Recombinant Bartonella tribocorum Lipoyl synthase (lipA)

What is Bartonella tribocorum Lipoyl synthase (lipA) and what is its biochemical function?

Bartonella tribocorum Lipoyl synthase (lipA) is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the final step in lipoic acid biosynthesis, specifically the insertion of sulfur atoms into octanoyl chains. The enzyme has the EC designation 2.8.1.8 and is also known by several alternative names including Lip-syn, LS, Lipoate synthase, Lipoic acid synthase, and Sulfur insertion protein LipA . The full-length protein consists of 320 amino acids and contains essential iron-sulfur clusters that are critical for its catalytic activity.

Functionally, Lipoyl synthase performs a key post-translational modification by adding lipoic acid to specific proteins involved in oxidative metabolism. This modification is essential for the activity of several multienzyme complexes, including pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, which are central to energy metabolism in bacteria.

What are the key structural domains in Bartonella tribocorum Lipoyl synthase and how do they relate to function?

Based on sequence analysis of Bartonella tribocorum Lipoyl synthase, the protein contains several key structural elements typical of radical SAM enzymes:

• CX₃CX₂C motif: Found in the N-terminal region (around positions 36-44: EAGCPNIGEC), this conserved cysteine-rich motif is responsible for coordinating the [4Fe-4S] cluster essential for radical SAM activity .

• Iron-sulfur binding domains: Additional cysteine-rich regions (such as CACFCNVA at positions 96-103) likely coordinate additional iron-sulfur clusters that serve as sulfur donors during catalysis .

• SAM binding domain: The central region of the protein contains the SAM binding pocket which positions the SAM molecule for electron transfer from the iron-sulfur cluster.

• Substrate binding region: The C-terminal domain likely contains regions involved in binding the octanoyl substrate.

The sequence "RTHKLVTVCE EAGCPNIGEC WSQRHASFMI LGEICTRACA FCNVATGIPL" contains critical residues for both metal coordination and catalytic activity .

How should recombinant Bartonella tribocorum Lipoyl synthase be stored and handled to maintain activity?

For optimal handling of recombinant Bartonella tribocorum Lipoyl synthase:

• Storage temperature: Store at -20°C for general use, or at -80°C for extended storage to maintain protein stability and activity .

• Aliquoting: Divide the protein into working aliquots to avoid repeated freeze-thaw cycles, which can compromise enzyme activity. Working aliquots can be stored at 4°C for up to one week .

• Reconstitution protocol:

  • Briefly centrifuge the vial before opening to bring contents to the bottom

  • Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (with 50% being recommended) to prevent freezing damage

  • Aliquot for long-term storage

• Special considerations: As an iron-sulfur protein, Lipoyl synthase is oxygen-sensitive. Handling under anaerobic or low-oxygen conditions may be necessary for studies requiring full enzymatic activity.

What experimental approaches are recommended for studying Bartonella tribocorum Lipoyl synthase activity?

For studying Bartonella tribocorum Lipoyl synthase activity, researchers should consider the following experimental approaches:

• Spectrophotometric assays: Monitor the formation of lipoylated proteins or the consumption of substrates using spectrophotometric methods.

• Reconstitution of iron-sulfur clusters:

  • Chemical reconstitution: Incubation with ferrous iron and sulfide under reducing conditions

  • Enzymatic reconstitution: Using cysteine desulfurase and iron sources

• Anaerobic enzyme assays: Due to the oxygen sensitivity of iron-sulfur clusters, activity assays should be performed under anaerobic conditions using:

  • Glove boxes

  • Sealed cuvettes with oxygen scavengers

  • Specialized anaerobic equipment

• Mass spectrometry: For direct detection of lipoylated products and intermediates in the reaction.

• EPR spectroscopy: To characterize the iron-sulfur clusters and radical intermediates formed during catalysis.

How can researchers verify the purity and activity of recombinant Bartonella tribocorum Lipoyl synthase preparations?

To verify the purity and activity of recombinant Bartonella tribocorum Lipoyl synthase:

• Purity assessment:

  • SDS-PAGE analysis: The recombinant protein should show >85% purity on SDS-PAGE

  • Size exclusion chromatography: To verify monodispersity and absence of aggregates

  • Western blot: Using antibodies specific to the protein or any affinity tags

• Activity verification:

  • Enzymatic assays measuring the insertion of sulfur into octanoyl substrates

  • Monitoring the formation of protein-bound lipoic acid

  • Spectroscopic analysis of iron-sulfur cluster integrity (UV-Vis and EPR)

• Structural integrity:

  • Circular dichroism to assess secondary structure

  • Thermal shift assays to evaluate protein stability

  • Iron and sulfide content analysis to verify cluster incorporation

A typical verification protocol should include both purity and activity assessments, as high purity does not necessarily correlate with enzymatic activity, especially for iron-sulfur proteins that may lose cluster integrity during purification.

