Recombinant Bartonella henselae Carbamoyl-phosphate synthase small chain (carA)

What is the functional role of CarA in B. henselae metabolism?

Carbamoyl phosphate synthetase (CPSase) plays a fundamental role in ammonia assimilation in all organisms, including B. henselae. The CarA protein represents the small chain of this enzyme complex, which catalyzes the synthesis of carbamoyl phosphate (CP), a critical precursor for pyrimidine nucleotides and arginine biosynthesis . In B. henselae, as an intracellular pathogen that colonizes endothelial cells and erythrocytes, CP synthesis is essential for bacterial replication and survival within host cells . The CarA subunit typically partners with CarB (the large chain) to form the complete CPSase enzyme complex. Based on comparative studies with other bacterial CPSases, the CarA subunit in B. henselae likely contributes to the glutaminase activity that hydrolyzes glutamine and transfers ammonia to the synthetase domain for carbamoyl phosphate production .

What expression systems are most effective for recombinant B. henselae CarA production?

For laboratory-scale production of recombinant B. henselae CarA, E. coli expression systems remain the most widely used approach due to their simplicity and high yield. When selecting an expression system, researchers should consider several factors:

• Vector selection: pET-based vectors with T7 promoters offer strong induction and high expression levels

• E. coli strain optimization: BL21(DE3) derivatives are preferred for their reduced protease activity

• Codon optimization: Adjusting for B. henselae codon bias can significantly improve expression in E. coli

• Induction conditions: Lower temperatures (16-25°C) often improve solubility of recombinant CarA

For functional studies requiring proper protein folding and activity, consider these methodological improvements:

These optimized conditions have been shown to increase the yield of soluble, properly folded recombinant proteins from fastidious bacteria like Bartonella species, though specific optimization for B. henselae CarA may require empirical testing.

What purification strategies work best for recombinant B. henselae CarA?

Purification of recombinant B. henselae CarA presents specific challenges due to its association with other cellular proteins and potential for forming protein complexes. An effective purification strategy typically involves:

• Affinity tagging: Fusion with a His6-tag enables one-step purification via immobilized metal affinity chromatography (IMAC)

• Buffer optimization: Including 10-15% glycerol helps maintain protein stability

• Size exclusion chromatography: Critical for separating monomeric, dimeric, and tetrameric forms

• Activity preservation: Addition of substrate analogs or stabilizing agents during purification

Chemical cross-linking followed by size exclusion chromatography can be particularly useful for studying the oligomeric state of B. henselae CarA, similar to the approach used for other CPSases . The recombinant protein purification approach should be designed to accommodate the known tendency of CarA to form complexes with CarB in its native state.

How can site-directed mutagenesis be applied to study the catalytic mechanism of B. henselae CarA?

Site-directed mutagenesis represents a powerful approach for elucidating the structure-function relationships of B. henselae CarA. Based on sequence alignments with better-characterized CPSases, several key residues can be targeted for mutagenesis studies:

• Active site residues: Conserved amino acids involved in ATP binding and ammonia transfer

• Subunit interface residues: Amino acids mediating CarA-CarB interactions

• Allosteric regulation sites: Residues potentially involved in feedback inhibition

When designing mutagenesis experiments, researchers should:

• Perform careful sequence alignment with CPSases of known structure

• Prioritize highly conserved residues first (>90% conservation across bacterial species)

• Use alanine scanning for initial assessment of residue importance

• Follow with more targeted substitutions based on initial results

• Generation of multiple single-point mutants

• Expression and purification under identical conditions

• Comprehensive kinetic characterization

• Structural analysis where possible

This approach has successfully elucidated catalytic mechanisms in related enzymes and can provide insights into the distinctive features of B. henselae CarA.

What are the kinetic parameters of recombinant B. henselae CarA and how do they compare to other bacterial CPSases?

Understanding the kinetic parameters of recombinant B. henselae CarA provides crucial insights into its enzymatic efficiency and substrate preferences. Based on studies of related CPSases, the following kinetic parameters should be determined:

For meaningful comparisons, kinetic studies should include:

The expected high apparent affinity for ATP and ammonia in B. henselae CarA would be similar to that observed in other specialized CPSases . These parameters should be determined under physiologically relevant conditions, ideally at 37°C and pH 7.4 to reflect the environment of the mammalian host.

How does the oligomeric state of B. henselae CarA affect its enzymatic function?

The oligomeric state of CPSases significantly impacts their enzymatic activity and regulation. For B. henselae CarA, determining whether it functions as a monomer, dimer, or higher-order oligomer is crucial for understanding its catalytic mechanism. Based on studies of related enzymes, CPSases typically form homodimers or tetramers .

Methodological approaches to study oligomerization include:

• Size exclusion chromatography to separate different oligomeric forms

• Chemical cross-linking to stabilize native oligomeric structures

• Analytical ultracentrifugation for precise determination of molecular weight

• Native PAGE to visualize different oligomeric states

Researchers should systematically investigate:

• Effect of protein concentration on oligomeric distribution

• Impact of substrates and allosteric regulators on oligomerization

• Correlation between oligomeric state and catalytic activity

• Temperature and pH dependency of oligomerization

The homodimeric/tetrameric structure observed in other small CPSases suggests B. henselae CarA likely functions in a similar oligomeric state, which would be consistent with the dimer-based CPSase activity and reaction mechanism documented in related enzymes .

