Recombinant Borrelia burgdorferi Flagellar biosynthetic protein FliQ (fliQ)

How does the structure of periplasmic flagella in B. burgdorferi differ from other bacteria?

B. burgdorferi possesses unique periplasmic flagella (PFs) that are anchored at each cell pole and extend through the periplasmic space, rather than projecting into the extracellular environment as in many other bacteria . The PFs consist of both FlaA and FlaB proteins, with FlaB forming the core and FlaA forming an outer sheath . Notably, B. burgdorferi expresses FlaA at lower levels compared to FlaB, unlike other spirochetes . The specific arrangement allows these spirochetes to move in a corkscrew manner through dense tissues. When studying flagellar components like FliQ, researchers should consider this unique structural arrangement and how it might influence the function and interactions of biosynthetic proteins within the flagellar assembly system.

What are the technical considerations for expressing recombinant FliQ protein from B. burgdorferi?

When expressing recombinant FliQ from B. burgdorferi, researchers should consider several methodological approaches based on successful expression of other flagellar proteins. For instance, based on the work with recombinant FlaA proteins from European borrelial strains, researchers might consider:

• Selecting an appropriate expression system (bacterial, yeast, or insect cell-based)

• Optimizing codon usage for the expression host

• Using affinity tags that minimize interference with protein folding

• Testing multiple purification conditions to ensure protein stability

The choice of purification method is critical, as demonstrated with FlaA, where using Triton X-100 versus Sarkosyl resulted in different protein recovery outcomes . When purifying FlaA with Triton X-100, researchers were able to isolate PFs with both FlaA and FlaB proteins, whereas using Sarkosyl resulted in no FlaA in the isolated PFs . Similar considerations may apply to FliQ purification.

What genetic approaches are most effective for studying FliQ function in B. burgdorferi?

Based on successful approaches with other flagellar proteins, the most effective genetic strategies for studying FliQ function would include:

• Transposon mutagenesis to generate FliQ-deficient mutants

• Site-directed mutagenesis to create specific amino acid substitutions

• Complementation studies using plasmid-based expression systems

• Conditional expression systems to study FliQ in different growth phases

Studies of FliH and FliI provide a methodological template, where transposon mutants exhibited altered morphology, reduced motility, and division defects . Similar phenotypic analyses would be valuable for FliQ mutants. Note that while genetic complementation may restore morphology and motility, it may not fully recover flagellar length and infectivity, as observed with FliH and FliI mutants .

How can cryo-electron tomography be applied to study the role of FliQ in flagellar assembly?

Cryo-electron tomography (cryo-ET) has proven valuable for examining the structural components of the flagellar motor in B. burgdorferi. Based on studies of FliH and FliI:

• Cryo-ET can visualize intact flagellar motors and identify structural differences between wild-type and mutant strains

• The technique allows detection of specific protein complexes within the flagellar apparatus

• Researchers can quantify flagellar number, length, and structural integrity

In studies of FliH and FliI, cryo-ET revealed that inactivation of either gene resulted in the loss of the FliH-FliI complex from otherwise intact flagellar motors . Similar approaches could determine whether FliQ disruption affects specific structural components of the flagellar motor or export apparatus.

What protein-protein interaction methods are suitable for identifying FliQ binding partners?

To characterize FliQ interactions within the flagellar export apparatus, researchers might employ:

• Co-immunoprecipitation with antibodies against FliQ or potential partners

• Bacterial two-hybrid systems to screen for interacting proteins

• Pull-down assays using recombinant FliQ as bait

• Chemical crosslinking followed by mass spectrometry to identify interaction networks

These approaches have been effective for characterizing protein complexes in bacterial systems. For example, understanding that FliH and FliI form a complex that functions in flagellar assembly provides a framework for investigating similar interactions involving FliQ.

How does FliQ contribute to the hierarchical assembly of flagellar structures in B. burgdorferi?

The flagellar assembly in bacteria typically follows a hierarchical order, with the export apparatus being an early structure formed during assembly. Research questions addressing FliQ's role might include:

• At what stage of flagellar assembly is FliQ required?

• Does FliQ function differ when synthesizing FlaA versus FlaB proteins?

• How is FliQ activity coordinated with other export apparatus components?

Given that spirochetes possess unique endoflagella and B. burgdorferi lacks the archetypal flagellar regulator σ28 , the assembly process likely has distinctive features requiring specialized experimental approaches to elucidate FliQ's specific contributions.

What is the impact of FliQ deficiency on B. burgdorferi pathogenesis in animal models?

