Recombinant Bartonella quintana Lipoprotein-releasing system ATP-binding protein LolD (lolD)

What is the function of LolD in Bartonella quintana's lipoprotein transport system?

LolD functions as the ATP-binding protein component of the LolCDE complex, an ABC transporter located in the inner membrane of B. quintana. The complete Lol pathway comprises:

• LolCDE complex (inner membrane)

• LolA (periplasmic chaperone)

• LolB (outer membrane receptor)

The LolCDE complex initiates lipoprotein transport by recognizing triacylated lipoproteins and transferring them to LolA. LolD specifically provides the energy for this process through ATP hydrolysis, which is essential for releasing lipoproteins from the inner membrane .

When a lipoprotein interacts with LolE, it causes allosteric modifications that increase LolD's affinity for ATP. As LolC attaches to LolA and ATP binds to LolD, the lipoprotein's interaction with LolE weakens, resulting in the lipoprotein's relocation to LolA upon ATP hydrolysis. The lipoprotein-LolA complex then attaches to LolB for final localization to the outer membrane .

What expression systems are commonly used for recombinant B. quintana LolD production?

Recombinant B. quintana LolD is typically produced using one of the following expression systems:

• E. coli expression system

• Yeast expression system

• Baculovirus expression system

• Mammalian cell expression system

Each system offers distinct advantages depending on research requirements. For functional studies requiring post-translational modifications, yeast or mammalian expression systems are often preferred. For structural studies requiring high protein yields, E. coli systems are commonly used .

Standard purification procedures yield recombinant LolD with greater than 85% purity as determined by SDS-PAGE analysis. Proper storage conditions include:

• Liquid form: 6 months at -20°C/-80°C

• Lyophilized form: 12 months at -20°C/-80°C

Researchers should avoid repeated freeze-thaw cycles and store working aliquots at 4°C for up to one week .

How does B. quintana LolD compare to LolD proteins in other bacterial species?

Comparative analysis shows conservation of LolD across various gram-negative bacteria, with functional homology but sequence variations. The table below summarizes key features of LolD proteins from different bacterial species:

These comparisons highlight evolutionary conservation of the lipoprotein transport mechanism across gram-negative bacteria while revealing species-specific adaptations .

What methodologies are most effective for studying LolD-dependent lipoprotein transport in B. quintana?

To effectively study LolD-dependent lipoprotein transport in B. quintana, researchers should implement a multi-faceted approach:

In vitro reconstitution assays:

• Purify recombinant LolCDE complex components (including LolD) and lipoprotein substrates

• Reconstitute into proteoliposomes

• Measure ATP hydrolysis rates using colorimetric phosphate release assays

• Assess lipoprotein transfer efficiency using fluorescently labeled lipoproteins

Genetic manipulation approaches:

• Generate conditional LolD mutants (complete knockouts are typically lethal)

• Use CRISPR-Cas9 or transposon mutagenesis to introduce point mutations in Walker A/B motifs

• Complement with wild-type or mutant LolD variants

Structural analyses:

• X-ray crystallography or cryo-EM of LolD alone and in complex with LolC/E

• Focus on ATP-binding states to understand conformational changes during transport cycle

Proteomic profiling:

• Identify lipoproteins affected by LolD dysfunction using comparative proteomics

• Analyze outer membrane fractions from wild-type vs. LolD-depleted conditions

The combination of these approaches provides comprehensive insights into LolD function in the context of B. quintana's unique pathogenic lifestyle .

How does ATP hydrolysis by LolD coordinate with the broader Lol pathway in B. quintana?

ATP hydrolysis by LolD serves as the energetic driver for lipoprotein release from the inner membrane through a coordinated mechanism:

Researchers investigating this coordination should employ ATPase assays with varying lipoprotein substrates and environmental conditions mimicking B. quintana's natural niches.

What strategies can resolve challenges in purifying functionally active recombinant B. quintana LolD?

