Recombinant Borrelia burgdorferi Flagellar M-ring protein (fliF)

What is the FliF protein in Borrelia burgdorferi and what role does it play in flagellar structure?

FliF is the primary structural component of the MS-ring (membrane and supramembrane ring) in the Borrelia burgdorferi flagellar motor. The MS-ring serves as the foundation for flagellar assembly, embedded in the cytoplasmic membrane and forming a circular hub that anchors the flagellar structure . In B. burgdorferi, this ring is particularly important because, unlike in many other bacteria, the flagella are located in the periplasmic space rather than externally. The MS-ring formed by FliF provides attachment sites for other flagellar components and contributes to the unique motility of this spirochete, which is essential for its pathogenesis and dissemination within mammalian hosts .

How does the FliF protein in B. burgdorferi differ from FliF proteins in other bacteria?

While the core function of forming the MS-ring is conserved, B. burgdorferi FliF has evolved specific adaptations related to the periplasmic location of its flagella. The MS-ring in B. burgdorferi is surrounded by a distinctive collar structure composed of multiple proteins (FlcA, FlcB, FlcC, FlbB, and FlcD/Bb0236) that is not found in most other bacterial flagellar systems . This collar effectively bridges the rotor and the 16 torque-generating stator complexes in each flagellar motor, contributing to the specialized motility of spirochetes in complex environments . Additionally, B. burgdorferi FliF must interact with flagellar proteins like FlbB, which forms a distinctive periplasmic ring around the MS-ring that acts as a scaffold supporting collar assembly and subsequent recruitment of stator complexes .

What is the molecular weight and structural characteristics of recombinant B. burgdorferi FliF?

The FliF protein in B. burgdorferi, when expressed as a recombinant protein fused to an MBP-tag (as described in the pull-down assays), forms a complex with a molecular weight dependent on the native FliF protein plus the 42.4 kDa contributed by the MBP tag . The native FliF forms oligomeric ring structures with specific symmetry that are crucial for flagellar function. Structural studies using cryo-electron tomography have revealed that the MS-ring formed by FliF serves as a central hub around which other flagellar components, including a 16-fold symmetric FlbB ring structure with 32-fold symmetry elements, are organized .

What are the optimal conditions for recombinant expression of B. burgdorferi FliF protein?

For optimal recombinant expression of B. burgdorferi FliF protein, the gene coding for FliF should be cloned into an expression vector such as pMALc-5x from New England Biolabs (NEB) to express MBP-FliF fusion protein . The vector should then be transformed into E. coli codon plus cells to account for codon usage differences between B. burgdorferi and E. coli. Protein expression should be induced following the manufacturer's instructions for the specific expression system being used. Based on approaches used for similar B. burgdorferi flagellar proteins, expression conditions typically involve induction with IPTG at concentrations between 0.1-1.0 mM when cultures reach mid-log phase, followed by growth at lower temperatures (16-25°C) to enhance proper folding of the recombinant protein .

What purification strategies yield the highest purity and activity for recombinant FliF protein?

A highly effective purification strategy for recombinant B. burgdorferi FliF involves:

• Expression as an MBP-fusion protein for enhanced solubility

• Cell lysis using French press to disrupt the E. coli cells harboring the expression plasmids

• Affinity chromatography using amylose resin, which binds specifically to the MBP tag

• Elution with maltose buffer according to manufacturer's instructions

• Optional: Secondary purification using size exclusion chromatography to separate monomeric from oligomeric species

This approach yields proteins with >90% purity, similar to that achieved with other B. burgdorferi flagellar proteins . For functional studies, it's crucial to verify that the recombinant protein maintains its ability to form oligomeric structures, which can be assessed through analytical ultracentrifugation or negative-stain electron microscopy.

How can researchers verify the structural integrity of purified recombinant FliF protein?

Verification of structural integrity for purified recombinant FliF protein can be accomplished through multiple complementary approaches:

• SDS-PAGE analysis to confirm the expected molecular weight (similar to the approach used for flagellin protein, which shows the predicted size of the fusion protein)

• Western blotting using antibodies against MBP or, if available, anti-FliF antibodies

• Circular dichroism spectroscopy to assess secondary structure content

• Limited proteolysis to evaluate the folding state of the protein

• Negative-stain electron microscopy to visualize oligomeric ring structures that would indicate proper folding and assembly capacity

• Functional binding assays with known interaction partners such as FlbB to confirm that the recombinant protein maintains its binding properties

What are the key protein interaction partners of FliF in the B. burgdorferi flagellar structure?

