Recombinant Bartonella henselae Dihydrodipicolinate synthase (dapA)

What is the role of DapA in Bartonella henselae metabolism?

DapA (dihydrodipicolinate synthase) catalyzes the first committed step in the diaminopimelate/lysine biosynthesis pathway in bacteria, including B. henselae. This enzyme specifically catalyzes the condensation of pyruvate and aspartate semialdehyde to form 4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid. This reaction is followed by the reduction step catalyzed by DapB (dihydrodipicolinate reductase), which has been characterized in B. henselae . The entire pathway is essential for peptidoglycan synthesis and bacterial survival, as humans lack this pathway, making it a potential antibiotic target for treating B. henselae infections.

How does B. henselae DapA compare to DapA enzymes from other bacterial species?

B. henselae DapA likely shares structural and functional similarities with other bacterial DapA enzymes, though specific differences may exist. Based on research on related enzymes like DapB from B. henselae, we can infer that DapA likely forms a homotetramer composed of identical subunits . Comparative analysis would typically involve sequence alignment, homology modeling, and structural comparisons with well-characterized DapA enzymes from model organisms such as E. coli or other pathogenic bacteria. Such analysis can reveal conserved catalytic residues and species-specific features that might influence substrate specificity or inhibitor binding.

What expression systems are most effective for producing recombinant B. henselae DapA?

Based on successful expression of other B. henselae proteins, E. coli BL21(DE3) R3 Rosetta cells represent an effective expression system for recombinant B. henselae DapA . The auto-induction method in a LEX Bioreactor has proven successful for related B. henselae proteins and likely provides good yields for DapA as well . The gene should be cloned into a suitable expression vector such as pAVA0421, which includes a cleavable hexahistidine tag for purification purposes. Expression optimization typically requires testing different growth temperatures (16-37°C), induction conditions, and media compositions to maximize protein solubility and yield.

What purification strategy yields the highest purity and activity for recombinant B. henselae DapA?

A four-step purification protocol similar to that used for B. henselae DapB would likely be effective: (1) Ni²⁺-affinity chromatography to capture the His-tagged protein; (2) cleavage of the hexahistidine tag using 3C protease; (3) reverse Ni²⁺-affinity chromatography to remove the cleaved tag; and (4) size-exclusion chromatography for final polishing . Buffer optimization is critical - a suitable starting condition would be 20 mM HEPES pH 7, 0.3 M NaCl, 5% glycerol, and 2 mM DTT, similar to conditions used for DapB . Enzyme activity should be monitored throughout purification using a spectrophotometric assay measuring the condensation of pyruvate and aspartate semialdehyde.

How can the oligomeric state of B. henselae DapA be determined accurately?

Multiple complementary techniques should be employed to determine the oligomeric state:

• Size-exclusion chromatography (SEC) with molecular weight standards provides initial estimation

• Dynamic light scattering (DLS) to measure the hydrodynamic radius

• Native PAGE to compare migration with known molecular weight standards

• Analytical ultracentrifugation for precise molecular weight determination

• Chemical crosslinking followed by SDS-PAGE to capture oligomeric interactions

• Crystallographic analysis of the protein structure

Based on related bacterial DapA enzymes and the tetrameric nature of B. henselae DapB, DapA likely exists as a tetramer in solution .

What crystallization conditions are optimal for B. henselae DapA structural determination?

While specific conditions for B. henselae DapA crystallization are not directly reported, the successful crystallization of B. henselae DapB provides a starting point. Initial screens should include conditions similar to those effective for DapB: 5% (w/v) PEG 4000, 200 mM sodium acetate, 100 mM sodium citrate tribasic pH 5.5 . A comprehensive screening approach should include:

• Commercial sparse matrix screens (e.g., Hampton Research, Molecular Dimensions)

• Systematic grid screens varying PEG concentration (2-20%), salt concentration, and pH (4.5-8.5)

• Addition of substrates or substrate analogs to stabilize the active site

• Microseeding techniques to improve crystal quality

• Optimization of protein concentration (typically 5-15 mg/ml)

• Crystallization at different temperatures (4°C and 20°C)

X-ray diffraction data collection to at least 2.5 Å resolution would be necessary for reliable structural determination.

