Recombinant Bartonella quintana L-lactate dehydrogenase [cytochrome] (lldD)

What is the basic function of Bartonella quintana L-lactate dehydrogenase [cytochrome] (lldD) and how does it differ from canonical LDH enzymes?

Bartonella quintana L-lactate dehydrogenase [cytochrome] (lldD) is an NAD-independent L-lactate dehydrogenase that catalyzes the oxidation of L-lactate to pyruvate, typically using FMN as a cofactor rather than NAD+. Unlike canonical LDH enzymes that are usually cytosolic and NAD-dependent, B. quintana lldD is membrane-associated and functions in the respiratory chain . This fundamental difference significantly affects experimental approaches when working with this enzyme, as traditional LDH assays measuring NADH oxidation are not applicable. Instead, researchers must employ alternative electron acceptors or direct pyruvate measurement techniques when characterizing its activity.

The enzyme plays a crucial role in L-lactate utilization as an energy source for B. quintana, which is particularly important for this pathogen during infection. Gene knockout studies in related bacteria have demonstrated that inactivation of lldD prevents growth on L-lactate as a sole carbon source, confirming its essential role in lactate metabolism .

What optimal conditions have been established for recombinant B. quintana lldD activity, and how do these compare to other bacterial L-iLDHs?

Research on L-iLDHs from various bacterial species suggests that recombinant B. quintana lldD likely exhibits optimal activity at pH 5.5-7.0 and temperatures around 30-37°C . Comparative studies with related bacterial L-iLDHs have shown that these enzymes typically have KM values for lactate in the range of 0.2-0.5 mM . While specific kinetic parameters for B. quintana lldD have not been extensively documented, related bacterial L-iLDHs have demonstrated catalytic efficiencies (kcat/KM) that make them valuable for various research and biotechnological applications.

When optimizing assay conditions for B. quintana lldD, researchers should consider buffer composition, pH stability, and potential inhibitors. Unlike NAD-dependent LDHs that show direct correlation between NADH oxidation and enzyme activity, monitoring L-iLDH activity often requires more complex assay systems utilizing artificial electron acceptors like dichlorophenolindophenol (DCIP) or ferricyanide.

How evolutionarily conserved is the lldD gene across Bartonella species, and what implications does this have for research targeting this enzyme?

The lldD gene appears highly conserved across Bartonella species as well as other alpha-proteobacteria. Sequence analysis has shown significant homology among lldD genes from various Bartonella species, suggesting evolutionary importance in the genus . This conservation extends to the protein's functional domains, particularly the FMN-binding site and catalytic residues.

For researchers, this conservation offers several advantages:

• Primers and probes designed for B. quintana lldD may be applicable for detecting related genes in other Bartonella species

• Antibodies raised against recombinant B. quintana lldD may cross-react with homologous proteins from related species

• Functional insights gained from studying B. quintana lldD may be extrapolated to understand lactate metabolism in other pathogenic Bartonella species

The high degree of conservation also suggests that lldD plays an essential role in Bartonella metabolism and survival, making it a potential target for broad-spectrum anti-Bartonella therapeutics .

What expression systems have proven most effective for producing active recombinant B. quintana lldD, and what are the critical optimization parameters?

For membrane-associated proteins like B. quintana lldD, E. coli-based expression systems have been successfully employed, with BL21(DE3) strains commonly used for recombinant production . Based on studies with similar bacterial L-iLDHs, the following critical parameters should be considered for optimization:

• Expression vector selection: pET series vectors containing T7 promoters have shown good results for bacterial L-iLDHs, with pET28a(+) being particularly suitable due to its His-tag options for purification .

• Induction conditions: IPTG concentration typically between 0.1-1.0 mM, with lower concentrations (0.1 mM) often yielding better results for membrane-associated proteins .

• Post-induction temperature: 37°C typically yields higher protein quantities but may lead to inclusion body formation. Lowering to 25-30°C often improves solubility .

• Induction duration: For membrane proteins like lldD, extended induction periods (24 hours) at lower temperatures may increase yield of properly folded protein .