Based on the available research, FliF in B. burgdorferi interacts with several key proteins:

• FlbB - Forms a distinctive periplasmic ring around the MS-ring and acts as a scaffold supporting collar assembly

• Collar proteins (FlcA, FlcB, FlcC, and FlcD/Bb0236) - Form complex, interconnected protein densities surrounding the MS-ring

• FliG - While not explicitly mentioned in the search results, FliG typically interacts with FliF in bacterial flagellar systems to form part of the rotor

• Stator complexes - The FliF-based MS-ring indirectly interacts with 16 torque-generating stator complexes through the collar protein network

Pull-down assays have specifically demonstrated interaction between recombinant FLAG-FlbB-His₆ and MBP-FliF proteins, which were co-purified using Ni-NTA agarose resin and detected by immunoblotting with Anti-MBP antibody .

How can researchers quantitatively assess the binding affinity between FliF and its interaction partners?

Researchers can quantitatively assess binding affinity between FliF and its interaction partners using multiple biophysical techniques:

• Surface Plasmon Resonance (SPR) - Immobilize purified FliF on a sensor chip and flow interaction partners at varying concentrations to determine association and dissociation rate constants (ka and kd) and calculate the equilibrium dissociation constant (KD)

• Isothermal Titration Calorimetry (ITC) - Directly measure the thermodynamic parameters of binding, providing not only binding affinity but also enthalpy and entropy changes

• Microscale Thermophoresis (MST) - Measure changes in the thermophoretic movement of fluorescently labeled FliF upon binding to interaction partners

• Bio-Layer Interferometry (BLI) - Similar to SPR but using optical interferometry to detect binding

• Pull-down assays with quantitative analysis - Similar to the methods described for FlbB-FliF interaction studies, where proteins are mixed in defined ratios, purified using affinity resin, and quantified by immunoblotting

For specificity control, researchers should include appropriate negative controls such as unrelated proteins expressed with the same tags.

What role does FliF play in the assembly and stability of the flagellar collar structure unique to spirochetes?

FliF forms the MS-ring that serves as the foundation for the unique flagellar collar structure in B. burgdorferi. This collar structure is crucial for the flat-wave morphology and motility of this Lyme disease spirochete . Evidence suggests that FliF provides a stable platform for the recruitment and assembly of collar proteins through direct or indirect interactions. The MS-ring formed by FliF is surrounded by a FlbB ring, which in turn acts as a scaffold supporting collar assembly .

The sequential assembly process likely proceeds as follows:

• FliF assembles into the MS-ring in the cytoplasmic membrane

• FlbB is recruited to form a distinctive periplasmic ring around the MS-ring

• Other collar proteins (FlcA, FlcB, FlcC, and FlcD/Bb0236) assemble onto this scaffold

• The complete collar structure facilitates recruitment of stator complexes

Without proper FliF function, this assembly sequence would be disrupted, preventing formation of functional periplasmic flagella and compromising the spirochete's distinctive motility, which is essential for its pathogenesis .

What are the most effective methods for generating site-directed mutations in recombinant FliF to study structure-function relationships?

For studying structure-function relationships in recombinant B. burgdorferi FliF, researchers can employ several effective site-directed mutagenesis approaches:

• QuikChange Mutagenesis - Design complementary primers containing the desired mutation and use high-fidelity DNA polymerase to amplify the entire plasmid containing the fliF gene

• Gibson Assembly - Generate DNA fragments with overlapping ends containing the desired mutations and assemble them in a single isothermal reaction

• Q5 Site-Directed Mutagenesis - Use non-overlapping primers where one or both contain the desired mutations, followed by KLD (kinase, ligase, DpnI) enzyme mix treatment

After generating mutations, express the mutant proteins using the same MBP-fusion system described for wild-type FliF . Crucial mutations to consider include:

• Conserved residues at the predicted interfaces with other flagellar proteins

• Residues involved in oligomerization

• Residues in predicted transmembrane domains

• Potential phosphorylation or glycosylation sites that might regulate function

Verify the impact of mutations using structural integrity assays (as described in 2.3) and functional interaction studies with binding partners like FlbB .

