Recombinant Bartonella henselae Succinyl-CoA ligase [ADP-forming] subunit beta (sucC)

What is Bartonella henselae Succinyl-CoA ligase and what is its role in bacterial metabolism?

Bartonella henselae Succinyl-CoA ligase [ADP-forming] subunit beta (sucC) is a critical enzyme in the tricarboxylic acid (TCA) cycle, catalyzing the reversible conversion of succinyl-CoA and ADP to succinate and ATP. As a mitochondrial matrix enzyme, it forms part of the Krebs cycle and plays an essential role in energy metabolism .

The enzyme consists of two subunits:

• Alpha subunit (encoded by SUCLA1/SUCLG1)

• Beta subunit (encoded by SUCLA2/SUCLG2, also known as sucC)

The beta subunit determines substrate specificity, dictating whether the enzyme utilizes ADP or GDP as a substrate . Research indicates that the enzyme may also interact with nucleoside diphosphate kinase, suggesting a potential role in nucleotide metabolism, which could impact bacterial replication and survival .

How is recombinant B. henselae SucC typically expressed and purified for research applications?

Recombinant B. henselae SucC is most commonly expressed in Escherichia coli expression systems. The general protocol involves:

• Cloning: The sucC gene is amplified from B. henselae genomic DNA using PCR with specific primers that include appropriate restriction sites .

• Vector construction: The amplified gene is typically cloned into an expression vector containing:

  • An inducible promoter (often T7)

  • A His-tag sequence (commonly 10x His at the N-terminus)

  • Appropriate selection markers

• Expression: Transformation into E. coli expression strains (commonly BL21(DE3)) followed by induction with IPTG .

• Purification: Using proprietary chromatographic techniques, primarily:

  • Immobilized metal affinity chromatography (IMAC)

  • Size exclusion chromatography (SEC)

The resulting protein is typically a single, non-glycosylated polypeptide chain with a molecular mass of approximately 45-48 kDa . Purification typically yields protein with >80-95% purity as determined by SDS-PAGE .

What are the optimal storage conditions for maintaining the stability of recombinant B. henselae SucC?

The stability of recombinant B. henselae SucC depends on proper storage conditions. Based on established protocols :

Important stability considerations:

• Avoid multiple freeze-thaw cycles as they significantly reduce protein activity

• Aliquoting before freezing is recommended for proteins that will be used multiple times

• The presence of cryoprotectants (glycerol or sucrose) is crucial for maintaining the protein's native conformation during freezing

How can recombinant B. henselae SucC be utilized in diagnostic development for Bartonellosis?

Recombinant B. henselae SucC has significant potential for diagnostic applications due to its immunogenic properties. Research has established that:

• Serological detection: SucC belongs to the family of highly immunoreactive proteins produced by B. henselae, making it valuable for developing serological assays for Cat Scratch Disease (CSD) . Studies have shown that up to 95% of patients with CSD develop antibodies against B. henselae antigens .

• ELISA development methodology:

  • Optimal coating concentration: 1-5 μg/mL of purified recombinant SucC in carbonate buffer (pH 9.6)

  • Blocking: 3-5% BSA or non-fat milk in PBS-T

  • Sample dilution: 1:100-1:400 for patient sera

  • Detection: Anti-human IgG/IgM conjugated to HRP

  • Substrate: TMB with measurement at 450 nm

• Western blot applications:

  • SucC-β has been shown to interact predominantly with IgG antibodies from patient sera

  • Optimal concentration: 10-20 μg of protein per lane on 10-12% SDS-PAGE

  • Transfer conditions: 100V for 1 hour in Tris-glycine buffer with 20% methanol

  • Blocking: 5% non-fat milk in TBS-T

• Distinguishing between Bartonella species:
  Research has shown that while the SucC-β peptide shares 81-93% identity with equivalent regions from other Bartonella species, specific epitopes can be identified for species-specific detection .

What challenges arise when working with recombinant B. henselae SucC and how can they be addressed?