What are potential applications of recombinant Bartonella tribocorum Lipoyl synthase in pathogenesis research?

Recombinant Bartonella tribocorum Lipoyl synthase has several potential applications in pathogenesis research:

• Study of metabolic adaptations during infection:

  • Lipoic acid-dependent metabolism may be crucial for bacterial survival in host environments

  • Understanding how lipoylation contributes to bacterial persistence during chronic infections

• Investigation of immune evasion mechanisms:

  • Bartonella species are known as "stealth pathogens" that evade host immune recognition

  • Lipoproteins and their modifications may play a role in TLR recognition, similar to how Bartonella species modify their LPS to evade TLR4 and flagellin to evade TLR5

• Drug target evaluation:

  • As an essential enzyme, LipA represents a potential target for new antimicrobials

  • Structure-based drug design targeting the unique features of Bartonella tribocorum LipA

• Comparative studies with other Bartonella species:

  • B. tribocorum LipA can be compared with homologs from B. henselae , B. quintana, and other species to understand conserved mechanisms of pathogenesis

  • Such studies may reveal why some Bartonella species cause persistent infections in specific hosts

• Host-pathogen interaction studies:

  • Using recombinant LipA to understand how lipoylated proteins interact with host cells

  • Investigating potential roles in bacterial entry, BCV (Bartonella-containing vacuole) formation, and delayed lysosomal fusion as observed with B. henselae

How does Bartonella tribocorum Lipoyl synthase compare to homologs from other Bartonella species?

A comparative analysis of Lipoyl synthase from different Bartonella species reveals important insights:

Key observations regarding comparative analysis:

• Conserved catalytic motifs: The CX₃CX₂C motif and other cysteine-rich regions for iron-sulfur cluster binding are highly conserved across Bartonella species, reflecting their essential enzymatic function.

• Variable regions: Differences in non-catalytic regions may reflect adaptations to different host environments or interactions with host-specific proteins.

• Functional implications: Despite high sequence conservation, subtle differences in Lipoyl synthase structure might contribute to the distinct pathogenicity profiles of different Bartonella species, similar to how variations in other proteins like BadA (Bartonella adhesin A) affect macrophage interactions in B. henselae .

What role might Lipoyl synthase play in Bartonella's ability to establish persistent infections?

Lipoyl synthase may significantly contribute to Bartonella's persistence mechanisms:

• Metabolic adaptation: By providing lipoic acid as a cofactor for key metabolic enzymes, LipA enables metabolic flexibility that may be crucial for adaptation to nutrient-limited host environments.

• Connection to immune evasion: While not directly mentioned in the search results, LipA-dependent lipoylation may influence the surface properties or metabolic state of Bartonella in ways that contribute to immune evasion. Bartonella species are known to evade immune recognition through several mechanisms:

  • Modified LPS that weakly activates TLR4 (1000–10,000-fold less active than Salmonella enterica LPS)

  • Altered flagellin that escapes TLR5 recognition while preserving motility

  • Possible TLR2-dependent recognition mechanisms

• Persistence in the primary niche: Bartonella species establish a primary niche (possibly endothelial cells), from which they are periodically seeded into the bloodstream. Lipoylated proteins may play roles in:

  • Bacterial survival in this niche

  • Regulation of the seeding process

  • Adaptation to different host cell types during infection

• Contribution to unique trafficking: B. henselae enters macrophages in a unique Bartonella-containing vacuole (BCV) that delays lysosomal fusion, allowing extended intracellular survival. Metabolic adaptations dependent on lipoylated proteins may contribute to this process .

What methodological challenges should researchers anticipate when studying Bartonella tribocorum Lipoyl synthase?

Researchers working with Bartonella tribocorum Lipoyl synthase should anticipate several methodological challenges:

• Oxygen sensitivity:

  • Iron-sulfur clusters are sensitive to oxygen, potentially leading to protein inactivation

  • Solution: Work under anaerobic conditions using glove boxes or sealed containers with oxygen scavengers

• Protein stability issues:

  • Repeated freeze-thaw cycles can compromise enzyme activity

  • Solution: Store working aliquots at 4°C for up to one week and prepare fresh aliquots as needed

• Iron-sulfur cluster reconstitution:

  • Heterologously expressed protein may contain incompletely formed clusters

  • Solution: In vitro reconstitution protocols using iron, sulfide, and reducing agents

• Assay development challenges:

  • Direct measurement of sulfur insertion is technically challenging

  • Solution: Develop coupled assays or use sensitive detection methods like mass spectrometry

• Expression and purification:

  • Expression in standard E. coli systems may result in poorly active enzyme

  • Solution: Consider specialized expression systems with iron-sulfur cluster assembly machinery or expression in yeast (as used for the commercial preparation)

• Functional validation:

  • Confirming that in vitro activity reflects physiological function

  • Solution: Complement lipA-deficient bacterial strains with the recombinant enzyme

How might inhibitors of Lipoyl synthase be designed as potential antimicrobials against Bartonella infections?