What are the optimal conditions for measuring B. henselae CarA enzymatic activity?

Establishing reliable assay conditions is critical for accurate characterization of B. henselae CarA activity. Based on enzyme assays developed for other CPSases, researchers should consider:

Buffer composition:

• HEPES or Tris buffer (50-100 mM, pH 7.5-8.0)

• Magnesium chloride (5-10 mM) as essential cofactor

• Potassium chloride (50-100 mM) for ionic strength

• DTT or β-mercaptoethanol (1-5 mM) to maintain reduced state

• Glycerol (5-10%) for protein stability

Reaction monitoring approaches:

• Coupled enzyme assays linking carbamoyl phosphate production to NADH oxidation

• Direct measurement of ATP consumption via luciferase assay

• Radiometric assays using 14C-labeled bicarbonate

• Colorimetric detection of inorganic phosphate release

The enzymatic assay should be validated by:

• Establishing linearity with respect to time and enzyme concentration

• Determining optimal substrate concentrations

• Confirming lack of interfering activities

• Verifying reproducibility across different protein preparations

When characterizing partial reactions, specific assay modifications are required:

• ATP hydrolysis: Detection of ADP formation using coupled enzymes

• Carbamate formation: Trapping and quantification of the unstable intermediate

• Carbamoyl phosphate synthesis: Product detection via conversion to citrulline

These methodological considerations ensure accurate assessment of enzyme activity and facilitate comparison with other CPSases.

How can structural biology techniques be applied to study B. henselae CarA?

Structural characterization of B. henselae CarA provides invaluable insights into its function and interactions. Multiple complementary approaches should be considered:

X-ray crystallography:

• Requires high-purity protein (>95%) and concentrated samples (10-15 mg/ml)

• Screening multiple crystallization conditions (temperature, pH, precipitants)

• Co-crystallization with substrates, inhibitors, or transition state analogs

• May require surface entropy reduction for improved crystal formation

Cryo-electron microscopy:

• Particularly valuable for studying CarA-CarB complex formation

• Sample preparation with minimal fixation to preserve native state

• Single-particle analysis for detailed structural determination

• Direct visualization of different conformational states

Molecular modeling:

• Homology modeling based on related CPSases with known structures

• Validation of models through mutagenesis of predicted key residues

• Molecular dynamics simulations to predict flexibility and domain movements

• Docking studies to predict substrate and inhibitor binding modes

Hydrogen-deuterium exchange mass spectrometry:

• Maps solvent accessibility and protein dynamics

• Identifies regions involved in substrate binding and conformational changes

• Provides insights into protein-protein interaction interfaces

• Complements static structural information from crystallography

The combination of these approaches provides a comprehensive structural understanding of B. henselae CarA, facilitating rational design of inhibitors and engineering of the enzyme for biotechnological applications.

What approaches are effective for studying B. henselae CarA expression and regulation in the pathogen?

Understanding how B. henselae regulates carA expression during infection provides insights into pathogenesis. Several methodological approaches are particularly valuable:

Transcriptomic analysis:

• RNA-seq to quantify carA expression under different conditions

• Comparison between in vitro culture and host cell infection models

• Identification of co-regulated genes in the same metabolic pathway

• Mapping of transcription start sites using 5′-RACE

Promoter analysis:

• Reporter gene fusions (e.g., lacZ, gfp) to monitor promoter activity

• Site-directed mutagenesis of predicted regulatory elements

• Electrophoretic mobility shift assays to identify DNA-binding proteins

• DNase footprinting to map regulator binding sites precisely

Regulation during infection:

• Infection of relevant cell types (endothelial cells, erythrocytes)

• Time-course analysis of gene expression during infection cycle

• Comparison between different physiological conditions

• Host factors influencing bacterial gene expression

Protein-level regulation:

• Western blot analysis using specific antibodies

• Pulse-chase experiments to determine protein stability

• Post-translational modification analysis

• Protein-protein interactions affecting CarA function

When studying B. henselae gene expression, researchers should be aware of the fastidious nature of this organism and the challenges of working with an intracellular pathogen . The availability of molecular diagnostic methods for B. henselae provides tools that can be adapted for research purposes .

Can recombinant B. henselae CarA be used for improved diagnostics of Bartonellosis?

Developing improved diagnostics for Bartonellosis represents an important research direction, particularly given the limitations of current methods. Recombinant B. henselae CarA has potential applications in diagnostic development:

Serological diagnostics:

• Evaluation of CarA as an antigen for antibody detection

• Comparison with current diagnostic antigens

• Development of ELISA, Western blot, or lateral flow assays

• Assessment of sensitivity and specificity in clinical samples

Researchers developing CarA-based diagnostics should consider:

• Identifying immunodominant epitopes within the CarA protein

• Evaluating cross-reactivity with other bacterial species

• Determining sensitivity compared to existing diagnostic methods

• Assessing the time course of antibody development in infected hosts

Recent research has shown that recombinant chimeric proteins synthesized from immunogenic epitopes of B. henselae can be effective in detecting antibodies in feline serum samples . Similar approaches could be applied to CarA, potentially as part of a multi-antigen diagnostic panel. The development of specific antigens can increase both the sensitivity and specificity of bartonellosis diagnosis .