What are the standard kinetic parameters for B. henselae DapA and how do they compare with orthologous enzymes?

While specific kinetic parameters for B. henselae DapA are not directly reported in the provided literature, standard enzyme kinetic analysis should include:

• Determination of Km values for both substrates (pyruvate and aspartate semialdehyde)

• Calculation of kcat and catalytic efficiency (kcat/Km)

• Assessment of pH and temperature optima

• Evaluation of metal ion requirements (typically divalent cations)

Kinetic assays typically involve spectrophotometric monitoring of product formation or coupled enzyme assays. Comparison with orthologous enzymes from other bacteria would provide insight into potential adaptations specific to B. henselae's ecological niche and pathogenic lifestyle.

How does intracellular localization affect DapA expression and activity in B. henselae?

Based on transcriptomic studies of B. henselae in different environments, significant changes in gene expression occur when bacteria transition from extracellular to intracellular locations . While specific data for DapA is not provided, similar enzymes involved in metabolic pathways may show altered expression patterns under these different conditions. Research methodologies to address this question should include:

• Comparative transcriptomic analysis of DapA expression in extracellular versus intracellular B. henselae using RT-qPCR and RNA-seq approaches

• Development of translational reporter fusions to monitor protein levels in different environments

• Extraction and purification of intracellular bacteria followed by enzyme activity assays

• Immunofluorescence microscopy to visualize enzyme localization

These approaches would help determine how the intracellular environment affects DapA expression and function, providing insights into B. henselae's metabolic adaptations during infection.

How essential is DapA for B. henselae survival and virulence during infection?

To determine the essentiality of DapA for B. henselae, several experimental approaches can be employed:

• Creation of conditional DapA mutants using inducible promoters

• Gene knockout attempts with lysine supplementation

• Growth curve analysis under different conditions

• Complementation studies with wild-type DapA

• In vitro infection models using endothelial cells and macrophages

• Animal infection models with wild-type and DapA-deficient strains

What is the potential of B. henselae DapA as an antibiotic target?

DapA represents a promising antibiotic target for several reasons:

• It catalyzes the first committed step in the lysine biosynthesis pathway

• The pathway is absent in mammals, reducing the risk of off-target effects

• Lysine is essential for bacterial peptidoglycan and protein synthesis

• The enzyme's structure likely contains druggable pockets for inhibitor binding

Development of DapA inhibitors would require:

• High-throughput screening of chemical libraries against purified enzyme

• Structure-based drug design utilizing crystallographic data

• Rational modification of substrate analogs

• Evaluation of inhibitor efficacy in cell culture models

• Assessment of inhibitor specificity against human enzymes

• Pharmacokinetic and pharmacodynamic studies

The fact that B. henselae can survive intracellularly presents an additional challenge, as inhibitors must penetrate host cells to reach the bacteria, particularly given the reduced antibiotic susceptibility of intracellular Bartonella reported previously .

How can RNA extraction protocols be optimized for studying DapA expression in intracellular B. henselae?

Extracting high-quality bacterial RNA from infected host cells presents significant challenges. Based on successful transcriptomic studies of intracellular B. henselae , the following methodology is recommended:

• Differential lysis protocols to separate bacterial from host cell contents

• Rapid processing to minimize RNA degradation

• Use of RNA stabilization reagents immediately upon cell lysis

• Selective depletion of host rRNA and mRNA

• DNase treatment to remove genomic DNA contamination

• Quality assessment using Bioanalyzer or similar platforms

• Specific RT-PCR primers designed for DapA with careful validation

When analyzing gene expression data, normalization to stable reference genes that maintain consistent expression under experimental conditions is essential. Additionally, multiple biological and technical replicates should be performed to ensure reproducibility.