How can cryo-electron tomography be optimized for studying the in situ structure of FliF within the B. burgdorferi flagellar motor?

Optimizing cryo-electron tomography (cryo-ET) for studying in situ FliF structure within B. burgdorferi flagellar motors requires attention to several key parameters:

• Sample preparation:

  • Use cell tips where flagellar motors are concentrated

  • Apply 4-5 μl of bacterial culture to glow-discharged, carbon-coated, copper grids

  • Plunge-freeze samples in liquid ethane using a vitrification device with controlled humidity

• Data collection:

  • Collect tilt series from approximately -64° to +64° with 2° increments

  • Use low-dose conditions (~100 electrons/Ų) for the entire tilt series

  • Implement dose-symmetric acquisition schemes to minimize radiation damage

• Tomogram reconstruction:

  • Align tilt series using fiducial gold markers

  • Use weighted back-projection or SIRT algorithms for reconstruction

  • Apply CTF correction to enhance resolution

• Subtomogram averaging:

  • Extract subvolumes containing flagellar motors

  • Apply alignment and averaging procedures to enhance the signal-to-noise ratio

  • Implement appropriate symmetry parameters during refinement

Using these optimized approaches, researchers have achieved resolutions of ~13Å for wild-type flagellar motors and ~44Å for mutant structures, allowing detailed visualization of the MS-ring formed by FliF and its interactions with surrounding proteins .

What considerations are important when developing antibodies against FliF for immunolocalization studies?

When developing antibodies against B. burgdorferi FliF for immunolocalization studies, researchers should consider:

• Antigen selection:

  • Use full-length recombinant MBP-FliF protein for polyclonal antibody production

  • Identify surface-exposed epitopes for monoclonal antibody development

  • Consider using synthetic peptides corresponding to unique regions of FliF

• Antibody validation:

  • Confirm specificity by Western blot against recombinant FliF and B. burgdorferi lysates

  • Test cross-reactivity with FliF proteins from related spirochetes

  • Perform immunoprecipitation to verify recognition of native FliF

• Immunolocalization optimization:

  • For immunogold electron microscopy, optimize fixation conditions to preserve flagellar structure

  • For immunofluorescence, test various permeabilization methods to allow antibody access to periplasmic flagella

  • Use known flagellar markers (like anti-flagellin antibodies) as co-localization controls

• Controls:

  • Include fliF knockout mutants as negative controls

  • Use pre-immune serum for background assessment

  • Perform peptide competition assays to confirm epitope specificity

These considerations will help ensure specific and reliable detection of FliF in its native context within the complex flagellar structure of B. burgdorferi.

How does mutation or deletion of fliF affect B. burgdorferi motility and virulence in animal models?

Mutation or deletion of the fliF gene in B. burgdorferi would have profound effects on motility and virulence:

These effects align with observations that B. burgdorferi motility is essential for dissemination and invasion in the mammalian host, which are in turn essential for pathogenesis . Unlike flagellar filament mutants where specific components like FlaB are degraded by proteases such as HtrA , MS-ring mutants would fail to initiate flagellar assembly altogether, resulting in a more fundamental defect in motility.

What is the relationship between FliF structure and the unique motility patterns of B. burgdorferi in different host environments?

The structure of FliF and the MS-ring it forms are fundamentally related to the unique motility patterns of B. burgdorferi in different host environments:

• Structural adaptations:

  • The MS-ring in B. burgdorferi must accommodate the periplasmic location of flagella

  • FliF interacts with spirochete-specific collar proteins that are crucial for the flat-wave morphology

  • The MS-ring must connect to 16 torque-generating stator complexes through this collar structure

• Environment-specific motility:

  • In low-viscosity environments (like bloodstream), the FliF-based motor structure enables translational movement

  • In high-viscosity environments (like connective tissues), the same motor structure facilitates cork-screw-like movement

  • The MS-ring structure allows for coordinated rotation of multiple flagellar motors at both cell poles

• Chemotactic adaptation:

  • The flagellar motor structure based on FliF enables response to chemotactic signals

  • This allows B. burgdorferi to navigate toward favorable environments and away from immune effectors

The distinct structure of the MS-ring and its integration with spirochete-specific features like the collar ultimately contribute to B. burgdorferi's ability to migrate through diverse host tissues, which is essential for its pathogenic lifecycle .