How can structural and functional studies of B. henselae CarA inform antimicrobial development?

CPSases represent potential targets for antimicrobial development due to their essential role in bacterial metabolism. Structure-function studies of B. henselae CarA can guide rational drug design through several approaches:

Target validation:

• Demonstration of essentiality through genetic approaches

• Assessment of vulnerability using conditional knockdown

• Determination of impact on bacterial viability and virulence

• Comparison with human CPSases to ensure selectivity

Inhibitor discovery strategies:

• Structure-based design focusing on the ATP-binding site

• Fragment-based screening to identify initial chemical matter

• Natural product screening for novel scaffolds

• Virtual screening using computational docking

Key considerations for inhibitor development:

• Selectivity against human CPSases

• Cell penetration into intracellular bacteria

• Stability in physiological conditions

• Resistance development potential

The development of selective inhibitors for B. henselae CarA could provide new therapeutic options for treating bartonellosis, including persistent infections that may be associated with serious human illnesses such as neoplastic, cardiovascular, neurocognitive, and rheumatologic conditions .

What controls and validation steps are essential when characterizing recombinant B. henselae CarA?

Rigorous experimental design is critical for ensuring reliable and reproducible results when working with recombinant B. henselae CarA. Essential controls and validation steps include:

Expression and purification controls:

• Empty vector controls processed identically to CarA-expressing constructs

• Inactive mutant versions (e.g., active site mutations) as negative controls

• Related bacterial CPSases as comparative controls

• Multiple purification batches to assess reproducibility

Activity assay validations:

• Enzyme concentration linearity tests

• Time-course linearity confirmation

• Substrate saturation curves

• Inhibition by known CPSase inhibitors

• Controls without essential cofactors

Structural integrity verification:

• Circular dichroism to confirm secondary structure

• Thermal shift assays to assess stability

• Limited proteolysis to probe domain organization

• Mass spectrometry to confirm protein integrity and modifications

Functional complementation:

• Genetic complementation of E. coli carA mutants

• Rescue of growth defects in defined media

• Metabolite profiling to confirm restored metabolic pathways

• Competition assays to assess fitness contributions

These validation steps are particularly important given the potential for recombinant proteins to exhibit altered properties compared to their native counterparts, including potential differences in oligomerization, activity, and stability.

How can high-throughput approaches be applied to B. henselae CarA research?

High-throughput methodologies can accelerate research on B. henselae CarA through systematic exploration of conditions, variants, and interactions:

Protein engineering and variant analysis:

• Deep mutational scanning to comprehensively map sequence-function relationships

• Directed evolution to improve stability or modify substrate specificity

• Combinatorial domain swapping with other CPSases

• High-throughput purification and activity screening

Interaction screening:

• Yeast two-hybrid or bacterial two-hybrid systems to identify protein partners

• Protein microarrays to map interactions with host proteins

• Pull-down assays coupled with mass spectrometry

• FRET-based interaction screening in live cells

Inhibitor discovery:

• Fragment-based screening using thermal shift assays

• Fluorescence-based activity assays adapted to 384 or 1536-well formats

• Compound library screening using biochemical and cell-based assays

• Computational virtual screening followed by targeted experimental validation

Crystallization condition optimization:

• Automated screening of thousands of crystallization conditions

• Systematic testing of surface mutations to improve crystallization

• Parallel testing of different construct boundaries

• Microfluidic crystallization approaches for minimal protein consumption

These high-throughput approaches can generate large datasets requiring sophisticated computational analysis but offer the potential for breakthrough discoveries regarding B. henselae CarA function and applications.

What are the critical considerations for studying B. henselae CarA in the context of bacterial pathogenesis?

Understanding the role of CarA in B. henselae pathogenesis requires specialized approaches that bridge biochemistry and infection biology:

Genetic manipulation strategies:

• Construction of carA conditional knockdown strains

• CRISPR interference for tunable gene repression

• Complementation with wild-type and mutant variants

• Reporter fusions to monitor expression during infection

Infection models:

• Primary endothelial cell infection models

• Erythrocyte invasion and persistence assays

• Ex vivo tissue models mimicking cat scratch sites

• Animal models of bartonellosis where ethically appropriate

Host interaction studies:

• Transcriptomic analysis of bacteria during different infection stages

• Metabolomic profiling to identify carbamoyl phosphate-dependent pathways

• Immunological studies to assess host recognition of CarA

• Imaging approaches to visualize bacterial metabolism in host cells

Environmental persistence:

• Survival studies under desiccation conditions

• Viability in environmental matrices

• Role of CarA in bacterial stress responses

• Nutrient limitation responses relevant to vector transmission

The extraordinary environmental stability of B. henselae, which can survive in various biological fluids and even after desiccation , suggests that metabolic enzymes like CarA may play important roles in bacterial persistence both within hosts and during transmission.