Several challenges have been reported when working with recombinant B. henselae SucC:

• Expression challenges:

  • Problem: Low solubility due to protein aggregation

  • Solution: Expression at lower temperatures (16-20°C) after induction; inclusion of solubility enhancers like sorbitol (0.5-1 M) or arginine (50-100 mM) in lysis buffer

• Purification issues:

  • Problem: Co-purification of bacterial chaperones

  • Solution: Include ATP (5-10 mM) and MgCl₂ (10-20 mM) in wash buffers to dissociate chaperone complexes; consider using stringent washing with increased imidazole concentrations (50-75 mM)

• Activity measurement:

  • Problem: Inconsistent enzymatic activity

  • Solution: Standardize assay conditions with defined buffer composition (typically 50 mM Tris-HCl pH 7.5-8.0, 10 mM MgCl₂, 0.1 mM succinyl-CoA, 0.5 mM ADP); include a positive control enzyme in each assay run

• Cross-reactivity in immunoassays:

  • Problem: Cross-reactivity with other bacterial species

  • Solution: Use epitope mapping to identify B. henselae-specific regions; implement absorption steps with lysates from related bacteria; perform confirmatory tests with alternative antigens

What methods are most effective for enhancing B. henselae growth and protein expression?

Recent research has demonstrated that blood supplementation significantly enhances B. henselae growth and consequently protein expression . The following methodology has proven effective:

• Optimal growth media:

  • BAPGM (Bartonella alpha-Proteobacteria growth medium) supplemented with sheep blood showed superior performance compared to Brugge medium with blood

  • Blood supplementation increased bacterial DNA concentration by an order of magnitude on average after day 9

• Culture conditions:

  • Temperature: 35-36°C

  • Atmosphere: 5% CO₂

  • Humidity: 100%

  • Culture duration: 10-12 days for optimal growth with blood supplementation

• Growth kinetics with blood supplementation:

  Culture Medium	Growth Without Blood	Growth With Blood	Optimal Sampling
  BAPGM	Decreased over time	Increased through day 10	Days 7, 14, 21
  Brugge	Moderate growth	Initial decrease, then significant increase after day 9	After day 9

• Inoculation protocol:

  • For optimal results, colonies should be harvested from blood agar plates

  • 5-6 colonies suspended in 10 mL of culture medium

  • Incubation in a T-25 tissue flask

This enhanced growth methodology directly translates to improved protein yields when expressing recombinant SucC.

How does B. henselae SucC contribute to pathogenesis and what experimental approaches can investigate this relationship?

Studies suggest SucC may have multifunctional roles in B. henselae pathogenesis beyond its metabolic function :

• Host-pathogen interaction studies:

  • Co-immunoprecipitation experiments have identified SucC as interacting with host mitochondrial proteins

  • Confocal microscopy using fluorescently-tagged SucC has demonstrated its localization in infected cells

  • Yeast two-hybrid screening has revealed potential interactions with host metabolic enzymes

• Mouse model development:
  Recent research has established a SCID/Beige immunocompromised mouse model that provides sustained B. henselae infection lasting up to 30 days, compared to the limited infection in immunocompetent mice . This model enables:

  • Tracking bacterial dissemination

  • Evaluating protein expression in vivo

  • Testing therapeutic interventions

• Methodological approaches to investigate SucC's role:

  • Gene knockout/knockdown: CRISPR-Cas9 or transposon mutagenesis to create SucC-deficient strains

  • Protein-protein interaction mapping: Pull-down assays with recombinant SucC to identify host interaction partners

  • Complementation studies: Reintroducing wild-type or mutant SucC to assess functional recovery

  • Comparative proteomics: Analyzing protein expression changes in SucC-deficient vs. wild-type strains

• Enzymatic activity modulation:
  Research has shown that SucC activity can be modulated by environmental conditions relevant to pathogenesis, suggesting its role may extend beyond metabolism to environmental adaptation during infection .

How can researchers address contradictory data when studying B. henselae SucC function?

