Recombinant Borrelia burgdorferi Flagellar protein FliL (fliL)

Basic Understanding of FliL

• What is FliL and what is its fundamental role in Borrelia burgdorferi?

  FliL is a putative periplasmic flagellar protein encoded by the fliL gene (locus BB0279) in B. burgdorferi. It plays a critical role in coordinating and regulating the orientation of periplasmic flagella. Cryo-electron tomography (cryo-ET) studies have revealed that FliL is strategically localized between the stator and rotor components of the flagellar motor . When fliL is inactivated, the periplasmic flagella are frequently tilted toward the cell pole instead of their normal orientation toward the cell body, resulting in defective motility despite the maintenance of normal cell morphology . This indicates that FliL acts as a crucial regulatory component in the flagellar apparatus rather than merely as a structural element.

• How is the fliL gene organized within the B. burgdorferi genome?

  The fliL gene (gene locus BB0279) is a 537-bp gene located within a large motility operon in the B. burgdorferi genome. It is separated from the upstream motB gene by 48 bp and from the downstream fliM gene by 37 bp . This specific genomic organization is significant because:

  • The 48-bp intergenic sequence between motB and fliL contains a ribosomal binding site

  • Unlike many bacterial species where motility genes are regulated by hierarchical cascades, B. burgdorferi's fliL appears to be transcribed by RNA polymerase containing σ70

  • This gene arrangement indicates co-transcription with other flagellar genes, which has important implications for gene expression studies and mutational analysis

  Understanding this organization is crucial for designing non-polar mutations and complementation strategies that don't disrupt downstream gene expression.

• What methodologies are most effective for studying FliL function?

  Multiple complementary approaches have proven effective for investigating FliL function:

  • Gene inactivation techniques: A novel non-polar gene inactivation system involves replacing fliL with just the coding sequence of an antibiotic resistance gene (e.g., aadA for streptomycin resistance), allowing downstream genes to be expressed using the targeted gene's transcriptional machinery .

  • Complementation studies: In cis complementation using suicide vectors to insert the fliL gene with the flgB promoter at a heterologous chromosomal location can restore function and confirm phenotype specificity .

  • Motility assays: Swimming ability in 1% methylcellulose or soft agar provides quantifiable measurements of flagellar function .

  • Cryo-electron tomography (cryo-ET): This advanced imaging technique allows visualization of flagellar motor structures and precise determination of FliL localization .

  • Recombinant protein expression: Expression of FliL protein without the transmembrane domain in E. coli systems enables antibody production and interaction studies .

Advanced Research Topics in FliL Biology

• How is recombinant FliL optimally expressed and purified for structural and functional studies?

  Expression and purification of recombinant B. burgdorferi FliL presents several challenges due to its transmembrane domain affecting protein solubility. Research indicates the following effective approach:

  1. PCR amplification of the fliL coding sequence without the predicted transmembrane domain (omitting the first 150 bp)

  2. Cloning into an E. coli expression vector with a histidine tag (e.g., pTrc-His TOPO)

  3. Transformation into a suitable E. coli host strain (e.g., DH5α)

  4. Induction with 0.25 mM isopropyl β-d-thiogalactoside for 4h at 37°C

  5. Purification using Ni²⁺ affinity chromatography

  6. Dialysis against phosphate-buffered saline

  For antibody production, approximately 400 μg of purified protein has been successfully used to raise specific antisera in rabbits . The resulting antibodies can detect a 20-kDa protein band in wild-type B. burgdorferi that is absent in fliL mutants, confirming both the specificity of the antibody and the effectiveness of the expression system.

• What structural insights has cryo-electron tomography provided about FliL's role in flagellar function?

  Cryo-electron tomography (cryo-ET) has revolutionized our understanding of FliL's structural role in the B. burgdorferi flagellar motor:

  • Comparative analysis of flagellar motors from wild-type and mutant cells provides structural evidence that FliL is localized between the stator and rotor .

  • In fliL mutants, periplasmic flagella are frequently tilted toward the cell pole instead of their normal orientation toward the cell body .

  • The altered flagellar orientation in mutants directly correlates with defective motility phenotypes.

  • Complementation of the mutant restores both the structural density in the motor and normal flagellar orientation.

  • Unlike model organisms where the flagellar rod contacts the peptidoglycan layer, the B. burgdorferi flagellar system has a unique architecture: the rod is significantly smaller (17nm vs. 30nm in E. coli) and a spirochete-specific collar contacts the peptidoglycan sacculus while the hook penetrates this layer .

