Recombinant Bartonella henselae (Dimethylallyl)adenosine tRNA methylthiotransferase MiaB (miaB)

What is Bartonella henselae MiaB and what is its function?

Bartonella henselae MiaB is a bifunctional radical-S-adenosylmethionine (radical-AdoMet) enzyme that catalyzes the posttranscriptional methylthiolation of N-6-isopentenyladenosine in tRNAs . This enzyme plays a critical role in tRNA modification which affects protein translation efficiency and accuracy. Similar to other radical-S-adenosylmethionine enzymes, B. henselae MiaB contains essential [4Fe-4S] clusters that are crucial for its catalytic function . The enzyme's activity contributes to bacterial survival and potentially to virulence through its effects on protein synthesis regulation.

How does Bartonella henselae MiaB differ from MiaB in other bacterial species?

While MiaB shares conserved functional domains across bacterial species, the B. henselae version has specific structural characteristics that may correlate with this pathogen's unique lifecycle. Based on comparative analyses with better-characterized MiaB enzymes like that from Thermotoga maritima, B. henselae MiaB likely contains two distinct [4Fe-4S]²⁺ˊ¹⁺ clusters . One cluster is coordinated by three conserved cysteines in the radical-AdoMet motif (typically Cys150, Cys154, and Cys157), while the other is likely coordinated by three N-terminal conserved cysteines (typically Cys10, Cys46, and Cys79) . These clusters, though structurally similar, differ in redox properties and EPR characteristics when in the reduced [4Fe-4S]¹⁺ state.

What structural features of B. henselae MiaB are critical for investigating its role in pathogenicity?

The dual [4Fe-4S] cluster organization in B. henselae MiaB presents a critical structural feature for investigating pathogenicity relationships. The second [4Fe-4S] cluster may serve as a sacrificial sulfur donor in the methylthiolation reaction, similar to what has been proposed for related enzymes like biotin synthase and lipoate synthase . This unique structural arrangement affects the enzyme's catalytic efficiency, which in turn may influence bacterial persistence in host tissues. Researchers should focus on the cysteine-rich regions that coordinate these iron-sulfur clusters when designing site-directed mutagenesis experiments to probe structure-function relationships.

How does B. henselae MiaB contribute to bacterial persistence in the host during infection?

B. henselae exhibits remarkable persistence in host tissues despite apparent clearance of cultivatable bacteria. In a murine model, while organs were cleared of cultivatable B. henselae within 6 days, bacterial DNA remained detectable in liver tissue for at least 3 months . MiaB's role in tRNA modification likely contributes to this persistence by optimizing translation efficiency under stress conditions within the host. The enzyme may facilitate adaptation to changing nutrient availability and immune pressures during different phases of infection. Researchers should consider designing experiments that monitor MiaB activity levels during various stages of infection to correlate with bacterial persistence patterns.

What are the challenges in purifying active recombinant B. henselae MiaB?

Purifying active recombinant B. henselae MiaB presents several challenges:

• Iron-sulfur cluster integrity: The two [4Fe-4S] clusters are oxygen-sensitive and can degrade during purification, resulting in inactive enzyme .

• Reconstitution requirements: Proper enzymatic activity often requires reconstitution of iron-sulfur clusters under strictly anaerobic conditions.

• Protein solubility: Recombinant MiaB may form inclusion bodies in expression systems, necessitating optimization of solubilization and refolding protocols.

• Stability concerns: The purified enzyme may exhibit limited stability, requiring optimization of buffer conditions and storage protocols.

To overcome these challenges, researchers should perform purification under anaerobic conditions, use reducing agents like dithiothreitol or β-mercaptoethanol throughout the process, and optimize expression conditions (temperature, inducer concentration, and duration) to maximize soluble protein production.

What assays are most effective for measuring B. henselae MiaB enzymatic activity?

The following assays are effective for measuring B. henselae MiaB enzymatic activity:

• Mass spectrometry-based assays: These directly detect the methylthiolated tRNA product and are considered the gold standard. LC-MS/MS approaches can provide quantitative analysis of modified nucleosides after enzymatic digestion of tRNA.

• Radioactive assays: Using S³⁵-labeled substrates or C¹⁴-labeled S-adenosylmethionine allows tracking of the methylthio group transfer to tRNA substrates.

• Spectroscopic methods: UV-visible spectroscopy, resonance Raman spectroscopy, and Mössbauer spectroscopy can monitor the state of the iron-sulfur clusters before and after catalysis .