Obtaining functionally active recombinant B. quintana LolD presents several challenges that can be addressed using the following strategies:

Challenge 1: Protein misfolding and inclusion body formation

• Solution: Use fusion tags that enhance solubility (MBP, SUMO, or thioredoxin)

• Lower expression temperature (16-20°C) to slow folding and reduce aggregation

• Add solubility enhancers to culture media (sorbitol, glycerol, or arginine)

• Consider cell-free expression systems for difficult-to-express constructs

Challenge 2: Maintaining nucleotide-binding capability

• Solution: Include low concentrations of ATP or non-hydrolyzable ATP analogs during purification

• Avoid metal chelators that might strip essential Mg²⁺ ions

• Use size exclusion chromatography in the final purification step to ensure proper oligomeric state

Challenge 3: Ensuring proper complex formation with LolC/E for functional studies

• Solution: Co-express LolC, LolD, and LolE using polycistronic vectors

• Apply mild detergents for membrane protein extraction (n-dodecyl-β-D-maltoside or digitonin)

• Use affinity tags on only one component to pull down the intact complex

Challenge 4: Assessing functional activity

• Solution: Establish ATPase activity assays using colorimetric phosphate detection

• Develop liposome-based transport assays with fluorescently labeled lipoproteins

• Use surface plasmon resonance to measure ATP and lipoprotein binding kinetics

These approaches have been successfully applied to other bacterial ABC transporters and can be adapted for B. quintana LolD, taking into account its specific biochemical properties .

How might LolD function differ between B. quintana's transmission cycle between human host and louse vector?

B. quintana cycles between two distinct environments—the human bloodstream (37°C) and the body louse gut (28°C)—which likely impacts LolD function in several ways:

Temperature-dependent changes:

• LolD's ATPase activity may show different kinetics at 28°C versus 37°C

• ATP binding affinity could be modulated by temperature shifts

• Conformational flexibility of the protein may differ between environments

Adaptation to nutritional environments:

• In the hemin-restricted human bloodstream, LolD may prioritize transport of iron acquisition lipoproteins

• In the hemin-rich louse gut, LolD may focus on transporting stress-response lipoproteins that protect against hemin toxicity

Interaction with host/vector factors:

• Human serum components may modulate LolD activity

• Louse gut enzymes or antimicrobial peptides might interact with LolD or its substrates

Experimental approach to study these differences:

• Compare LolD expression levels at 28°C versus 37°C using RT-qPCR and Western blotting

• Measure ATPase activity of purified LolD at both temperatures

• Identify temperature-dependent lipoprotein substrates using comparative proteomics

• Assess LolD inhibition sensitivity under host versus vector conditions

This research would provide valuable insights into how B. quintana adapts its lipoprotein transport machinery during transmission .

What role might LolD play in B. quintana's resistance to host immune responses?

LolD's role in immune evasion stems from its function in transporting virulence-associated lipoproteins to the bacterial surface:

Contribution to membrane integrity:
LolD ensures proper lipoprotein localization, maintaining membrane integrity against host antimicrobial peptides and complement. Lipoproteins transported by the Lol system may serve as structural components that shield underlying pathogen-associated molecular patterns (PAMPs) from recognition by host pattern recognition receptors.

Transport of immune modulatory lipoproteins:
Several B. quintana lipoproteins likely interfere with TLR signaling pathways. For example, B. quintana lipopolysaccharide (LPS) functions as a potent TLR4 antagonist, inhibiting both mRNA transcription and release of TNF-α, IL-1β, and IL-6 in human monocytes . The transport of these immunomodulatory molecules to the bacterial surface depends on a functioning Lol system.

Interface with VirB/VirD4 Type IV Secretion System:
Recent research on Bartonella effector proteins (Beps) demonstrates their importance in immune evasion. While LolD doesn't directly participate in type IV secretion, proper membrane organization facilitated by the Lol system is likely necessary for assembly and function of the VirB/VirD4 complex .