  These structural findings collectively suggest that FliL functions as a crucial mechanical coupling protein that ensures proper force transmission between motor components and the flagellar filament.

• How does the non-polar gene inactivation system for fliL differ from traditional mutagenesis approaches?

  The novel gene inactivation system for fliL in B. burgdorferi represents a significant methodological advancement over traditional approaches:

  1. Traditional approaches typically use antibiotic resistance cassettes containing promoters (such as PflgB-Kan, PflgB-aadA, or PflgB-aacC1), which can cause polar effects on downstream genes when inserted into operons .

  2. Promoterless cassettes (PL-Kan or PL-aadA) with intact ribosomal binding sites and transcription start sites were developed to reduce polar effects, but still contained some regulatory elements .

  3. The novel approach for fliL uses only the coding sequence of the antibiotic resistance gene (from ATG start to TTG stop codon), replacing the target gene while maintaining the operon's transcriptional integrity .

  The implementation involves:

  • Overlapping PCR to create a construct with the upstream gene (motB), the aadA coding sequence, and the downstream gene (fliM)

  • Transformation of B. burgdorferi and selection with streptomycin

  • Confirmation by PCR and Western blotting

  This method allows for specific inactivation of fliL while maintaining expression of downstream genes in the operon, creating a true non-polar mutation that enables precise study of FliL function without confounding effects on other genes.

• What is the relationship between FliL and other components of the type III secretion system in B. burgdorferi?

  FliL functions within the context of a complex flagellar apparatus that includes a type III secretion system (T3SS) for flagellar protein export. Several relationships have been established:

  • While FliL is involved in flagellar orientation, other T3SS components like FliH and FliI function in the export and assembly of flagellar structural proteins .

  • Mutations in fliH and fliI result in loss of the FliH-FliI complex from flagellar motors, leading to reduced flagellar numbers and truncated flagella .

  • In contrast to FliH/FliI mutants which exhibit rod-shaped or string-like morphology and division defects, fliL mutants maintain normal cell morphology despite motility defects .

  • Both FliL and FliH/FliI mutants show severe defects in flagellar function and reduced infectivity in mouse models .

  • Other flagellar proteins like the hook protein (encoded by flgE), the switch protein (fliG2), and the filament protein (flaB) have different roles - mutations in these genes result in complete loss of periplasmic flagella and rod-shaped cells .

  This complex relationship between different flagellar proteins highlights the modular nature of the flagellar apparatus and the specific role of FliL in orientation rather than assembly or export.

• How do flagellar proteins like FliL contribute to B. burgdorferi pathogenesis in Lyme disease?

  Flagellar proteins, including FliL, play critical roles in B. burgdorferi pathogenesis through multiple mechanisms:

  1. Motility-dependent dissemination: Functional flagella are essential for B. burgdorferi to migrate from the tick midgut to salivary glands during feeding, and from the site of inoculation to distant tissues in mammals . FliL specifically ensures proper flagellar orientation for effective motility.

  2. Host colonization: Non-motile B. burgdorferi mutants typically show reduced or absent infectivity in mouse models, indicating the essential nature of motility for establishing infection .

  3. Immune recognition: Some flagellar proteins like FlaA (flagellin A) are immunogenic and recognized by antibodies in 71-86% of patients with neuroborreliosis or Lyme arthritis . While FliL itself hasn't been characterized as a major immunogen, the flagellar system as a whole represents a significant interface with host immunity.

  4. Diagnostic relevance: Recombinant flagellar proteins have been developed as diagnostic antigens for detecting antibodies in Lyme disease patients, with varying sensitivity and specificity depending on the protein and disease stage .

  5. Persistence mechanisms: The National Institute of Allergy and Infectious Diseases (NIAID) has identified persistent symptoms associated with Lyme disease as a research priority, including investigation of the bacterial genetic basis for persistence . Motility mechanisms may contribute to the ability of the bacterium to reach protected niches within the host.

  Understanding the specific contribution of FliL to these pathogenic processes could potentially inform new diagnostic and therapeutic approaches to Lyme disease.

• What structural and functional variations exist in FliL across different Borrelia species and strains?

  While direct comparative data on FliL across Borrelia species is limited in the provided search results, important insights can be drawn from studies of other flagellar proteins and genome analysis:

  • Recent genomic mapping of 47 strains of known and potential Lyme disease-causing Borrelia provides a framework for comparative analysis of genes including fliL .

  • Some flagellar proteins show significant variation between Borrelia species. For example, FlaA (flagellin A) shows 92-93% sequence identity across B. burgdorferi sensu stricto, B. afzelii, and B. garinii .