• Coupled enzyme assays: These indirectly measure MiaB activity by linking it to a detectable signal through secondary enzymatic reactions.

For optimal results, researchers should use purified unmodified tRNA substrates and ensure anaerobic conditions throughout the assay to maintain iron-sulfur cluster integrity.

How should immunological studies be designed to investigate host responses to B. henselae MiaB?

Design of immunological studies investigating host responses to B. henselae MiaB should consider:

• Animal model selection: C57BL/6 mice have been successfully used to study B. henselae infection and immune responses . This model shows granulomatous inflammation in liver tissue, reaching maximum intensity during the fourth week of infection and resolving within 12 weeks .

• T-cell response assessment: Measure proliferative responses of spleen cells from infected mice using heat-killed B. henselae or purified recombinant MiaB . Include conditions with anti-CD4 and anti-CD8 antibodies to determine the roles of different T-cell subsets.

• Cytokine profiling: Analyze cytokine release patterns to characterize the Th cell responses. Consider measuring IFN-γ, IL-4, IL-10, and IL-17 to determine Th1/Th2/Th17 balance.

• Antibody response characterization: Develop ELISAs using recombinant MiaB to detect specific antibody responses, and analyze IgG isotypes to further characterize the immune response type .

What are the optimal conditions for expressing and purifying recombinant B. henselae MiaB with preserved enzymatic activity?

The following protocol outlines optimal conditions for expression and purification of active recombinant B. henselae MiaB:

• Expression system selection:

  • E. coli BL21(DE3) with pET-based vectors containing the MiaB gene with a His-tag for purification .

  • Consider co-expression with iron-sulfur cluster assembly proteins (ISC or SUF system) to improve yield of active protein.

• Culture conditions:

  • Grow cultures in iron-supplemented media (50-100 μM ferric ammonium citrate).

  • Induce at OD₆₀₀ of 0.6-0.8 with 0.1-0.2 mM IPTG.

  • Lower the temperature to 18-20°C after induction.

  • Add L-cysteine (0.5 mM) to promote iron-sulfur cluster formation.

• Purification strategy:

  • Perform all steps in an anaerobic chamber with degassed buffers containing 5 mM DTT.

  • Use immobilized metal affinity chromatography (IMAC) followed by gel filtration.

  • Include 10% glycerol in all buffers to stabilize the protein.

  • Consider iron-sulfur cluster reconstitution after purification using ferrous ammonium sulfate, sodium sulfide, and a reducing agent under strict anaerobic conditions.

• Activity verification:

  • Measure iron and sulfide content to confirm the presence of [4Fe-4S] clusters.

  • Verify enzymatic activity using tRNA methylthiolation assays.

  • Monitor UV-visible absorption spectra for characteristic features of iron-sulfur clusters.

How can recombinant B. henselae MiaB be used to develop improved diagnostic tools for Bartonellosis?

Recombinant B. henselae MiaB represents a potential diagnostic target for improving Bartonellosis detection. Current serological methods such as immunofluorescent antibody assays (IFAs), Western blot (WB), and enzyme-linked immunosorbent assays (ELISAs) for diagnosing canine and human Bartonelloses frequently generate false negative results . Researchers can develop diagnostic approaches using recombinant MiaB through:

When developing these tools, researchers should determine optimal cutoff values through ROC curve analysis using serum samples from confirmed cases and controls. For example, recombinant whole Pap31 demonstrated 72% sensitivity and 61% specificity at a cutoff value of 0.215 for human Bartonelloses , providing a benchmark for MiaB-based diagnostic development.

What role does B. henselae MiaB play in bacterial virulence and host-pathogen interactions?

B. henselae MiaB's role in virulence and host-pathogen interactions can be investigated through several approaches:

• Gene knockout studies: Create MiaB-deficient mutants and assess their ability to:

  • Survive in various host cell types

  • Persist in animal models

  • Induce granulomatous inflammation in liver tissue

  • Trigger host immune responses

• Transcriptional analysis: Compare gene expression patterns between wild-type and MiaB-deficient strains under different growth conditions.

• Proteomics: Identify proteins whose expression is altered in MiaB-deficient strains, particularly those involved in adhesion, invasion, and immune evasion.

• Immunological studies: Investigate the specific immune responses triggered by recombinant MiaB, including T-cell proliferation and cytokine production .

The chronology of infection in the murine model provides an excellent framework for these studies. Following intraperitoneal infection with B. henselae, viable bacteria are cleared from organs within 6 days, but bacterial DNA remains detectable in liver tissue for at least 3 months . This persistence correlates with the development of granulomatous lesions that reach maximal density 4-12 weeks post-infection . Researchers should design time-course experiments to examine MiaB expression and activity during these distinct phases of infection.