How do post-translational modifications of FliF impact flagellar assembly and function in B. burgdorferi?

While the search results don't directly address post-translational modifications (PTMs) of FliF in B. burgdorferi, we can provide a methodological approach to studying this important aspect:

• Potential PTMs to investigate:

  • Phosphorylation - May regulate assembly or motor function

  • Glycosylation - Could affect interactions with other flagellar components

  • Proteolytic processing - Might be involved in maturation or regulation

• Analytical approaches:

  • Mass spectrometry analysis of purified native FliF from B. burgdorferi

  • Comparison of PTM patterns between FliF isolated from different growth conditions

  • Site-directed mutagenesis of potential modification sites followed by functional assessment

• Functional impacts to assess:

  • Effects on MS-ring assembly and stability

  • Changes in protein-protein interactions with collar components

  • Alterations in flagellar filament assembly efficiency

  • Impacts on motor rotation and switching

• Regulatory significance:

  • PTMs may serve as mechanisms for B. burgdorferi to adapt its motility to different host environments

  • Modifications could be part of the regulatory network that coordinates flagellar gene expression with assembly

Understanding these modifications would provide insights into the sophisticated regulation of flagellar function that enables B. burgdorferi to navigate and survive in diverse host environments.

How does B. burgdorferi FliF compare structurally and functionally to homologs in other spirochetes and non-spirochete bacteria?

A comparative analysis of B. burgdorferi FliF with homologs in other bacteria reveals several important distinctions:

Key structural differences include:

• In B. burgdorferi, FliF must accommodate the unique periplasmic location of flagella

• B. burgdorferi FliF interacts with spirochete-specific collar proteins essential for its flat-wave morphology

• The MS-ring in B. burgdorferi connects to 16 torque-generating stator complexes through the collar structure

These structural adaptations reflect the specialized motility requirements of B. burgdorferi for invasion and dissemination within mammalian hosts .

What insights from other bacterial flagellar systems can be applied to studying B. burgdorferi FliF?

Several insights from well-studied bacterial flagellar systems can be applied to B. burgdorferi FliF research:

• Structural biology approaches:

  • High-resolution structures of FliF from E. coli and Salmonella can guide modeling of B. burgdorferi FliF domains

  • Symmetry determination methods used in other systems can be applied to understand the B. burgdorferi MS-ring

• Assembly mechanisms:

  • Knowledge about FliF oligomerization in model organisms can inform studies of B. burgdorferi MS-ring formation

  • Understanding of membrane insertion and anchoring from other systems may apply to B. burgdorferi FliF

• Regulatory insights:

  • Transcriptional and post-translational regulatory mechanisms identified in other bacteria may have parallels in B. burgdorferi

  • Export and assembly checkpoints known from model systems can guide investigation of B. burgdorferi flagellar assembly

• Experimental techniques:

  • FRET-based protein interaction assays developed for other flagellar systems

  • Single-molecule approaches to study motor function and dynamics

  • In vitro reconstitution methods to study isolated components

How can structural predictions from AlphaFold or similar tools be validated for B. burgdorferi FliF protein?

Validating structural predictions from AlphaFold or similar tools for B. burgdorferi FliF requires a multi-faceted approach:

• Computational validation:

  • Assess confidence scores (pLDDT values) from AlphaFold predictions

  • Compare predictions from multiple tools (AlphaFold, RoseTTAFold, etc.)

  • Use evolutionary coupling analysis to verify predicted contacts

• Experimental validation:

  • Limited proteolysis to probe accessible regions and domain boundaries

  • Circular dichroism to verify secondary structure content matches predictions

  • Cross-linking mass spectrometry to validate predicted proximity of amino acids

  • Cryo-EM of purified recombinant FliF to verify oligomeric structure

• Mutational analysis:

  • Design mutations based on predicted structures and assess their impact on:

    • Protein stability and folding

    • Oligomerization capacity

    • Interaction with binding partners like FlbB

• In situ validation:

  • Compare predicted structures with in situ structures obtained by cryo-ET

  • Fit predicted atomic models into subtomogram averages of flagellar motors

  • Assess whether models explain observed densities and connections

This approach, similar to that used for FlbB structural modeling , provides robust validation of computational predictions and ensures that structural models accurately represent the native FliF protein.