What experimental approaches can distinguish between transcriptional and post-transcriptional regulation of DapA in B. henselae?

To comprehensively understand DapA regulation in B. henselae, multiple levels of regulation should be investigated:

• Transcriptional regulation:

  • Promoter mapping using 5' RACE

  • Reporter gene fusions to monitor promoter activity

  • ChIP-seq to identify transcription factor binding sites

  • In vitro DNA-protein binding assays

• Post-transcriptional regulation:

  • RNA stability assays using rifampicin to inhibit transcription

  • Northern blot or qRT-PCR to measure mRNA half-life

  • Analysis of potential regulatory RNA elements (riboswitches, sRNAs)

  • Polysome profiling to assess translation efficiency

• Post-translational regulation:

  • Western blotting to assess protein levels versus mRNA levels

  • Mass spectrometry to identify potential modifications

  • Pulse-chase experiments to determine protein turnover rates

The comparative analysis between extracellular and intracellular conditions would be particularly informative, as B. henselae is known to undergo significant transcriptional reprogramming upon host cell entry .

How can protein-protein interactions between DapA and other enzymes in the lysine biosynthesis pathway be effectively studied in B. henselae?

Multiple complementary approaches should be employed to study protein-protein interactions:

• Co-immunoprecipitation using antibodies against DapA

• Bacterial two-hybrid systems

• Proximity-based labeling approaches (BioID, APEX)

• FRET/BRET analyses with fluorescently tagged proteins

• Surface plasmon resonance to measure binding kinetics

• Isothermal titration calorimetry for thermodynamic parameters

• Native gel electrophoresis to detect stable complexes

• Crosslinking mass spectrometry to map interaction interfaces

These methods could reveal whether DapA forms a metabolon with other enzymes in the lysine biosynthesis pathway, potentially including DapB, which has been confirmed to exist as a tetramer in B. henselae . Metabolon formation would allow substrate channeling and increased efficiency of the pathway, which could be particularly important during intracellular growth when resources may be limited.

How has the DapA enzyme evolved in Bartonella species compared to other alphaproteobacteria?

Evolutionary analysis of DapA would provide insights into adaptation and specialization of this enzyme in Bartonella. Recommended approaches include:

• Comprehensive phylogenetic analysis comparing DapA sequences from:

  • Multiple Bartonella species

  • Other alphaproteobacteria

  • More distant bacterial lineages

• Calculation of selection pressure (dN/dS ratios) on different regions of the protein

• Identification of:

  • Conserved catalytic residues

  • Bartonella-specific sequence motifs

  • Positively selected residues potentially involved in adaptation

• Homology modeling to map sequence variations onto the protein structure

• Functional validation of identified variations through site-directed mutagenesis

Such analysis could reveal whether DapA has undergone specific adaptations in Bartonella related to its unique lifestyle as a facultative intracellular pathogen that can persist in mammalian hosts and arthropod vectors.

What methodological approaches can address inconsistencies between in vitro and in vivo findings about DapA function?

When discrepancies arise between in vitro enzymatic studies and in vivo observations about DapA function, several approaches can help resolve these inconsistencies:

• Development of cell-free expression systems from B. henselae to better approximate the native cellular environment

• Use of permeabilized cells to study enzyme activity in a near-native context

• Creation of fluorescent biosensors to monitor enzyme activity or substrate/product levels in living bacteria

• Application of metabolomics to track lysine pathway intermediates in different growth conditions

• Integration of transcriptomic, proteomic, and metabolomic data for systems biology modeling

• Development of inducible gene expression systems to titrate DapA levels and observe effects

• Consideration of spatial and temporal regulation using advanced microscopy techniques

These approaches acknowledge that enzymes may behave differently in the complex cellular environment compared to purified systems, particularly given B. henselae's ability to adapt to diverse microenvironments during its infection cycle .