When facing contradictory results in B. henselae SucC research, implement this systematic approach to resolve discrepancies :

• Comprehensive data examination:

  • Thoroughly analyze all findings to identify specific points of contradiction

  • Pay special attention to outliers that may have influenced results

  • Compare data with existing literature on SucC from other bacterial species

• Methodological evaluation:

  • Expression systems: E. coli strain variations (BL21 vs. Rosetta) can affect protein folding

  • Purification methods: Different chromatographic techniques may yield proteins with varying activity levels

  • Activity assays: Variations in buffer composition, pH, temperature, and substrate concentrations can significantly impact results

• Standardization protocol:

  • Create reference standards for enzyme activity measurements

  • Establish uniform experimental conditions across laboratories

  • Implement positive and negative controls in each experimental setup

• Alternative explanations to consider:

  • Post-translational modifications affecting enzyme activity

  • Protein-protein interactions unique to specific experimental conditions

  • Conformational changes due to buffer components or handling

• Data reconciliation framework:

  Contradiction Type	Investigation Approach	Resolution Method
  Activity discrepancies	Test multiple substrates (ADP vs. GDP)	Determine substrate preference
  Structural variations	Circular dichroism or thermal shift assays	Identify stability conditions
  Expression yield differences	Optimize codon usage and expression conditions	Standardize production protocol
  Varied immunoreactivity	Epitope mapping	Identify conserved antigenic regions

What are the most promising research directions for using recombinant B. henselae SucC in vaccine development?

While B. henselae SucC has not been extensively studied as a vaccine candidate, several approaches show promise based on its characteristics and research with related proteins :

• Epitope-based vaccine development:

  • Immunoinformatic analysis has identified immunodominant epitopes in SucC that could be incorporated into epitope-based vaccines

  • Peptide-based approaches using specific SucC regions can potentially elicit protective immunity without safety concerns of whole-protein vaccines

• Experimental approaches for vaccine evaluation:

  • T-cell stimulation assays: Measuring IFN-γ production by PBMCs exposed to recombinant SucC or derived peptides

  • Antibody neutralization tests: Evaluating whether anti-SucC antibodies can neutralize B. henselae infection in cell culture models

  • Challenge studies: Using the newly developed SCID/Beige mouse model to assess protection

• Advantages of SucC as a vaccine component:

  • High conservation across B. henselae strains, potentially providing broad protection

  • Strong immunogenicity, as demonstrated by high antibody titers in infected individuals

  • Potential for cross-protection against related Bartonella species due to sequence similarity

• Formulation considerations:

  • Adjuvant selection is critical for enhancing immunogenicity

  • Delivery systems (liposomes, nanoparticles) can improve antigen presentation

  • Combination with other B. henselae immunogens like Pap31 may provide synergistic protection

What is known about the structure-function relationship of B. henselae SucC and how does it compare to homologs in other species?

The structure-function relationship of B. henselae SucC has been partially elucidated through comparative studies with homologs from other species:

• Structural features:

  • The protein consists of a single polypeptide chain of approximately 45-48 kDa

  • Contains a conserved nucleotide-binding domain responsible for ADP binding

  • Features a CoA-binding domain critical for interaction with succinyl-CoA

  • Requires interaction with the alpha subunit (SucD) to form a functional heterodimer

• Comparative analysis with other species:

  • Shares significant homology with SucC from other alpha-proteobacteria

  • Particularly high conservation within the Bartonella genus

  • Key catalytic residues are preserved across species, while surface-exposed regions show greater variability

• Functional insights:

  • The beta subunit determines whether the enzyme utilizes ADP or GDP as a substrate

  • Mutations in homologous human genes (SUCLA2/SUCLG2) cause severe metabolic disorders, highlighting the enzyme's critical role

  • The enzyme's reverse reaction (ATP synthesis) may be particularly important under energy-limited conditions during infection

• Species-specific variations:

  Species	Identity with B. henselae SucC	Key Functional Differences
  B. bacilliformis	81-93%	Minor variations in substrate affinity
  B. quintana	~90%	Similar kinetic properties
  E. coli	65-70%	Different regulatory mechanisms
  Human	40-45%	GDP preference rather than ADP

What enzymatic assays are most appropriate for characterizing the activity of recombinant B. henselae SucC?