A systematic optimization approach addressing these parameters sequentially has been shown to significantly impact both yield and activity of recombinant L-iLDHs. Researchers should monitor both protein expression levels and enzyme activity throughout optimization efforts, as conditions maximizing expression do not always maximize functional enzyme recovery.

What purification strategies are most effective for recombinant B. quintana lldD, and how can researchers address common purification challenges?

Purification of recombinant B. quintana lldD presents unique challenges due to its membrane association. Based on successful purification strategies for similar enzymes, a multi-step approach is recommended:

• Membrane fraction isolation: Differential centrifugation to separate membrane fractions (30,000-100,000 × g), followed by solubilization using mild detergents like n-dodecyl β-D-maltoside (DDM) or Triton X-100 at 0.5-2% .

• Affinity chromatography: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA columns with imidazole gradient elution (20-250 mM) .

• Ion exchange chromatography: Typically using Q-Sepharose or DEAE columns as a secondary purification step to remove contaminants .

• Size exclusion chromatography: Final polishing step to achieve high purity, usually with Superdex 200 columns .

Common challenges include:

• Insufficient solubilization from membranes (address by optimizing detergent type/concentration)

• Loss of activity during purification (include stabilizing agents like glycerol 10-20% and reducing agents)

• Aggregation (consider adding low concentrations of compatible solutes like arginine 50-100 mM)

• Co-purification of contaminants (implement more stringent washing steps during IMAC)

For inclusion body recovery, a systematic approach for solubilization and refolding has shown up to 50% recovery of active enzyme in similar L-iLDH studies .

How can researchers verify the structural integrity and functional activity of purified recombinant B. quintana lldD?

A comprehensive validation approach should include:

• Purity assessment:

  • SDS-PAGE analysis (expected molecular weight ~55-60 kDa)

  • Western blot using anti-His antibodies or specific antibodies raised against B. quintana lldD

• Structural integrity evaluation:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Fluorescence spectroscopy to monitor FMN binding

  • Size exclusion chromatography to confirm quaternary structure

• Functional activity assays:

  • Spectrophotometric assays measuring L-lactate oxidation (decrease in absorbance at 340 nm when using DCIP or ferricyanide as electron acceptors)

  • Pyruvate formation measurement using 2,4-dinitrophenylhydrazine derivatization

  • Oxygen consumption measurement using oxygen electrodes when testing respiratory chain function

• Kinetic parameter determination:

  • KM for L-lactate (typically 0.2-0.5 mM in related L-iLDHs)

  • Substrate specificity profiling using various 2-hydroxy acids

  • pH and temperature optima determination

The established protocol for LDH activity assay involves:

• Reaction mixture containing enzyme, substrate (L-lactate, typically 0.2-20 mM), and electron acceptor

• Monitoring spectrophotometric changes at appropriate wavelengths

• Calculating activity based on extinction coefficient of the electron acceptor

What evidence supports the role of lldD in B. quintana pathogenesis and survival within host cells?

Multiple lines of evidence suggest lldD plays a critical role in B. quintana pathogenesis:

• Host cell survival: B. quintana has been demonstrated to survive in neutrophil-like cells (nHL-60) for up to 72 hours despite their antimicrobial properties . The ability to utilize host-derived lactate via lldD may contribute to this intracellular persistence.

• Metabolic adaptation: During infection, B. quintana encounters various microenvironments with fluctuating oxygen levels. The NAD-independent lldD allows the pathogen to utilize lactate as an alternative energy source when oxidative phosphorylation is limited .

• Gene conservation: The high conservation of lldD across pathogenic Bartonella species suggests its importance for the pathogen's lifestyle .

• Homologous systems: Knockout studies of lldD in other bacterial pathogens have demonstrated significantly reduced virulence and impaired intracellular survival , suggesting similar roles in B. quintana.

While direct experimental evidence specifically for B. quintana lldD is still emerging, the enzyme appears to be part of the metabolic adaptation mechanisms that allow this fastidious pathogen to survive in diverse host environments, including erythrocytes and endothelial cells where lactate metabolism may provide a competitive advantage .

How does B. quintana lldD potentially contribute to the bacterium's ability to establish chronic infections and evade host immune responses?