Designing inhibitors of Bartonella tribocorum Lipoyl synthase as potential antimicrobials should consider the following strategic approaches:

• Targeting the SAM binding site:

  • SAM analogs with modifications that allow binding but prevent radical generation

  • Compounds that compete with SAM binding but lack the ability to participate in radical chemistry

• Iron-sulfur cluster disruption:

  • Small molecules that bind near the cluster coordination sites

  • Compounds that alter the redox state of the iron-sulfur clusters

• Substrate binding site targeting:

  • Octanoyl substrate analogs with modifications that prevent catalysis

  • Compounds that mimic transition state structures during sulfur insertion

• Allosteric inhibitors:

  • Molecules that bind to non-catalytic regions and induce conformational changes

  • Compounds that prevent essential protein dynamics required for catalysis

• Rational design considerations:

  • Use the protein sequence information from Bartonella tribocorum Lipoyl synthase to create homology models if crystal structures are unavailable

  • Focus on regions with amino acid sequences "RTHKLVTVCE EAGCPNIGEC" and "CACFCNVATGIPL" which likely contain essential catalytic elements

  • Develop selectivity by targeting regions that differ between bacterial and mammalian lipoyl synthases

• Screening approaches:

  • High-throughput biochemical assays using reconstituted enzyme

  • Fragment-based screening to identify initial binding compounds

  • Virtual screening using computational models of the enzyme structure

What insights can comparative studies of Lipoyl synthase provide about Bartonella species adaptation to different hosts?

Comparative studies of Lipoyl synthase across Bartonella species can reveal important insights about host adaptation:

• Evolutionary analysis:

  • Sequence divergence patterns in LipA may correlate with host specificity patterns

  • Comparison between B. tribocorum (infects rats), B. henselae (cats and humans), and B. quintana (humans) could reveal host-specific adaptations

• Functional variations:

  • Different Bartonella species might have evolved variations in LipA activity or regulation

  • These differences could reflect metabolic adaptations to distinct host environments

• Integration with pathogenesis mechanisms:

  • Similar to how Bartonella species have evolved different mechanisms for immune evasion (e.g., modified LPS and flagellin) , LipA may show species-specific features

  • The role of LipA in supporting the "stealth pathogen" lifestyle of Bartonella could vary between species that cause different clinical manifestations

• Host-specific metabolism:

  • Variations in LipA might reflect different metabolic requirements during:

    • Intraerythrocytic bacteremia (which may last weeks, months, or years)

    • Persistence in the primary niche

    • Adaptation to arthropod vectors for transmission

• Research methodology:

  • Express and characterize LipA from multiple Bartonella species

  • Compare enzymatic properties, substrate specificity, and activity under different conditions

  • Correlate biochemical differences with genomic analyses and host range information

How might Lipoyl synthase activity be connected to the unique intracellular trafficking of Bartonella in host cells?

The connection between Lipoyl synthase activity and Bartonella's unique intracellular trafficking offers an intriguing research direction:

• Potential roles in Bartonella-containing vacuole (BCV) formation:

  • B. henselae enters macrophages in a unique vacuolar compartment (BCV) that lacks typical early endocytic markers

  • BCV shows delayed fusion with lysosomes (24 hours versus 2 hours for heat-killed bacteria)

  • Lipoylated proteins might influence membrane properties or signaling events that modify this trafficking

• Metabolic adaptation during intracellular survival:

  • Lipoic acid-dependent metabolic pathways could be essential for bacterial survival in specialized intracellular niches

  • The delayed lysosomal targeting observed with B. henselae might be supported by metabolic states dependent on lipoylated proteins

• Connection to identified trafficking factors:

  • Four genes affecting BCV trafficking have been identified in B. henselae:

    • Putative virulence-associated protein VapA5

    • Putative heme-binding protein HbpD

    • D-serine/D-alanine/glycine transport protein CycA

    • An uncharacterized protein

  • Potential functional or regulatory relationships between these proteins and lipoylated enzymes merit investigation

• Experimental approaches:

  • Develop LipA-deficient Bartonella strains and assess their intracellular trafficking

  • Identify and characterize lipoylated proteins involved in Bartonella-host interactions

  • Study how inhibition of lipoic acid metabolism affects the formation and trafficking of BCVs

• Comparative analysis with other pathogens:

  • Other bacterial pathogens that modify endocytic trafficking might share mechanistic features

  • Lipoic acid metabolism has been implicated in virulence for other intracellular pathogens, suggesting potential common mechanisms

What are common pitfalls in activity assays for Lipoyl synthase and how can they be addressed?

