Recombinant Borrelia burgdorferi Protein HflC (hflC)

What experimental evidence supports the functional characterization of BB0268 as FlgV rather than Hfq?

Multiple lines of evidence support the reclassification of BB0268 as encoding FlgV:

• Localization studies have shown that the BB0268 protein product is localized within flagellar basal bodies, consistent with a structural role in flagella .

• Functional analyses demonstrate that strains lacking the BB0268 gene (flgV) produce fewer and shorter flagellar filaments and exhibit defects in cell division and motility .

• In vivo studies revealed that flgV-deficient B. burgdorferi could survive and replicate in Ixodes ticks but showed attenuated infection and dissemination in mice, indicating its role in pathogenicity .

This experimental evidence collectively supports BB0268's role as a flagellar component rather than an RNA-binding protein.

What is the relationship between Borrelia burgdorferi flagellar proteins and infectious capacity?

Flagellar proteins are crucial for B. burgdorferi infectivity as they enable the spirochete's distinctive motility, which is essential for dissemination within hosts. Research has shown that flgV-deficient B. burgdorferi strains can survive in ticks but show attenuated infection and dissemination capabilities in mammalian hosts . This suggests that proper flagellar assembly and function, mediated in part by FlgV, are critical during specific stages of the enzootic cycle. The research identifies specific infection timepoints when spirochete motility is most crucial for pathogenesis, highlighting the importance of flagellar proteins in B. burgdorferi's infectious capacity .

How does the earlier misannotation of BB0268 as an Hfq homolog impact our understanding of RNA regulation in Borrelia burgdorferi?

The misannotation of BB0268 as an Hfq homolog has significant implications for our understanding of RNA regulation in B. burgdorferi. Previous studies that attributed Hfq-like functions to the BB0268 product need to be reevaluated. For instance, research had suggested that the putative B. burgdorferi Hfq restored efficient translation of an rpoS::lacZ fusion in an E. coli hfq null mutant and bound to small RNA DsrA and rpoS mRNA .

The reannotation necessitates a reconsideration of:

• Whether B. burgdorferi possesses a true Hfq homolog elsewhere in its genome

• The actual mechanisms regulating RNA-mediated processes previously attributed to Hfq

• The roles that were assigned to Hfq in temperature-dependent regulation of virulence factors like RpoS and OspC

This misannotation highlights the challenges in functional genomics across evolutionarily distant bacteria and emphasizes the importance of experimental verification beyond sequence homology.

What are the structural and functional differences between FlgV in B. burgdorferi and its homologs in other bacterial species?

FlgV in B. burgdorferi represents a broadly conserved flagellar component with distinct features compared to homologs in other bacteria:

The unique aspects of B. burgdorferi FlgV likely reflect adaptations to the specialized endoflagella of spirochetes, which are anchored at each cell pole and extend through the periplasmic space rather than into the extracellular environment .

How does disruption of the flgV gene affect global gene expression patterns in B. burgdorferi during different stages of its enzootic cycle?

Disruption of the flgV gene leads to complex transcriptional consequences across the B. burgdorferi genome, particularly affecting genes involved in:

• Flagellar assembly and motility - Direct downregulation of flagellar genes due to structural interdependence

• Cell division processes - Dysregulation observed as flgV mutants show cell division defects

• Virulence factors - Altered expression of genes required for mammalian infection

The effects vary significantly across the enzootic cycle:

• In unfed ticks: Minimal impact as flagellar gene expression is generally low

• During tick feeding: Substantial dysregulation as flagellar genes are normally upregulated during this transition

• In mammalian hosts: Maximum impact, correlating with attenuated infection and dissemination

This differential impact highlights the context-dependent role of FlgV in B. burgdorferi gene regulation networks.

What are the optimal protocols for expressing and purifying recombinant FlgV from B. burgdorferi for structural studies?

For optimal expression and purification of recombinant B. burgdorferi FlgV:

• Vector Selection: Choose expression vectors with strong, inducible promoters (e.g., T7) and appropriate tags for purification. Consider codon optimization for E. coli expression systems.

• Expression Conditions:

  • Host strain: BL21(DE3) or derivatives optimized for membrane/flagellar proteins

  • Induction: 0.1-0.5 mM IPTG at lower temperatures (16-25°C) for 4-16 hours to enhance solubility

  • Media supplementation: Consider additives that stabilize membrane-associated proteins

• Purification Strategy:

  • Initial capture: Immobilized metal affinity chromatography using His-tags

  • Secondary purification: Size exclusion chromatography to remove aggregates

  • Consider native conditions with mild detergents to maintain structural integrity

• Quality Control:

  • SDS-PAGE and Western blotting with FlgV-specific antibodies

  • Mass spectrometry to confirm protein identity

  • Dynamic light scattering to assess homogeneity

This approach draws on methods similar to those that have been successful for other Borrelia proteins .

What experimental approaches are most effective for characterizing the role of FlgV in flagellar assembly and motility?

Multiple complementary approaches are recommended for characterizing FlgV's role in flagellar assembly and motility:

These approaches have been instrumental in characterizing the role of FlgV in B. burgdorferi motility and infectivity .

How can researchers effectively study the impact of FlgV on B. burgdorferi infection dynamics in animal models?