The potential contributions of lldD to chronic infection establishment include:

The research challenges in this area include developing appropriate in vitro and in vivo models that can accurately represent the complex host-pathogen interactions involving B. quintana metabolism during chronic infection.

What experimental approaches can researchers use to study the specific contribution of lldD to B. quintana virulence in different infection models?

To investigate lldD's role in B. quintana virulence, researchers should consider these methodological approaches:

• Genetic manipulation approaches:

  • Construction of lldD knockout mutants using homologous recombination or CRISPR-Cas9 systems

  • Complementation studies to confirm phenotype specificity

  • Creation of point mutations in catalytic residues to distinguish enzymatic from structural functions

• Cellular infection models:

  • Human endothelial cell infection assays to assess bacterial persistence and proliferation

  • nHL-60 neutrophil-like cell models to study survival in immune cells

  • Erythrocyte infection models to evaluate intraerythrocytic survival

• Transcriptional analysis:

  • RT-qPCR to measure lldD expression during different stages of infection

  • RNA-seq to identify co-regulated genes in the lactate utilization pathway

  • Promoter reporter assays to identify environmental signals triggering lldD expression

• Metabolic studies:

  • 13C-labeled lactate tracing to confirm utilization by B. quintana during infection

  • Measurement of extracellular and intracellular lactate levels during infection

  • Oxygen consumption analysis to assess respiratory chain involvement

• Functional assays:

  • Gentamicin protection assays to quantify intracellular survival

  • Cytotoxicity assays to measure host cell viability during infection

  • Beta-lactamase reporter systems to study effector protein translocation

These approaches can be applied to increasingly complex models, from in vitro cellular systems to potential animal models, though B. quintana's narrow host range presents challenges for the latter.

How can recombinant B. quintana lldD be utilized for developing improved diagnostic tools for Bartonella infections?

Recombinant B. quintana lldD offers several promising avenues for improving Bartonella diagnostics:

• Serological diagnostics:

  • Development of enzyme-linked immunosorbent assays (ELISAs) using purified recombinant lldD as antigen

  • Evaluation of anti-lldD antibodies as biomarkers for active vs. past infections

  • Potential for distinguishing B. quintana from other Bartonella species based on antibody cross-reactivity patterns

• Molecular diagnostics:

  • Design of lldD-specific PCR primers for sensitive and specific detection

  • Development of multiplex PCR assays targeting lldD alongside other Bartonella genes

  • Loop-mediated isothermal amplification (LAMP) assays for point-of-care testing

• Functional diagnostics:

  • Development of activity-based assays to detect metabolically active Bartonella in clinical samples

  • Correlation between lldD enzyme activity and bacterial load or disease severity

Current diagnostic challenges for B. quintana include long cultivation times (12-45 days) and serological cross-reactivity between Bartonella species . Focusing on specific virulence factors like lldD could potentially improve both sensitivity and specificity of detection methods.

Research has shown increasing cases of B. quintana infection in Canada and the United States , highlighting the need for improved diagnostics. The enzyme's conservation across Bartonella species makes it a promising target for broad Bartonella detection, while sequence variations might enable species-specific identification.

What are the potential challenges in targeting B. quintana lldD for therapeutic development, and how might these be addressed?

Several challenges exist in targeting lldD for therapeutic development:

• Structural challenges:

  • Membrane association complicates structural studies necessary for rational drug design

  • Limited crystallographic data on bacterial L-iLDHs hinders structure-based approaches

  • Potential conformational changes during catalysis create moving targets for inhibitor design

• Selectivity issues:

  • Homology with human LDH enzymes raises concerns about cross-reactivity and side effects

  • Conservation across bacterial species may limit specificity for B. quintana

  • Need to differentiate from other FAD/FMN-dependent dehydrogenases

• Pharmacological challenges:

  • Compound access to intracellular bacteria within erythrocytes and endothelial cells

  • Effective therapeutic concentrations in relevant tissues (e.g., heart valves for endocarditis)

  • Potential for resistance development through metabolic adaptation

• Validation challenges:

  • Limited animal models for B. quintana infection to test therapeutic efficacy

  • Need for appropriate biomarkers to monitor treatment response

  • Long treatment durations required for chronic Bartonella infections (e.g., 23 weeks reported in one case)

Potential approaches to address these challenges:

• Utilizing computational modeling to predict structure in absence of crystallographic data

• Focusing on unique structural features of B. quintana lldD compared to human LDH

• Developing combination therapies targeting multiple metabolic pathways

• Exploring adjuvant therapies that enhance immune clearance alongside metabolic inhibition

What methodological approaches can be used to screen for potential inhibitors of B. quintana lldD, and how should activity be validated?

