Recombinant Treponema hyodysenteriae Flagellar filament core protein flaB2 (flaB2)

How does flaB2 relate structurally and functionally to other flagellar core proteins in Treponema hyodysenteriae?

Treponema hyodysenteriae (now also known as Brachyspira hyodysenteriae) flagella are composed of at least three FlaB-related core proteins (FlaB1, FlaB2, and FlaB3) which share extensive immunological and N-terminal sequence similarities . These proteins collectively form the internal core structure of the periplasmic flagella. While flaB2 is specifically characterized as a 34 kDa core protein, FlaB1 appears as a 38-kDa protein in electrophoretic analyses .

Despite their similarities, these proteins are encoded by distinct genes that can be individually targeted through mutagenesis, suggesting they serve non-redundant functions in flagellar assembly and bacterial motility . Current research indicates that these flagellar proteins play critical roles in maintaining periplasmic flagellar structural integrity, bacterial motility, and intestinal colonization capabilities .

What are the optimal storage and reconstitution protocols for recombinant flaB2 protein to maintain its structural integrity?

For optimal preservation of recombinant flaB2 protein, the following storage and reconstitution protocols are recommended:

Storage conditions:

• Store at -20°C for regular use

• For extended storage, conserve at -20°C or -80°C

• Avoid repeated freezing and thawing cycles

• Working aliquots may be stored at 4°C for up to one week

Reconstitution procedure:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is the default recommendation)

• Aliquot for long-term storage at -20°C/-80°C

The shelf life of properly stored recombinant flaB2 is approximately 6 months in liquid form and 12 months in lyophilized form when maintained at -20°C/-80°C .

What methodological approaches are most effective for studying the glycosylation patterns of flaB2?

Based on current research, several complementary methodological approaches have proven effective for investigating flaB2 glycosylation:

• Two-dimensional gel electrophoresis with dual staining:

  • Perform 2D-gel electrophoresis of purified periplasmic flagella (PFs)

  • Follow with both Coomassie blue staining (for protein detection) and specialized glycosylation staining (for glycan detection)

  • This combined approach allows simultaneous visualization of the protein and its glycosylation status

• Deglycosylation assays to determine glycan linkage type:

  • Treatment with PNGase F to test for N-linked glycosylation (negative results with flaB proteins)

  • β-elimination under alkaline conditions to remove O-linked glycans

  • Comparative analysis of treated and untreated samples using glycosylation staining

• Genetic approaches:

  • Creation of deletion mutants (e.g., ΔflaB2) through allelic exchange mutagenesis

  • Analysis of glycosylation patterns in mutant strains to confirm protein-glycan relationships

Research has demonstrated that flaB2, along with other FlaB proteins, undergoes O-linked glycosylation, as evidenced by negative results with PNGase F treatment and positive deglycosylation with β-elimination procedures .

What strategic approaches are most effective for creating flaB2 mutants, and what phenotypic changes should researchers anticipate?

Creating effective flaB2 mutants requires sophisticated genetic manipulation techniques:

Recommended mutant creation strategy:

• Clone the flaB2 gene with sufficient flanking regions

• Disrupt the gene by replacing an internal fragment with an antibiotic resistance cassette (e.g., chloramphenicol or kanamycin resistance genes)

• Introduce the disrupted gene construct into Treponema hyodysenteriae through electroporation

• Allow for recovery and phenotypic expression before selecting transformants with appropriate antibiotics

• Verify successful mutation through PCR analysis and immunoblotting to confirm the absence of the flaB2 protein

Anticipated phenotypic changes:
While the search results don't specifically detail flaB2 mutant phenotypes, research on related flagellar protein mutants suggests several likely outcomes:

• Altered motility: Flagellar mutants typically display modified swimming patterns or reduced motility in vitro, though they may retain some movement capability

• Reduced colonization ability: Studies with flaB1 mutants demonstrated compromised ability to colonize the mouse cecum, suggesting flaB2 mutants would likely show similar deficits

• Decreased virulence: Flagellar protein mutants show significantly reduced ability to cause disease in animal models

• Potential compensatory mechanisms: Other flagellar proteins may partially compensate for the loss of flaB2, resulting in less severe phenotypes than might be expected from complete flagellar dysfunction

How can researchers effectively assess the impact of flaB2 mutations on flagellar assembly and bacterial motility?

To comprehensively evaluate the effects of flaB2 mutations on flagellar structure and function, researchers should employ a multi-methodological approach:

Structural analysis techniques:

• Electron microscopy examination: To determine whether mutants remain capable of assembling morphologically normal periplasmic flagella, as observed with some flagellar protein mutants

• SDS-PAGE and immunoblotting: To confirm the absence of flaB2 protein and assess potential effects on the expression of other flagellar proteins

• 2D-gel electrophoresis with glycosylation staining: To evaluate changes in the glycosylation patterns of remaining flagellar proteins

Functional assessment methods:

• In vitro motility assays: Quantitative analysis of swimming behavior in semi-solid media or via direct microscopic observation

• Murine colonization models: Assessment of bacterial load in the cecum following experimental infection

• Competitive colonization experiments: Co-inoculation of wild-type and mutant strains to directly compare colonization efficiency

• Swine virulence studies: Evaluation of disease development in the natural host animal

Research with flaA1 and flaB1 mutants demonstrated that even single flagellar protein mutations can significantly affect both in vitro motility and in vivo colonization capabilities, highlighting the critical role of these proteins in bacterial pathogenesis .

What is the significance of O-linked glycosylation in flaB2, and how does it affect protein stability and function?