Data from immune stimulation experiments:
B. quintana LPS completely inhibited E. coli LPS-induced TNF-α production at ratios of at least 10:1 and inhibited cytokine induction approximately 50% at a 1:1 ratio . This immunomodulatory capability depends on proper surface presentation of these molecules, a process requiring functional LolD.

How can researchers effectively design inhibitors targeting B. quintana LolD for potential therapeutic development?

Designing effective LolD inhibitors requires a systematic approach targeting unique features of the B. quintana protein:

Structure-based design strategies:

• Target the ATP-binding pocket with nucleotide analogs modified to enhance specificity

• Identify allosteric sites unique to bacterial LolD versus human ABC transporters

• Design peptides that mimic the LolC-LolD interface to prevent complex formation

• Develop compounds that lock LolD in non-productive conformational states

Key structural features to target:

• Walker A and B motifs responsible for ATP binding and hydrolysis

• Interface regions between LolD and other Lol components

• Potential B. quintana-specific sequence regions identified through comparative analysis

Screening methodologies:

• High-throughput ATPase inhibition assays using purified recombinant LolD

• Bacterial cell-based screening using reporter systems linked to outer membrane lipoprotein localization

• Fragment-based screening to identify chemical scaffolds with affinity for LolD

• In silico screening leveraging homology models based on related ABC transporter structures

Validation approaches:

• Assess impact on lipoprotein localization using subcellular fractionation

• Measure growth inhibition specifically under conditions requiring outer membrane lipoproteins

• Evaluate effects on B. quintana survival in macrophage infection models

• Test for synergy with existing antibiotics that target cell envelope functions

This inhibitor development strategy takes advantage of the essential nature of LolD while acknowledging the challenges of targeting an intracellular bacterial protein with drug-like molecules .

What are the optimal conditions for assessing B. quintana LolD ATPase activity in vitro?

Establishing optimal conditions for B. quintana LolD ATPase activity measurement requires careful consideration of multiple parameters:

Buffer composition:

• HEPES or Tris buffer (50 mM, pH 7.5)

• MgCl₂ (5-10 mM) as a cofactor for ATP hydrolysis

• KCl or NaCl (100-150 mM) for ionic strength

• Glycerol (5-10%) for protein stability

• Reducing agent (1-2 mM DTT or 0.5-1 mM TCEP)

Substrate conditions:

• ATP concentration: 0.1-5 mM (generate Michaelis-Menten curves)

• Consider including lipid bilayers or nanodiscs with embedded LolC/E

• Test requirement for lipoproteins as stimulators of activity

Reaction conditions:

• Temperature: Test both 28°C (louse vector) and 37°C (human host)

• Time points: 0, 5, 10, 15, 30, 60 minutes

• Protein concentration: 0.1-1 μM purified LolD

Detection methods:

• Malachite green assay for inorganic phosphate detection

• Coupled enzyme assay (pyruvate kinase/lactate dehydrogenase) for continuous monitoring

• Radioactive [γ-³²P]ATP for highest sensitivity

• NADH-coupled assay for high-throughput applications

Controls:

• Include negative controls: heat-inactivated LolD, ATPase inhibitors

• Positive controls: well-characterized ABC transporter ATPase domains

• Walker A/B mutants of LolD to establish background activity levels

Researchers should optimize these conditions specifically for B. quintana LolD, as parameters established for E. coli or other bacterial LolD proteins may not be directly transferable .

What methods enable accurate detection of lipoprotein mislocalization in B. quintana with compromised LolD function?