A comprehensive screening and validation pipeline for lldD inhibitors would include:

• Primary screening methods:

  • High-throughput spectrophotometric assays measuring inhibition of L-lactate oxidation

  • Virtual screening using homology models of B. quintana lldD

  • Fragment-based screening to identify chemical scaffolds with binding potential

  • Repurposing screens of approved drugs with known safety profiles

• Secondary validation assays:

  • Dose-response curves to determine IC50 values

  • Mechanism of inhibition studies (competitive, noncompetitive, uncompetitive)

  • Time-dependence studies to identify slow-binding or irreversible inhibitors

  • Selectivity profiling against human LDH and other related dehydrogenases

• Cellular validation:

  • Growth inhibition of B. quintana under conditions requiring lactate utilization

  • Cell infection models to assess intracellular activity

  • Cytotoxicity assessment in mammalian cell lines

  • Measurement of lactate consumption in infected vs. uninfected cells

• Advanced validation:

  • Bacterial resistance development assessment through serial passaging

  • Pharmacokinetic and pharmacodynamic studies in appropriate models

  • Combination studies with established antibiotics (doxycycline, azithromycin)

  • Target engagement studies using thermal shift assays or activity-based protein profiling

The established protocol for LDH inhibition assays typically involves:

• Preincubation of enzyme with test compounds

• Initiation of reaction with substrate addition

• Continuous monitoring of activity using appropriate detection systems

• Analysis of inhibition patterns to determine mechanism and potency

What are the critical factors affecting the stability and storage of recombinant B. quintana lldD, and how can researchers optimize these conditions?

Optimizing stability and storage conditions for recombinant B. quintana lldD requires consideration of several factors:

• Buffer composition optimization:

  • pH stability profile determination (likely optimal between pH 7.0-8.0 based on related enzymes)

  • Ionic strength effects (typically 50-150 mM NaCl provides optimal stability)

  • Effect of divalent cations (Mg²⁺, Ca²⁺) on enzyme stability

  • Inclusion of stabilizing agents (10-20% glycerol, 1-5 mM β-mercaptoethanol or DTT)

• Storage temperature effects:

  • Short-term stability at 4°C (typically 1-2 weeks)

  • Long-term storage at -20°C or -80°C with cryoprotectants

  • Freeze-thaw cycle effects on activity (aliquoting recommended)

  • Lyophilization potential with appropriate excipients

• Cofactor considerations:

  • FMN retention during storage (supplementation may be required)

  • Protection from light to prevent cofactor degradation

  • Oxidation state maintenance (reducing environments beneficial)

• Solubility enhancement:

  • For membrane-associated proteins like lldD, detergent selection is critical

  • Detergent concentration above critical micelle concentration but below inhibitory levels

  • Alternative solubilization agents (nanodisc technology, amphipols) for long-term stability

Studies with similar enzymes suggest that recombinant lldD retains >80% activity for at least 2 months when stored at -80°C in a buffer containing 50 mM phosphate (pH 7.5), 100 mM NaCl, 10% glycerol, and 1 mM DTT . Activity should be verified using standardized assays before experimental use.

How can researchers effectively address inconsistencies in recombinant B. quintana lldD activity data across different experimental setups?