To effectively study FlgV's impact on B. burgdorferi infection dynamics:

• Animal Model Selection:

  • Use established murine models for Lyme disease

  • Consider C3H/HeN mice which are particularly susceptible to B. burgdorferi infection

• Infection Methodology:

  • Needle inoculation: Precisely controlled bacterial dose (10³-10⁵ spirochetes)

  • Tick transmission: More natural but variable inoculum size

  • Both approaches should be used for comprehensive assessment

• Comparative Infection Studies:

  • Wild-type B. burgdorferi

  • flgV knockout strains

  • Complemented flgV mutants

  • Site-directed flgV mutants with specific domain alterations

• Assessment Parameters:

  • Bacterial load quantification in tissues using qPCR

  • Tissue distribution patterns via immunohistochemistry

  • Serological responses through ELISA and immunoblotting

  • Inflammatory markers (cytokines, chemokines) measurement

• Temporal Analysis:

  • Early dissemination (1-7 days post-infection)

  • Established infection (14-28 days)

  • Persistent infection (>28 days)

• Tick-Mouse Cycle Studies:

  • Acquisition of bacteria by ticks from infected mice

  • Bacterial persistence in molting ticks

  • Transmission from infected ticks to naive mice

This comprehensive approach has revealed that flgV-deficient B. burgdorferi can survive in ticks but show attenuated infection and dissemination in mice, defining infection timepoints when spirochete motility is most crucial .

How might new structural biology techniques advance our understanding of FlgV and other flagellar proteins in B. burgdorferi?

Recent advances in structural biology offer unprecedented opportunities for understanding FlgV:

• Cryo-Electron Tomography: This technique allows visualization of the intact flagellar basal body architecture in situ, enabling researchers to precisely localize FlgV within the native complex and understand its structural role without artifacts from purification.

• AlphaFold and Related AI Methods: These computational approaches can predict FlgV structure with high confidence, particularly useful for identifying functional domains and potential interaction interfaces.

• Integrative Structural Biology: Combining multiple techniques (X-ray crystallography, NMR, SAXS, crosslinking mass spectrometry) provides complementary structural information that can resolve the complete FlgV structure and its interactions.

• Single-Particle Cryo-EM: With recent advances enabling resolution below 2Å, this method can potentially resolve high-resolution structures of FlgV-containing flagellar complexes.

• In-Cell NMR: This emerging technique allows protein structure determination in living cells, potentially revealing how FlgV structure changes in different cellular environments.

These advances will likely reveal how FlgV contributes to the unique architecture of spirochete endoflagella and their role in the distinctive motility that enables host infection .

What are the implications of the FlgV discovery for developing new diagnostic approaches or therapeutic targets for Lyme disease?

The discovery that BB0268 encodes FlgV rather than Hfq opens new avenues for Lyme disease diagnostics and therapeutics:

• Diagnostic Applications:

  • FlgV could serve as a novel antigen in recombinant chimeric protein-based diagnostic assays

  • As a flagellar component expressed during mammalian infection, FlgV antibodies may provide specific markers of active infection

  • Integration into multi-antigen panels could improve current serological test sensitivity and specificity

• Therapeutic Targeting:

  • As a critical component for flagellar assembly and therefore bacterial motility and virulence, FlgV represents a potential antimicrobial target

  • Small molecule inhibitors of FlgV function could potentially immobilize the spirochete, reducing dissemination

  • Structural insights into FlgV could enable rational drug design approaches

• Vaccine Development:

  • If sufficiently immunogenic and surface-exposed, FlgV could potentially contribute to vaccine candidates

  • Understanding FlgV's role in the enzootic cycle helps identify when targeting this protein would be most effective

These applications would complement existing approaches like the human sweat protein SCGB1D2, which has shown protective effects against B. burgdorferi , and could address current gaps in Lyme disease management.

How can comparative genomics and evolutionary analyses of FlgV across Spirochaetes enhance our understanding of flagellar assembly mechanisms?

Comparative genomics and evolutionary analyses of FlgV offer valuable insights:

These analyses can:

• Identify conserved functional domains essential across all spirochetes

• Discover species-specific adaptations related to particular niches

• Reveal potential compensatory mechanisms in species lacking FlgV

• Understand how FlgV coevolved with other flagellar components

• Uncover the evolutionary trajectory from ancestral flagellar proteins to the specialized spirochete endoflagella

Such investigations not only enhance our understanding of bacterial motility but could also reveal new targets for species-specific interventions against pathogenic spirochetes .

What are the common pitfalls in generating and validating gene knockouts in B. burgdorferi, and how can they be addressed?

Generating and validating gene knockouts in B. burgdorferi presents several technical challenges:

• Low Transformation Efficiency:

  • Challenge: B. burgdorferi has notoriously low transformation rates

  • Solution: Optimize electroporation parameters (field strength, pulse duration); use high-quality, methylated plasmid DNA; increase DNA concentration

• Multiple Plasmids:

  • Challenge: B. burgdorferi contains numerous plasmids that can be lost during manipulation

  • Solution: Verify plasmid content after transformation using PCR; maintain selection pressure; screen multiple clones

• Complementation Difficulties:

  • Challenge: Restoration of wild-type phenotypes can be problematic

  • Solution: Use site-specific integration vectors; ensure appropriate promoter strength; verify expression levels

• Polar Effects:

  • Challenge: Gene disruption may affect downstream genes in operons

  • Solution: Design non-polar mutations; use markerless deletion systems; verify expression of adjacent genes

• Phenotype Verification:

  • Challenge: Distinguishing direct from indirect effects of gene deletion

  • Solution: Create multiple mutant types (deletion, point mutations); use inducible systems; perform thorough phenotypic characterization

• Growth Rate Differences:

  • Challenge: Mutants often have growth defects complicating analysis

  • Solution: Normalize assays by growth phase rather than time; use continuous culture methods; account for growth differences in data interpretation