Detecting lipoprotein mislocalization in B. quintana with compromised LolD function requires sensitive and specific techniques:

Subcellular fractionation approaches:

• Differential ultracentrifugation to separate inner and outer membranes

• Sucrose density gradient centrifugation for higher resolution separation

• Triton X-114 phase partitioning to isolate amphipathic membrane proteins

  • This approach was successfully used with B. quintana to identify membrane-associated hemin-binding proteins

Immunological detection methods:

• Western blotting of fractionated samples using antibodies against known lipoproteins

• Immunofluorescence microscopy to visualize lipoprotein localization in situ

• Immunoelectron microscopy for highest resolution localization

  • This technique confirmed surface exposure of B. quintana HbpA

Proteomics approaches:

• Comparative proteomic analysis of membrane fractions from wild-type vs. LolD-compromised B. quintana

• Enrichment of lipoproteins using palmitate labeling or biotinylation

• Quantitative mass spectrometry to assess changes in lipoprotein distribution

Reporter systems:

• Fusion of lipoproteins to fluorescent proteins or enzymatic reporters

• β-lactamase fusions to measure surface accessibility

• Split-GFP complementation to assess compartment-specific localization

Validation controls:

• Use known inner and outer membrane markers to confirm fractionation quality

• Include temperature-sensitive LolD mutants as reference standards

• Establish profiles for specific inhibitors of different lipoprotein transport steps

These approaches provide complementary information, and combining multiple methods yields the most comprehensive analysis of lipoprotein mislocalization resulting from LolD dysfunction .

How can researchers differentiate between direct and indirect effects of LolD disruption on B. quintana pathogenesis?

Differentiating direct from indirect effects of LolD disruption requires sophisticated experimental designs:

Genetic complementation strategies:

• Generate conditional LolD depletion strains of B. quintana

• Rescue with plasmid-encoded wild-type LolD to identify reversible phenotypes

• Perform point-specific mutations in ATP-binding motifs to identify activity-dependent effects

• Create chimeric LolD proteins with domains from related species to map functional regions

Temporal analysis approaches:

• Use time-course experiments following LolD depletion to separate immediate from delayed effects

• Analyze transcriptome changes at multiple time points to identify compensatory responses

• Measure membrane integrity parameters to distinguish structural from signaling phenotypes

• Monitor bacterial viability to separate bactericidal from virulence-attenuating effects

Biochemical bypass experiments:

• Identify lipoproteins dependent on LolD for localization using proteomics

• Express these proteins with alternative localization signals to bypass the Lol system

• Assess whether pathogenesis is restored when specific lipoproteins are correctly localized

• Use these results to distinguish essential structural roles from virulence factor roles

In vivo infection models with specific readouts:

• Macrophage infection assays measuring distinct parameters:

  • Bacterial adhesion and invasion (early events)

  • Intracellular survival (intermediate events)

  • Host cell cytokine production (response events)

• Animal models with tissue-specific analyses

Transcriptomic/proteomic correlation:
Compare datasets from:

• LolD-depleted bacteria

• Bacteria treated with envelope stress-inducing antibiotics

• Bacteria exposed to host immune factors

Overlapping and distinct gene/protein expression patterns will help distinguish direct LolD-dependent effects from general stress responses .

What experimental design best evaluates the interaction between B. quintana LolD and potential inhibitory compounds?

A comprehensive experimental design for evaluating LolD-inhibitor interactions should proceed through the following stages:

Stage 1: Initial Compound Screening

• Primary biochemical assay: ATPase activity inhibition using purified LolD

  • ATP concentration: At Km value (typically 0.1-0.5 mM)

  • Protein concentration: 0.1-0.5 μM

  • Compound concentration range: 0.1-100 μM

  • Controls: Known ATPase inhibitors (orthovanadate, AMPPNP)

  • Readout: IC50 determination

• Counter-screening:

  • Test against human ATP-utilizing enzymes to assess selectivity

  • Screen against related bacterial ABC transporters to determine specificity

Stage 2: Mechanism of Action Studies

• Binding affinity determination:

  • Surface plasmon resonance (SPR)

  • Isothermal titration calorimetry (ITC)