Based on findings with other flagellar proteins, FliQ-deficient mutants would likely show attenuated virulence. Research approaches should consider:

• Mouse infection models using needle inoculation

• Tick feeding experiments to assess survival and replication in the vector

• Tissue distribution studies to evaluate dissemination capabilities

• Quantitative PCR to measure bacterial burdens in different organs

Studies of FlgV-deficient B. burgdorferi have shown that such mutants can survive and replicate in Ixodes ticks but are attenuated for infection and dissemination in mice . Similar experiments with FliQ mutants would help determine if FliQ has comparable importance for infection.

How does post-translational modification affect FliQ function in the flagellar export apparatus?

FlaA in B. burgdorferi undergoes post-translational glycosylation , raising questions about modifications of other flagellar proteins including FliQ:

• Does FliQ undergo post-translational modifications?

• What enzymes are responsible for any such modifications?

• How do modifications affect protein-protein interactions or export apparatus function?

Methodological approaches would include mass spectrometry to identify modifications, site-directed mutagenesis to eliminate modification sites, and functional assays to assess the impact on flagellar assembly.

How does FliQ structurally and functionally compare to the newly characterized FlgV protein?

Recent research has identified FlgV (BB0268) as a structural flagellar component that modulates flagellar assembly in B. burgdorferi . Comparative studies between FliQ and FlgV would investigate:

• Structural similarities and differences between the proteins

• Their respective localization within the flagellar apparatus

• The phenotypic effects of single versus double mutations

• Conservation patterns across bacterial species

Such comparisons would provide insights into the specialized roles of different flagellar proteins in B. burgdorferi. For instance, FlgV is broadly conserved in the flagellar superoperon alongside σ28 in many Spirochaetae, Firmicutes, and other phyla , suggesting functional importance across diverse bacteria.

What is the relationship between FliQ and the FliH-FliI complex in flagellar export?

The FliH-FliI complex plays a crucial role in B. burgdorferi flagellar assembly, with mutations resulting in truncated flagella . Research questions regarding FliQ's relationship to this complex include:

• Does FliQ physically interact with FliH and/or FliI?

• Is FliQ dependent on the FliH-FliI complex for proper localization?

• Can overexpression of FliQ compensate for FliH or FliI deficiency?

Experimental approaches would include co-immunoprecipitation, bacterial two-hybrid assays, and genetic suppressor screens to identify functional relationships.

How does the regulation of FliQ expression differ from that of FlaA and FlaB?

Understanding the regulation of flagellar gene expression is crucial for comprehending the assembly process. For FliQ, important questions include:

• Is FliQ regulated at the transcriptional or translational level?

• What environmental signals modulate FliQ expression?

• How does FliQ expression compare temporally with other flagellar proteins?

Research on FlaA has shown that it is likely regulated at the translational level in B. burgdorferi, as a flagellar mutant still synthesized flaA message but failed to produce FlaA protein . Similar regulatory mechanisms might control FliQ expression.

Can recombinant FliQ be used as a diagnostic antigen for Lyme disease?

While some flagellar proteins have shown potential as diagnostic antigens, the utility of FliQ would require investigation:

• Is FliQ immunogenic during human infection?

• Does it elicit antibodies that can be detected in early disease stages?

• How specific are anti-FliQ antibodies for B. burgdorferi versus other spirochetes?

Studies of recombinant FlaA have produced contradictory results regarding its diagnostic utility. Gilmore et al. reported promising results using recombinant FlaA for detecting antibodies in erythema migrans patients, while Ge et al. failed to demonstrate a useful serologic role for another rFlaA construct . Similar comparative studies would be needed to evaluate FliQ's diagnostic potential.

What timepoints during infection are most dependent on FliQ-mediated motility?

Understanding when flagellar function is most critical during infection provides insights into pathogenesis:

• Is FliQ particularly important for initial infection, dissemination, or persistence?

• Does its importance differ between tick and mammalian hosts?

• Could targeting FliQ function represent a therapeutic approach?

Research on FlgV has defined specific infection timepoints when spirochete motility is most crucial , and similar temporal analyses would be valuable for understanding FliQ's role in the infection process.

How conserved is FliQ across Borrelia species and is there potential for broad-spectrum targeting?

Characterizing FliQ conservation would address:

• Is FliQ highly conserved across Borrelia species causing different clinical manifestations?

• Are there species-specific structural or functional differences?

• Could FliQ be targeted to inhibit multiple pathogenic Borrelia species?

Western blot analysis using Treponema pallidum anti-FlaA serum has shown that FlaA is antigenically well conserved across several spirochete species , and similar conservation analyses for FliQ would inform its potential as a broad-spectrum target.