Researchers working with Lipoyl synthase activity assays should be aware of these common pitfalls and their solutions:

• Oxygen sensitivity issues:

  • Problem: Loss of activity due to iron-sulfur cluster oxidation

  • Solution: Perform all steps under strict anaerobic conditions; use oxygen scavengers in buffers

• Incomplete iron-sulfur cluster incorporation:

  • Problem: Suboptimal activity due to poorly formed clusters

  • Solution: Implement chemical or enzymatic reconstitution protocols before activity assays

• Substrate limitations:

  • Problem: Low activity due to inappropriate substrate presentation

  • Solution: Test multiple substrate forms (free octanoic acid, octanoyl-ACP, protein-bound octanoyl groups)

• Electron donation system issues:

  • Problem: Insufficient electron supply for radical SAM chemistry

  • Solution: Optimize reducing systems (dithionite, flavodoxin/flavodoxin reductase/NADPH)

• Detection sensitivity:

  • Problem: Difficulty quantifying low levels of lipoylated products

  • Solution: Develop more sensitive detection methods (MS-based approaches, antibody-based detection)

• Buffer incompatibilities:

  • Problem: Buffer components interfering with activity or detection

  • Solution: Systematically test different buffer compositions and pH values

• Data interpretation challenges:

  • Problem: Distinguishing enzymatic from non-enzymatic reactions

  • Solution: Include proper controls (heat-inactivated enzyme, reactions without key components)

What are the implications of studying recombinant Bartonella tribocorum Lipoyl synthase versus the native enzyme?

Understanding the differences between recombinant and native Lipoyl synthase is crucial for proper experimental design and data interpretation:

• Post-translational modifications:

  • Recombinant protein expressed in yeast may have different post-translational modifications than the native bacterial enzyme

  • This could affect activity, stability, or protein-protein interactions

• Iron-sulfur cluster formation:

  • Heterologous expression systems may produce protein with incompletely formed or incorrectly assembled iron-sulfur clusters

  • The recombinant protein may require additional reconstitution steps not needed for the native enzyme

• Protein folding considerations:

  • Expression in non-native hosts may result in subtle structural differences

  • The recombinant protein has a purity of >85% according to SDS-PAGE , suggesting potential contaminants

• Tag effects:

  • The recombinant protein may contain affinity tags (though the specific tag type is determined during manufacturing)

  • These tags could influence activity, especially if located near catalytic residues

• Experimental design implications:

  • Benchmark recombinant enzyme against native enzyme when possible

  • Consider complementation studies in lipA-deficient bacteria to validate functional equivalence

  • Be cautious when extrapolating in vitro findings to in vivo situations

• Advantages of recombinant protein:

  • Availability in larger quantities than could be purified from native sources

  • Consistent preparation that reduces experimental variability

  • Opportunity for protein engineering to facilitate specific experimental approaches

How can researchers design meaningful experiments to investigate the role of Lipoyl synthase in Bartonella pathogenesis?

To design meaningful experiments investigating Lipoyl synthase in Bartonella pathogenesis:

• Genetic manipulation approaches:

  • Create lipA deletion mutants and assess:

    • Growth characteristics in different media

    • Ability to infect host cells and establish BCVs

    • Capacity to evade immune recognition

    • Persistence in experimental infection models

• Complementation studies:

  • Rescue phenotypes with:

    • Wild-type lipA

    • Catalytically inactive mutants

    • lipA from other bacterial species

  • This approach can establish causality between enzymatic activity and observed phenotypes

• Proteomic identification of lipoylated proteins:

  • Identify all proteins modified by LipA in Bartonella

  • Determine how the lipoylation profile changes during different infection stages

  • Compare with other Bartonella species to identify common and species-specific targets

• Integrative approaches:

  • Connect LipA activity to known pathogenesis mechanisms:

    • Delayed BCV-lysosome fusion

    • Ability to establish the primary niche

    • Relapsing bacteremia characteristic of Bartonella infections

    • Transition between arthropod vector and mammalian host

• Small molecule studies:

  • Use specific inhibitors of LipA to:

    • Assess effects on in vitro growth

    • Determine impact on host cell interactions

    • Evaluate potential as antimicrobial agents

• Host response studies:

  • Investigate how lipoylation affects:

    • Recognition by host pattern recognition receptors

    • Inflammatory cytokine production

    • Activation of innate and adaptive immunity

  • Compare with known immune evasion mechanisms like TLR4/TLR5 avoidance