  • Microscale thermophoresis (MST)

• Binding site identification:

  • Hydrogen-deuterium exchange mass spectrometry

  • Photoaffinity labeling with compound analogs

  • Competition assays with ATP and known binding site probes

• Mode of inhibition analysis:

  • Enzyme kinetics varying both inhibitor and ATP concentrations

  • Determine competitive, non-competitive, or mixed inhibition patterns

Stage 3: Functional Impact Assessment

• Lipoprotein transport assays:

  • In vitro reconstituted systems with purified components

  • Permeabilized cell assays measuring lipoprotein transfer

• Cellular activity evaluation:

  • Growth inhibition in B. quintana cultures

  • Synergy testing with antibiotics targeting cell envelope

• Membrane integrity studies:

  • Outer membrane permeability assays

  • Lipoprotein localization analysis by fractionation and immunoblotting

Stage 4: Structure-Activity Relationship Development

• Compound optimization:

  • Synthesize analogs based on binding mode predictions

  • Test derivatives for improved potency and selectivity

  • Assess physicochemical properties relevant for antimicrobial activity

This multi-stage approach ensures comprehensive characterization of potential LolD inhibitors, from initial discovery through mechanistic understanding and optimization .

How do functional differences between B. quintana and B. henselae LolD proteins correlate with their host adaptation strategies?

B. quintana and B. henselae LolD proteins show interesting functional differences that align with their distinct host adaptation strategies:

Genomic context differences:
B. quintana has undergone genome reduction compared to B. henselae (1.58 Mb vs. 1.93 Mb) , suggesting streamlining of various systems, potentially including the Lol pathway. This reduction correlates with B. quintana's more specialized human-louse transmission cycle compared to B. henselae's broader host range.

Protein sequence variations:
While both proteins maintain core ATP-binding motifs, differences in peripheral regions may influence:

• Interaction specificity with other Lol components

• Substrate recognition profiles

• Regulatory mechanisms governing activity

Expression pattern differences:
In B. quintana, LolD expression may be more tightly regulated to correspond with its transition between human host (37°C) and louse vector (28°C) . In contrast, B. henselae may maintain more consistent expression across various conditions to accommodate its broader host range.

Substrate specificity differences:
The repertoire of lipoproteins transported by LolD likely differs between species:

• B. quintana may prioritize transport of lipoproteins involved in hemin management, given its extraordinarily high hemin requirement (the highest reported for any bacterium)

• B. henselae may transport a broader range of lipoproteins involved in cat-human host adaptation

Experimental approaches to investigate these differences:

• Generate chimeric LolD proteins exchanging domains between B. quintana and B. henselae

• Compare ATPase activity profiles across temperature ranges mimicking distinct host environments

• Identify species-specific lipoprotein substrates using comparative proteomics

• Assess cross-complementation capability using genetic rescue experiments

These investigations would provide valuable insights into how adaptations in the Lol system contribute to the distinct ecological niches occupied by these related Bartonella species .

What insights can structural comparisons between B. quintana LolD and other bacterial ATP-binding cassette (ABC) transporters provide?

Structural comparisons between B. quintana LolD and other bacterial ABC transporters can yield valuable insights into its function and potential targeting strategies:

Conserved structural elements:

• Nucleotide-binding domain (NBD) architecture:

  • Walker A motif (P-loop): Forms interactions with ATP phosphate groups

  • Walker B motif: Coordinates Mg²⁺ ion for ATP hydrolysis

  • Signature motif (C-loop): Completes ATP binding site in the functional dimer

  • Q-loop: Senses ATP binding/hydrolysis and communicates with transmembrane domains

• Dimerization interface:

  • ABC transporters function as dimers with two ATP binding sites

  • Interface residues determine specificity of homodimer vs. heterodimer formation

Species-specific structural adaptations:
B. quintana LolD likely exhibits unique features compared to other bacterial ABC transporters:

• Surface residues involved in LolC/E interaction

• Conformational flexibility adapted to B. quintana's environmental transitions

• Potential regulatory sites responsive to host/vector conditions

Structural implications for function:

• Nucleotide-binding pocket variants:

  • Subtle amino acid differences can alter ATP binding affinity and hydrolysis rates

  • May explain differences in transport efficiency between bacterial species

• Power transmission mechanism:

  • Structural changes upon ATP binding/hydrolysis are transmitted to LolC/E

  • Species-specific variations may optimize lipoprotein release kinetics

Structural biology approaches:

• Homology modeling based on solved structures of related ABC transporters

• Cryo-EM analysis of the entire LolCDE complex from B. quintana

• Hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

• Small-angle X-ray scattering (SAXS) to determine solution structure

These comparative analyses can identify unique structural features of B. quintana LolD that may be exploited for specific inhibitor design while providing fundamental insights into ABC transporter evolution in pathogenic bacteria .

How does the role of LolD in B. quintana virulence compare with other essential membrane biogenesis pathways?

LolD's contribution to B. quintana virulence can be compared with other membrane biogenesis pathways to understand their relative importance:

LolD vs. Bam (β-barrel assembly machinery) complex:

• Similarity: Both are essential for outer membrane protein localization

• Difference: Lol system transports lipoproteins, while Bam inserts β-barrel proteins

• Virulence impact: Bam mutations typically cause more severe membrane defects than Lol mutations

LolD vs. Lpt (lipopolysaccharide transport) system:

• Similarity: Both transport major outer membrane components

• Difference: Lpt transports LPS, which in B. quintana acts as a TLR4 antagonist

• Virulence impact: Given B. quintana LPS's role in immune evasion, Lpt defects may more directly affect immune interactions

LolD vs. Tat (twin-arginine translocation) pathway:

• Similarity: Both translocate fully-folded proteins

• Difference: Tat transports proteins across the inner membrane while Lol relocates lipoproteins from inner to outer membrane

• Virulence impact: Tat substrates include many virulence factors, but fewer essential proteins

LolD vs. VirB/VirD4 type IV secretion system:

• Similarity: Both are involved in protein transport

• Difference: VirB/VirD4 secretes effector proteins directly into host cells

• Virulence impact: VirB/VirD4 is specifically dedicated to virulence while Lol has broader physiological roles

Comparative susceptibility to inhibition:
The table below summarizes the comparative vulnerability of these systems:

This comparison highlights LolD as an attractive target—essential for bacterial viability yet with fewer existing inhibitors compared to other systems .

What genetic and experimental techniques can be adapted from other bacterial systems to study B. quintana LolD?

Several genetic and experimental techniques can be adapted from other bacterial systems to study B. quintana LolD:

Genetic manipulation approaches:

• Conditional expression systems:

  • Tetracycline-regulated promoters (successfully used in Bartonella)

  • Riboswitches for fine-tuned gene expression control

  • Degron tags for inducible protein degradation

• CRISPR-Cas9 genome editing:

  • Recently adapted for Bartonella species

  • Allows precise mutations in chromosomal lolD

  • Can create scarless mutations in ATP-binding motifs

• Transposon mutagenesis screens:

  • Identify genetic interactions with lolD

  • Map suppressors of lolD defects

  • Discover synthetic lethal interactions

Biochemical and imaging techniques:

• Reconstituted liposome systems:

  • In vitro reconstitution of LolCDE complex in proteoliposomes

  • Fluorescence-based transport assays using labeled lipoproteins

  • Adapted from E. coli Lol studies but optimized for B. quintana components

• Super-resolution microscopy:

  • Track lipoprotein localization in live bacteria

  • Visualize LolD-dependent transport events

  • Study co-localization with other membrane biogenesis machineries

• Chemical genetics approaches:

  • Small molecule screening for LolD inhibitors

  • Photocrosslinking to identify binding partners

  • Activity-based protein profiling to assess functional state

Adaptation considerations for B. quintana:

These adapted techniques would provide comprehensive insights into LolD function while addressing the specific challenges posed by B. quintana's fastidious nature and unique lifecycle .