  • Different Borrelia species exhibit varied tissue tropism: B. burgdorferi is associated with arthritis, B. afzelii with dermatologic symptoms, and B. garinii with neurologic manifestations . These differences may partially relate to variations in motility proteins including FliL.

  • The expression of some B. burgdorferi proteins varies with temperature and environment. For example, BB0405 is significantly downregulated at 37°C compared to 33°C in B. afzelii but not in B. burgdorferi sensu stricto . Similar regulatory differences might exist for FliL.

  • Functional conservation can be tested through heterologous complementation experiments, examining whether FliL from one species can restore function in another species' mutant.

  Understanding these variations has implications for developing broad-spectrum diagnostic tests and therapeutic approaches effective against diverse Borrelia species.

• What potential exists for targeting FliL in novel therapeutic approaches to Lyme disease?

  FliL represents a potential therapeutic target for several reasons:

  1. Essential for pathogenesis: Defects in motility significantly reduce or eliminate infectivity in animal models , suggesting that targeting FliL function could impair the bacterium's ability to establish infection or disseminate within hosts.

  2. Unique to bacteria: FliL has no human homolog, potentially allowing for selective targeting without direct host toxicity.

  3. Structural information available: Cryo-ET studies have localized FliL within the flagellar motor , potentially enabling structure-based drug design.

  4. Alternative to conventional antibiotics: As antibiotic resistance and persistence in Lyme disease remain concerns , targeting motility through FliL inhibition could represent a novel therapeutic paradigm.

  5. Reduced selection pressure: Anti-virulence approaches that impair motility rather than killing bacteria directly might reduce selection pressure for resistance.

  Potential therapeutic strategies could include:

  • Small molecule inhibitors of FliL function

  • Peptide-based inhibitors that disrupt FliL interactions with other flagellar components

  • Combination approaches with conventional antibiotics

  Research is needed to develop high-throughput screening assays for FliL inhibitors and to evaluate their efficacy in animal models of Lyme disease.

Current Research Trends and Technical Considerations

• How are new genetic tools advancing our understanding of fliL and other flagellar genes?

  Recent advances in genetic tools are significantly enhancing research on B. burgdorferi flagellar genes including fliL:

  • Development of non-polar gene inactivation systems allows specific targeting of individual genes without disrupting downstream expression, crucial for genes in operons like fliL .

  • In cis complementation approaches using heterologous chromosomal sites enable restoration of function while maintaining physiological expression levels .

  • A powerful new visualization tool has been adapted to study spirochetes, allowing observation of living cells .

  • These tools enable identification and characterization of essential genes and pathways that could become targets for drugs and vaccines .

  • Pull-down assays with recombinant proteins expressed with different tags (6xHis, MBP, FLAG) in E. coli enable biophysical interaction studies between flagellar components .

  • The development of web-based software and comprehensive genomic data from 47 strains of Borrelia provides resources for comparative genomic studies of flagellar genes .

  These methodological advances collectively enhance our ability to dissect the complex flagellar structure and function in B. burgdorferi, potentially leading to new interventions for Lyme disease.

• What are the emerging challenges and frontiers in FliL research?

  Several significant challenges and frontier areas remain in understanding FliL and its role in B. burgdorferi:

  1. Structural characterization: While cryo-ET has provided insights into FliL's location in the flagellar motor, high-resolution structures through X-ray crystallography or cryo-EM would enhance our understanding of its function and interactions.

  2. Protein-protein interactions: Identifying the specific binding partners of FliL is essential for understanding its mechanical role in flagellar orientation. Pull-down assays and other protein interaction methods need to be optimized for FliL .

  3. Regulatory mechanisms: How fliL expression is regulated during different phases of the B. burgdorferi life cycle remains poorly understood. Temperature, pH, and host factors may all influence expression patterns.

  4. In vivo dynamics: Developing tools to visualize FliL function in living cells remains challenging but would provide crucial insights into its dynamic role during motility.

  5. Translational applications: Moving from basic understanding to applications in diagnostics, vaccines, or therapeutics represents a significant frontier.

  6. Host-pathogen interface: Understanding how FliL-dependent motility affects interactions with host immune components and tissue barriers requires integrated approaches combining bacterial genetics and immunology.

  7. Persistent infection: The role of motility in establishing persistent infection or contributing to post-treatment Lyme disease syndrome (PTLDS) remains an important area for investigation.

  Addressing these challenges requires interdisciplinary approaches combining structural biology, genetics, cell biology, and immunology.