How can FliF be engineered as a platform for displaying heterologous antigens in B. burgdorferi-based vaccine development?

Engineering FliF as a platform for heterologous antigen display offers several advantages for vaccine development:

• Design considerations:

  • Identify surface-exposed loops in the FliF structure that can accommodate insertions

  • Use structural modeling to predict optimal insertion sites that won't disrupt MS-ring formation

  • Create genetic constructs that fuse antigenic epitopes to these permissive sites

• Expression strategies:

  • Develop complementation systems where engineered FliF replaces native FliF

  • Create dual-expression systems where both native and engineered FliF are co-expressed

  • Optimize expression levels to ensure proper flagellar assembly

• Validation methods:

  • Verify MS-ring assembly and flagellar function in engineered strains

  • Confirm surface exposure of inserted epitopes using immunogold electron microscopy

  • Assess immune responses to the displayed antigens in animal models

• Potential applications:

  • Display protective antigens from B. burgdorferi for Lyme disease vaccines

  • Create multivalent vaccines by displaying antigens from multiple pathogens

  • Develop diagnostic tools using engineered FliF displaying specific epitopes

This approach takes advantage of the organized, multimeric structure of FliF while utilizing B. burgdorferi's natural adjuvant properties to potentially enhance immune responses.

What are the challenges and solutions for studying the dynamics of FliF assembly and turnover in live B. burgdorferi cells?

Studying FliF assembly and turnover dynamics in live B. burgdorferi presents several challenges and potential solutions:

• Challenges:

  • The small size of B. burgdorferi cells (approximately 0.2-0.5 μm in diameter)

  • The periplasmic location of flagella, making them less accessible

  • Limited genetic tools compared to model organisms

  • The complex structure of the flagellar motor with multiple interacting components

• Solutions for visualization:

  • Fluorescent protein fusions with FliF that retain function

  • Split-fluorescent protein complementation to study FliF-partner interactions

  • Photoactivatable or photoconvertible FliF fusions for pulse-chase experiments

  • Super-resolution microscopy techniques (PALM, STORM) to overcome size limitations

• Solutions for turnover studies:

  • Inducible expression systems to control FliF production

  • Protein degradation tags to trigger selective FliF removal

  • Quantitative proteomic approaches using stable isotope labeling

  • Fluorescence recovery after photobleaching (FRAP) with functional fluorescent fusions

• Solutions for assembly studies:

  • Single-molecule tracking of labeled FliF components

  • Correlative light and electron microscopy to connect dynamics with structure

  • Microfluidic systems for rapid environmental changes to trigger assembly

These approaches would provide unprecedented insights into the dynamic processes of flagellar motor assembly in this important pathogen.

How can multi-omics approaches integrate structural, functional, and regulatory data about FliF to create comprehensive models of B. burgdorferi flagellar assembly?

A multi-omics approach to create comprehensive models of B. burgdorferi flagellar assembly centered on FliF would integrate:

• Structural omics:

  • Cryo-ET data of intact flagellar motors in different assembly states

  • Atomic structures from X-ray crystallography or cryo-EM of isolated components

  • Computational models refined against experimental data

  • Cross-linking mass spectrometry data defining protein-protein interfaces

• Functional omics:

  • Transcriptomics to identify co-regulated flagellar genes

  • Proteomics to quantify stoichiometry and post-translational modifications

  • Phenomics relating flagellar structure variants to motility phenotypes

  • Interactomics mapping the complete network of protein-protein interactions

• Regulatory omics:

  • ChIP-seq to identify transcription factor binding sites controlling flagellar gene expression

  • RNA-seq under different conditions to map environmental regulation

  • Phosphoproteomics to identify signaling networks controlling assembly

  • Metabolomics to link metabolic state to flagellar function

• Integration strategies:

  • Mathematical modeling of assembly pathways with kinetic parameters

  • Machine learning approaches to predict assembly outcomes from multi-omics data

  • Systems biology frameworks connecting regulation to structure and function

  • 4D visualization tools to represent the temporal dimension of assembly

This integrated approach would provide a holistic understanding of how B. burgdorferi coordinates the complex process of flagellar assembly, with FliF serving as the foundation for this elaborate nanomachine that is essential for pathogenesis .