How can structural analysis of B. henselae MiaB inform the development of targeted antimicrobial strategies?

Structural analysis of B. henselae MiaB can guide the development of targeted antimicrobial strategies through:

• Crystal structure determination: Obtain high-resolution structures of MiaB in different states (substrate-bound, intermediate, product-bound) to identify catalytic pockets and allosteric sites for drug targeting.

• Molecular dynamics simulations: Model the conformational changes during catalysis to identify transition states that could be targeted by inhibitors.

• Structure-based drug design: Use the iron-sulfur cluster binding sites as unique targets for small molecule inhibitors, as these features are distinct from human enzymes.

• Fragment-based screening: Identify small molecules that bind to critical regions of MiaB and develop them into potential inhibitors.

The dual [4Fe-4S] cluster arrangement in MiaB provides a particularly attractive target . The first cluster, coordinated by conserved cysteines in the radical-AdoMet motif (Cys150, Cys154, and Cys157), is essential for AdoMet binding and radical generation. The second cluster, coordinated by N-terminal cysteines (Cys10, Cys46, and Cys79), may serve as a sulfur donor . Inhibitors that disrupt either cluster formation or their redox cycling could potentially suppress B. henselae growth while having minimal effects on human cells that lack this radical-AdoMet enzyme.

What are the key considerations when designing a murine model to study B. henselae MiaB function in vivo?

When designing a murine model to study B. henselae MiaB function in vivo, researchers should consider:

• Mouse strain selection: C57BL/6 mice have been successfully used for B. henselae infection studies . BALB/c mice may provide different immunological profiles and should be considered for comparative studies.

• Bacterial preparation: To establish effective infection:

  • Use B. henselae strains that have undergone passages through mice prior to experimental infection to enhance virulence .

  • Harvest bacteria during logarithmic growth phase.

  • Administer intraperitoneally in doses ranging from 5×10⁷ to 5×10⁸ CFU .

• Infection monitoring protocol:

  • Track bacterial loads in spleen, liver, and other tissues over time.

  • Use both culture methods and PCR detection, as cultivatable bacteria are typically cleared within 6 days while bacterial DNA persists for months .

  • Monitor granulomatous inflammation in liver tissue, which peaks around 4 weeks post-infection .

• Experimental timeline:

  • Short-term studies (1-2 weeks): Focus on initial bacterial replication and dissemination.

  • Mid-term studies (3-12 weeks): Examine granuloma formation and immune response development.

  • Long-term studies (up to 20 weeks): Investigate chronic effects and resolution of inflammation.

• Genetic approaches:

  • Use wild-type bacteria versus MiaB-deficient mutants to assess the specific role of this enzyme.

  • Consider conditional knockout systems to control MiaB expression at different infection stages.

What strategies can be employed to improve the yield and stability of recombinant B. henselae MiaB protein?

To improve yield and stability of recombinant B. henselae MiaB protein:

• Codon optimization:

  • Adjust the MiaB gene sequence to match the codon usage bias of the expression host.

  • Optimize GC content and remove potential secondary structures in the mRNA.

• Fusion partners:

  • Use solubility-enhancing fusion tags such as MBP (maltose-binding protein), SUMO, or TrxA (thioredoxin).

  • Include a TEV or PreScission protease site for tag removal.

• Expression conditions optimization:

  • Screen multiple temperatures (15-30°C) and IPTG concentrations (0.1-1.0 mM).

  • Test auto-induction media which often improves yields for iron-sulfur proteins.

  • Supplement media with iron and sulfur sources.

• Stabilization approaches:

  • Include osmolytes like glycerol (10-20%) or trehalose (0.5 M) in purification buffers.

  • Test different pH ranges (typically pH 7.0-8.5) to identify optimal stability conditions.

  • Add reducing agents (DTT, β-mercaptoethanol, or TCEP) to prevent oxidative damage.

• Storage considerations:

  • Flash-freeze purified protein in liquid nitrogen with 20% glycerol.

  • Store at -80°C in small aliquots to avoid freeze-thaw cycles.

  • For short-term storage, maintain at 4°C under anaerobic conditions.

• Iron-sulfur cluster preservation:

  • Perform all purification steps under strict anaerobic conditions.

  • Include iron-sulfur cluster reconstitution steps using ferrous iron and sulfide under reducing conditions.

  • Monitor cluster integrity using UV-visible spectroscopy.