Addressing inconsistencies requires systematic troubleshooting and standardization:

• Standardization of enzyme preparation:

  • Implement consistent purification protocols with defined quality control metrics

  • Quantify specific activity in standardized units (μmol/min/mg protein)

  • Establish minimum purity requirements (typically >90% by SDS-PAGE)

  • Verify FMN saturation through spectroscopic analysis

• Reaction condition standardization:

  • Define standard assay conditions (temperature, pH, ionic strength)

  • Standardize substrate concentrations relative to KM values

  • Establish consistent data collection timeframes

  • Implement appropriate controls (positive and negative) in each experiment

• Analytical validation:

  • Verify linearity of response under experimental conditions

  • Establish detection limits and quantification ranges

  • Assess and correct for potential interfering factors

  • Implement statistical approaches for outlier identification

• Documentation and reporting standards:

  • Detailed methodology reporting including buffer compositions

  • Clear specification of enzyme source and preparation method

  • Comprehensive data presentation including raw data availability

  • Use of reference standards when available

Data variability across studies on bacterial L-iLDHs has often been attributed to:

• Differences in membrane solubilization efficiency

• Varying levels of cofactor saturation

• Inhibitory effects of purification reagents (imidazole, high salt)

• Storage-related activity losses

Implementing a standardized quality control pipeline with defined acceptance criteria can significantly reduce inter-laboratory variability in activity measurements .

What cutting-edge techniques are being applied to study the structure-function relationship of B. quintana lldD and similar bacterial dehydrogenases?

Advanced techniques being applied to study bacterial L-iLDHs include:

• Structural biology approaches:

  • Cryo-electron microscopy for membrane protein structures without crystallization

  • NMR spectroscopy for dynamic structural elements

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes

  • Small-angle X-ray scattering (SAXS) for solution structure determination

• Functional genomics techniques:

  • CRISPR interference (CRISPRi) for partial gene repression to study essentiality

  • Transposon sequencing (Tn-seq) to identify genetic interactions

  • RNA-seq for transcriptional response to environmental conditions

  • Ribosome profiling for translational regulation analysis

• Advanced biochemical methods:

  • Microscale thermophoresis for binding affinity measurements

  • Surface plasmon resonance for real-time interaction analysis

  • Isothermal titration calorimetry for thermodynamic profiling

  • Single-molecule enzymology to detect conformational dynamics

• Computational approaches:

  • Molecular dynamics simulations of enzyme-substrate interactions

  • Quantum mechanics/molecular mechanics (QM/MM) for reaction mechanism elucidation

  • Machine learning for prediction of substrate specificity

  • Systems biology modeling of metabolic pathways involving lldD

• In vivo techniques:

  • FRET-based biosensors for intracellular enzyme activity

  • Chemical proteomics for target engagement verification

  • Metabolic flux analysis using stable isotope labeling

  • In vivo imaging of fluorescently tagged enzyme localization

These advanced techniques are helping to bridge the gap between structural information and functional understanding of bacterial dehydrogenases, potentially revealing novel aspects of B. quintana lldD that could be exploited for therapeutic or diagnostic purposes.

Table of Comparative Properties: Bacterial L-lactate Dehydrogenases

*Estimated values based on related bacterial L-iLDHs; specific values for B. quintana lldD require experimental verification

What is the recommended protocol for cloning, expressing, and purifying active recombinant B. quintana lldD?

Based on successful approaches with related enzymes, the following protocol is recommended:

Cloning procedure:

• Amplify the lldD gene using PCR with high-fidelity polymerase and specific primers containing appropriate restriction sites (BamHI and HindIII recommended) .

• Primer design example:

  • Forward: 5'-GGATCCATG[N-terminal sequence of B. quintana lldD]-3'

  • Reverse: 5'-AAGCTT[C-terminal sequence of B. quintana lldD]-3'

• Clone the PCR product into pET28a(+) expression vector, which provides an N-terminal His-tag for purification .

• Transform the construct into E. coli DH5α for plasmid maintenance.

• Verify construct by sequencing before transformation into expression strain.

Expression protocol:

• Transform verified construct into E. coli BL21(DE3) cells .

• Culture transformed cells in LB broth with kanamycin (50 μg/ml) at 37°C.

• Induce expression with 0.1 mM IPTG when OD600 reaches 0.6-0.8.

• Continue incubation at 30°C for 16-24 hours with shaking at 150 rpm.

• Harvest cells by centrifugation at 5,000 × g for 10 minutes at 4°C.

Purification protocol:

• Resuspend cell pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM PMSF, 1 mg/ml lysozyme).

• Disrupt cells by sonication or French press.