Several enzymatic assays have been developed to characterize SucC activity, each with specific advantages:

• Coupled spectrophotometric assays:

  • Forward reaction (succinyl-CoA → succinate):

    • Couples ADP formation to pyruvate kinase and lactate dehydrogenase reactions

    • Monitors NADH oxidation at 340 nm

    • Components: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 0.2 mM NADH, 1 mM phosphoenolpyruvate, 10 U/mL pyruvate kinase, 15 U/mL lactate dehydrogenase, 0.1 mM succinyl-CoA, 0.5 mM ADP

  • Reverse reaction (succinate → succinyl-CoA):

    • Couples succinyl-CoA formation to DTNB reaction

    • Monitors thionitrobenzoate formation at 412 nm

    • Components: 50 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 0.2 mM DTNB, 0.1 mM CoA, 1 mM ATP, 10 mM succinate

• Direct product quantification:

  • HPLC-based assay:

    • Directly measures succinyl-CoA consumption or formation

    • Provides more accurate kinetic parameters

    • Typical conditions: C18 reverse-phase column, mobile phase of 100 mM sodium phosphate (pH 4.5) with acetonitrile gradient, detection at 254 nm

• Isotope-based assays:

  • [³²P]-ADP incorporation:

    • Measures formation of [³²P]-ATP

    • Highly sensitive for low enzyme concentrations

    • Requires radioactive handling precautions

• Recommended controls and validation:

  • Include commercially available succinyl-CoA synthetase as positive control

  • Verify linearity of enzyme activity with protein concentration

  • Confirm substrate saturation with Michaelis-Menten plots

  • Typical specific activity: 2-10 μmol/min/mg protein under optimal conditions

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

Site-directed mutagenesis provides a powerful approach to investigate the catalytic mechanism of B. henselae SucC:

• Experimental design for mutagenesis studies:

  • PCR-based all-recombinant cloning: A rapid and efficient methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation

  • Primer design: Forward and reverse primers containing the desired mutation with ~15-20 nucleotides flanking the mutation site

  • PCR conditions: High-fidelity polymerase (e.g., Q5) with optimized annealing temperatures (typically 59-64°C)

• Key residues for targeted mutagenesis:
  Based on homology with well-characterized succinyl-CoA synthetases:

  • Histidine residues in the active site (involved in phosphoryl transfer)

  • Lysine residues in the nucleotide-binding domain

  • Aspartate residues coordinating the magnesium ion

  • Arginine residues interacting with the substrate carboxyl group

• Functional analysis of mutants:

  • Steady-state kinetics: Determine kcat and Km values for wild-type and mutant enzymes

  • Isothermal titration calorimetry: Measure binding affinities for substrates

  • Thermal stability assays: Assess structural integrity using circular dichroism or differential scanning fluorimetry

  • pH-rate profiles: Identify ionizable groups essential for catalysis

• Structure-guided mutagenesis strategy:

  Domain	Target Residues	Expected Effect	Validation Method
  Nucleotide binding	Conserved P-loop	Altered ADP/GDP specificity	Nucleotide preference assay
  CoA binding	Hydrophobic pocket	Changed acyl-CoA affinity	ITC binding studies
  Subunit interface	α/β interaction surface	Disrupted heterodimer formation	Size-exclusion chromatography
  Catalytic site	His-Asp dyad	Reduced phosphoryl transfer	Product formation rate

How does B. henselae SucC compare to other immunogenic proteins as a diagnostic target?

B. henselae SucC shows distinctive characteristics compared to other immunogenic proteins used in diagnostics:

• Comparative immunoreactivity:

  Protein	Molecular Weight	Primary Antibody Response	Sensitivity in Diagnostics	Cross-reactivity
  SucC (SucB)	45-48 kDa	IgG	High	Moderate with other Bartonella
  Pap31	31 kDa	IgM	Very high (97.2%)	Low (species-specific)
  GroEL	60 kDa	IgG	Moderate	High with other bacteria
  SCS-α	30-31 kDa	IgM/IgG	High (34.5% for IgG)	Moderate

• Diagnostic performance characteristics:

  • SucC (SucB) shows high immunoreactivity, with up to 95% of Cat Scratch Disease patients developing antibodies against B. henselae antigens

  • Unlike Pap31, which is highly specific to particular Bartonella species, SucC shows some cross-reactivity with homologs from other Bartonella species (81-93% identity)

  • The immunogenic protein dihydrolipoamide-succinyltransferase (SucB) is considered one of the most important antigens for Cat Scratch Disease diagnosis

• Advantages of SucC as a diagnostic target:

  • Stable protein that can be produced recombinantly with high purity

  • Strong immunogenicity leading to detectable antibody responses

  • Conserved across B. henselae strains, reducing false negatives

• Limitations compared to other targets:

  • Less species-specific than Pap31, which shows potential to discriminate between B. bacilliformis and other Bartonella species

  • May show cross-reactivity with SCS-β from other bacterial genera, such as Brucella or Anaplasma

What are the most effective protocols for detecting anti-SucC antibodies in patient samples?