What are the most promising therapeutic targets within the FliF-based flagellar assembly pathway for controlling B. burgdorferi infection?

The FliF-based flagellar assembly pathway offers several promising therapeutic targets for controlling B. burgdorferi infection:

• FliF-FlbB interface:

  • The interaction between FliF and FlbB is critical for collar assembly and flagellar function

  • Small molecules disrupting this interface could prevent proper flagellar assembly

  • This target is particularly attractive due to the demonstrated physical interaction between these proteins

• MS-ring assembly:

  • Compounds that interfere with FliF oligomerization could prevent MS-ring formation

  • These would act early in the flagellar assembly process, having maximal impact

  • The unique features of the B. burgdorferi MS-ring could allow for specificity

• FliF-specific regions:

  • Targeting spirochete-specific regions of FliF not present in commensal bacteria

  • This approach could minimize disruption of beneficial microbiota

  • Monoclonal antibodies against these regions could be effective in early infection

• Regulatory pathways:

  • Targeting regulatory mechanisms controlling fliF expression

  • Disrupting export or assembly checkpoints specific to B. burgdorferi

  • Triggering premature degradation of FliF through proteolytic pathways

These approaches would directly impact B. burgdorferi motility, which is essential for its dissemination and invasion in the mammalian host, processes that are critical for pathogenesis .

What unresolved questions about FliF structure and function should be prioritized in future research?

Several critical questions about B. burgdorferi FliF structure and function remain unresolved and should be prioritized in future research:

• Structural questions:

  • What is the atomic-resolution structure of B. burgdorferi FliF, and how does it differ from non-spirochete homologs?

  • How does FliF oligomerize to form the MS-ring, and what determines its diameter and curvature?

  • What specific interfaces mediate FliF interaction with unique spirochete components like FlbB and the collar proteins?

• Assembly questions:

  • What is the temporal sequence of FliF assembly into the MS-ring?

  • How is FliF targeted to the membrane and what chaperones are involved?

  • What quality control mechanisms ensure proper MS-ring assembly?

• Functional questions:

  • How does the MS-ring structure contribute to the unique rotational properties of spirochete flagella?

  • What role does FliF play in the directional switching of flagellar motors?

  • How do post-translational modifications regulate FliF function under different conditions?

• Evolutionary questions:

  • How has FliF evolved specifically in spirochetes to accommodate periplasmic flagella?

  • What structural adaptations in FliF contribute to the distinctive motility patterns of B. burgdorferi?

  • How do sequence variations in FliF across Borrelia strains relate to differences in motility and virulence?

Addressing these questions would significantly advance our understanding of this fundamental protein and potentially lead to new therapeutic approaches targeting B. burgdorferi motility.

How might emerging technologies in structural biology and synthetic biology transform our ability to study and manipulate B. burgdorferi FliF?

Emerging technologies are poised to revolutionize our understanding and manipulation of B. burgdorferi FliF:

• Structural biology advances:

  • Cryo-electron tomography with improved detectors and phase plates could achieve sub-nanometer resolution of in situ flagellar motors

  • Integrative structural biology combining cryo-ET, X-ray crystallography, and AlphaFold predictions to build complete atomic models

  • Time-resolved cryo-EM to capture intermediates in MS-ring assembly

  • Advanced image processing algorithms to extract more information from existing data

• Synthetic biology approaches:

  • CRISPR-Cas systems optimized for B. burgdorferi to enable precise genetic manipulation

  • Optogenetic control of FliF expression or degradation to study assembly dynamics

  • Synthetic flagellar motors with modified or hybrid components to understand design principles

  • Cell-free expression systems to study flagellar assembly in controlled environments

• Nanotechnology integration:

  • Single-molecule force spectroscopy to measure mechanical properties of FliF interactions

  • Nanobody-based probes for super-resolution imaging of flagellar structures

  • Microfluidic devices mimicking host environments to study contextual flagellar function

  • DNA origami scaffolds to template and study controlled FliF assembly

• Computational advances:

  • Molecular dynamics simulations of complete MS-ring structure in membrane environments

  • Machine learning approaches to predict functional consequences of FliF mutations

  • Systems biology models integrating structural, genetic, and proteomic data