• Centrifuge at 10,000 × g for 20 minutes to remove cell debris.

• Ultracentrifuge supernatant at 100,000 × g for 1 hour to isolate membrane fraction.

• Solubilize membrane pellet in solubilization buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 1% DDM, 10% glycerol).

• Apply solubilized protein to Ni-NTA column equilibrated with binding buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.05% DDM, 10% glycerol, 10 mM imidazole).

• Wash with binding buffer containing 20 mM imidazole.

• Elute with elution buffer (binding buffer with 250 mM imidazole).

• Dialyze against storage buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.05% DDM, 10% glycerol).

Activity verification:
Measure enzyme activity using a spectrophotometric assay with DCIP or ferricyanide as electron acceptors, monitoring absorbance changes at appropriate wavelengths .

What experimental design considerations are critical when studying B. quintana lldD's role in host-pathogen interactions?

When investigating lldD's role in host-pathogen interactions, researchers should consider:

• Selection of appropriate cellular models:

  • Human endothelial cells (HUVECs or HMECs) to study vascular manifestations

  • HL-60 cells differentiated with retinoic acid as neutrophil models

  • CD34+ progenitor-derived erythroid cells for erythrocyte infection models

  • Appropriate controls including uninfected cells and cells infected with defined mutants

• Bacterial strain considerations:

  • Wild-type B. quintana strains with verified virulence

  • Isogenic lldD knockout mutants (when available)

  • Complemented mutants to confirm phenotype specificity

  • Reporter strains for tracking intracellular localization and activity

• Infection parameters optimization:

  • Multiplicity of infection (MOI) titration (typically 50-100 bacteria per cell)

  • Infection duration optimization based on experimental endpoints

  • Synchronization methods for studying specific infection stages

  • Consideration of co-infection models to study interaction with other pathogens

• Readout selection:

  • Quantification of intracellular bacteria (CFU counts, fluorescence, qPCR)

  • Host cell response parameters (cytokine production, cell death, phenotypic changes)

  • Metabolic alterations (lactate consumption/production, pH changes)

  • Gene expression changes in both host and pathogen

• Technical considerations:

  • Appropriate statistical design with sufficient biological and technical replicates

  • Inclusion of time-course studies to capture dynamic interactions

  • Use of multiple complementary techniques to confirm observations

  • Validation in increasing complexity models when possible

Research has demonstrated that B. quintana can inhibit apoptosis and modulate host cell death pathways , suggesting that measurement of cell viability using LDH release assays is particularly relevant when studying this pathogen's interactions with host cells.

How should researchers approach the development of selective inhibitors targeting B. quintana lldD while minimizing cross-reactivity with human LDH?

A strategic approach to developing selective inhibitors includes:

• Structural comparison and targeting unique features:

  • Identify differences in catalytic site architecture between B. quintana lldD and human LDH

  • Focus on the FMN binding site present in bacterial L-iLDH but absent in human NAD-dependent LDH

  • Target membrane-association domains unique to bacterial L-iLDH

  • Exploit differences in quaternary structure (bacterial L-iLDHs are often monomeric or dimeric while human LDHs are tetrameric)

• Rational design approach:

  • Develop homology models based on related bacterial L-iLDHs

  • Perform virtual screening targeting unique binding pockets

  • Design substrate analogs that exploit differences in substrate specificity

  • Consider allosteric inhibitors targeting non-conserved regulatory sites

• Screening strategies:

  • Differential screening against both B. quintana lldD and human LDH isoforms

  • Calculation of selectivity indices for each compound

  • Counter-screening against other human dehydrogenases to identify potential off-targets

  • Biochemical selectivity verification using purified enzymes

• Medicinal chemistry optimization:

  • Structure-activity relationship studies focusing on selectivity enhancement

  • Improvement of physicochemical properties for cellular penetration

  • Modification of scaffolds to reduce binding to human LDH isoforms

  • Optimization of pharmacokinetic parameters while maintaining selectivity

• Biological validation:

  • Cellular models to confirm target engagement and specificity

  • Assessment of effects on bacterial vs. host cell metabolism

  • Evaluation of toxicity profiles in multiple human cell types

  • Confirmation of activity against intracellular bacteria