Several protocols have been developed for detecting anti-SucC antibodies in patient samples:

• ELISA optimization for anti-SucC detection:

  • Antigen coating: 2-5 μg/mL of purified recombinant SucC in carbonate buffer (pH 9.6), incubated overnight at 4°C

  • Blocking: 3% BSA or 5% non-fat milk in PBS-T (0.05% Tween-20), 1-2 hours at room temperature

  • Sample dilution: 1:100-1:200 for serum samples in blocking buffer

  • Detection system: HRP-conjugated anti-human IgG or IgM (1:5000-1:10000)

  • Substrate: TMB with reaction stopped using 2N H₂SO₄, read at 450 nm

  • Controls: Include known positive and negative sera, as well as buffer-only wells

• Western blot protocol:

  • Sample preparation: 0.5-1 μg recombinant SucC per lane on 12% SDS-PAGE

  • Transfer: Semi-dry or wet transfer to PVDF membrane (100V for 1 hour)

  • Blocking: 5% non-fat milk in TBS-T (0.1% Tween-20), 1 hour at room temperature

  • Primary antibody: Patient serum diluted 1:100-1:500 in blocking buffer, overnight at 4°C

  • Secondary antibody: HRP-conjugated anti-human IgG/IgM (1:5000), 1 hour at room temperature

  • Development: Enhanced chemiluminescence detection

• Line immunoassay approach:

  • Recombinant SucC immobilized on nitrocellulose strips

  • Multiple antigens can be tested simultaneously

  • Provides semi-quantitative results based on band intensity

  • Higher throughput than Western blotting, more discriminatory than ELISA

• Performance optimization:

  • For maximum sensitivity, combine IgG and IgM detection

  • Consider using reduced sample dilution (1:50) for early-stage infections

  • Implement confirmation tests with alternative B. henselae antigens

  • When possible, complement serological testing with PCR detection of bacterial DNA

What techniques can be used to investigate the role of SucC in B. henselae metabolism and virulence?

Multiple experimental approaches can elucidate SucC's role in B. henselae metabolism and virulence:

• Gene manipulation techniques:

  • Conditional knockdown: Using antisense RNA or CRISPR interference to reduce SucC expression without completely eliminating it

  • Allelic replacement: Generating point mutations in the chromosomal sucC gene to study specific residues

  • Complementation studies: Reintroducing wild-type or mutant sucC genes to confirm phenotypes

• Metabolic studies:

  • Isotope tracing: Using ¹³C-labeled substrates to track carbon flow through the TCA cycle

  • Metabolomics: Analyzing changes in metabolite profiles when SucC function is altered

  • Respirometry: Measuring oxygen consumption as a readout of TCA cycle function

• Host-pathogen interaction studies:

  • Cell infection models: Comparing invasion and persistence of wild-type and SucC-deficient B. henselae in endothelial cells

  • Bacterial survival assays: Testing resistance to oxidative stress and nutrient limitation

  • SCID/Beige mouse model: Recently developed immunocompromised mouse model allowing for sustained B. henselae infection (up to 30 days)

• Protein localization and trafficking:

  • Immunofluorescence microscopy: Using anti-SucC antibodies to track protein localization

  • Subcellular fractionation: Determining whether SucC is strictly cytoplasmic or associated with membranes

  • Bacterial two-hybrid system: Identifying protein interaction partners that might link metabolism to virulence

• Expression analysis:

  • qRT-PCR: Measuring sucC transcript levels under different growth conditions

  • Proteomics: Comparing proteome changes in response to SucC manipulation

  • Reporter gene fusions: Monitoring sucC promoter activity during infection