How might understanding B. quintana LolD function contribute to improved diagnostic approaches for Bartonella infections?

Understanding B. quintana LolD function can enhance diagnostic approaches for Bartonella infections in several ways:

LolD-dependent surface lipoprotein biomarkers:
The Lol system transports lipoproteins to the bacterial surface, where they become accessible targets for diagnostic assays. Identifying LolD-dependent surface lipoproteins unique to B. quintana can lead to more specific diagnostic tests. For example, hemin-binding proteins transported by the Lol system represent promising diagnostic targets .

Comparative diagnostic sensitivity:
Recent studies evaluating B. quintana detection methods show variable sensitivity:

LAMP assays targeting B. quintana genes have demonstrated superior sensitivity (1-10 copies/μL) compared to qPCR, with 100% specificity when testing against other Bartonella species .

Applications for diagnostically challenging cases:
Patients with endocarditis showed significantly lower PCR cycle threshold values for B. quintana in tissue samples compared to blood samples (mean Ct values of 19.2 vs. 35.7 for the yopP gene target), highlighting the importance of appropriate sample selection .

Potential for novel diagnostic approaches:

• Development of lipoprotein-specific monoclonal antibodies for direct antigen detection

• Aptamer-based tests targeting LolD-dependent surface molecules

• Host antibody profiling against B. quintana-specific lipoproteins

• Multiplexed PCR assays including lolD and lipoprotein-encoding genes

These approaches could improve early detection of B. quintana infections, particularly important given the rising incidence in vulnerable populations and the diagnostic challenges posed by this fastidious organism .

What research approaches can determine whether targeting LolD represents a viable therapeutic strategy against B. quintana infections?

Evaluating LolD as a therapeutic target against B. quintana requires a systematic research approach:

Target validation studies:

• Genetic essentiality assessment:

  • Generate conditional LolD-depletion strains in B. quintana

  • Demonstrate growth inhibition upon LolD depletion

  • Confirm lethality cannot be bypassed through compensatory mechanisms

• Cellular vulnerability analysis:

  • Identify minimal LolD activity threshold required for viability

  • Determine whether partial inhibition is sufficient for therapeutic effect

  • Assess resistance development frequency and mechanisms

Drug discovery approaches:

• High-throughput screening:

  • Develop biochemical assays measuring LolD ATPase activity

  • Screen compound libraries against purified recombinant LolD

  • Validate hits in cellular assays measuring lipoprotein transport

• Structure-based design:

  • Generate structural models of B. quintana LolD based on related ABC transporters

  • Identify unique binding pockets suitable for selective targeting

  • Design compounds predicted to bind these sites using in silico approaches

Therapeutic evaluation pipeline:

• In vitro efficacy studies:

  • Determine minimum inhibitory concentrations (MICs) against B. quintana

  • Assess bactericidal versus bacteriostatic effects

  • Evaluate activity against intracellular bacteria

• Ex vivo infection models:

  • Test efficacy in human macrophage infection models

  • Evaluate activity in endothelial cell infection models relevant to B. quintana pathogenesis

  • Assess cytotoxicity against mammalian cells

• Animal model studies:

  • Leverage mouse models using B. tribocorum (related Bartonella species adaptable to mice)

  • Develop humanized mouse models for B. quintana

  • Evaluate pharmacokinetics, efficacy, and toxicity

Therapeutic potential assessment criteria:

• Therapeutic index (ratio of toxic to effective dose) >10

• Bacterial specificity (minimal activity against beneficial microbiota)

• Low resistance development frequency (<10⁻⁸)

• Activity against both growing and dormant B. quintana