hits	IC50 < 10 μM against B. quintana AcpS	Biochemical assays
  Lead compounds	IC50 < 1 μM, selectivity > 100-fold vs. human enzymes	Enzyme panel testing
  Optimized leads	Cellular activity (MIC < 1 μg/mL), low cytotoxicity	Bacterial and mammalian cell culture
  Preclinical candidates	In vivo efficacy, favorable PK/PD properties	Animal models of infection

This systematic approach provides a roadmap for the development of selective B. quintana AcpS inhibitors with therapeutic potential against infections caused by this challenging pathogen.