Research has demonstrated that flaB2, along with the other flagellar filament core proteins (FlaB1 and FlaB3), undergoes O-linked glycosylation . This post-translational modification appears to play critical roles in protein stability and function:

Evidence for O-linked glycosylation:

• Deglycosylation experiments show that while PNGase F (which removes N-linked glycans) has no effect on FlaB proteins, β-elimination treatment (which removes O-linked glycans) successfully removes the glycoreactive moieties

• After β-elimination treatment, glycosylated FlaB proteins are no longer detected by glycosylation staining

Functional significance:

• Protein stability: Following β-elimination treatment, FlaB proteins become "moderately degraded; whereas a non-specific protein associated with the PFs remained unaffected," suggesting that glycosylation directly contributes to the structural stability of these proteins within the assembled filaments

• Filament integrity: The glycosylation status likely influences the proper assembly and maintenance of the complex flagellar structure

• Motility effects: Since flagellar structure directly impacts bacterial motility, glycosylation indirectly affects the swimming capabilities of the organism

• Potential immunological significance: Although not specifically mentioned in the search results, bacterial protein glycosylation often plays roles in immune evasion or modulation

The specific glycan structures attached to flaB2 have not been fully characterized in the provided search results, representing an area for further research investigation.

How do the glycosylation patterns of flaB2 compare with those of flaB1 and flaB3, and what analytical techniques best reveal these differences?

Comparative glycosylation analysis techniques:

When analyzing glycosylation differences between flaB proteins, researchers should consider:

• Glycosylation site variations: Different proteins may have glycans attached at different amino acid positions

• Glycan structure differences: The specific sugar compositions and linkages may vary between proteins

• Glycosylation density: The number of glycans per protein molecule may differ

• Functional consequences: Whether these differences result in distinct functional properties for each protein

While the current research demonstrates that all three FlaB proteins are O-glycosylated, more detailed comparative analysis would provide valuable insights into how these modifications contribute to the specific roles of each protein within the flagellar structure .

How can mutational analysis of flaB2 contribute to our understanding of Treponema hyodysenteriae pathogenesis and host colonization?

Mutational analysis of flaB2 provides critical insights into the molecular mechanisms of Treponema hyodysenteriae pathogenesis through several research applications:

Key research applications:

• Structure-function relationships: By creating targeted mutations in different regions of flaB2, researchers can determine which domains are essential for flagellar assembly, motility, and bacterial colonization

• Motility-colonization correlation: Analysis of flaB2 mutants with varying degrees of motility impairment can help establish the quantitative relationship between swimming capability and intestinal colonization efficiency

• Glycosylation site mapping: Site-directed mutagenesis of potential glycosylation sites can reveal how specific modifications contribute to protein stability and function

• Protein interaction networks: Mutations affecting interaction surfaces between flaB2 and other flagellar proteins can illuminate the complex assembly process of periplasmic flagella

Research findings with flagellar mutants:
Studies with flagellar mutants have demonstrated that "chemotactically regulated or motility-regulated mucus association appears to play a key role in establishing infection by S. hyodysenteriae" . Isogenic flagellar mutant strains unable to express FlaA1 or FlaB1 were found to be "significantly less motile and less able to colonize mice and pigs than was wild-type S. hyodysenteriae" and were "rendered avirulent for swine" . These findings highlight the essential connection between flagellar function and bacterial pathogenesis.

What methodological considerations are critical when expressing and purifying recombinant flaB2 for structural and functional studies?

Successful expression and purification of recombinant flaB2 requires careful attention to several methodological considerations:

Expression system optimization:

• E. coli is the recommended expression host for recombinant flaB2 production

• Expression should target the full length protein (region 1-285)

• The tag type may vary depending on the manufacturing process and specific experimental needs

Purification considerations:

• Purified recombinant flaB2 should achieve >85% purity as assessed by SDS-PAGE

• Proteins should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol is recommended for long-term storage stability

Critical quality control measures:

• Protein identity verification: Confirm protein identity through mass spectrometry or N-terminal sequencing to ensure correspondence with the expected sequence

• Functional assessment: Evaluate the structural integrity and functionality of the purified protein through appropriate functional assays

• Glycosylation analysis: When expressed in bacterial systems, recombinant flaB2 may lack the native O-linked glycosylation present in Treponema hyodysenteriae, which could affect certain functional studies

• Endotoxin removal: For immunological studies, ensuring low endotoxin levels is essential to prevent confounding inflammatory responses

Experimental design considerations:
When using recombinant flaB2 for research, it's important to consider that bacterially-expressed recombinant proteins may differ from native proteins in terms of post-translational modifications, which could affect experimental outcomes, particularly in studies focused on protein-protein interactions or structural analyses.

How does the structure and function of Treponema hyodysenteriae flaB2 compare with flagellar proteins in other pathogenic spirochetes?

Flagellar proteins represent conserved but diversified structures across pathogenic spirochetes, with important comparative aspects:

Structural comparisons:
The flagellar filament core proteins in Treponema hyodysenteriae, including flaB2, share the fundamental architectural role of forming the internal core of periplasmic flagella that is characteristic of spirochetes . The flagella are composed of at least three FlaB-related core proteins (FlaB1, FlaB2, and FlaB3) which share extensive immunological and N-terminal sequence similarities .

Unlike externally flagellated bacteria, spirochetes including Treponema/Brachyspira species possess periplasmic flagella with a distinctive architecture consisting of a central core of FlaB proteins surrounded by FlaA sheath proteins . This architecture is conserved across spirochetes, though the specific number and properties of component proteins may vary between species.

Functional implications:
While direct comparative data is limited in the search results, research on flagellar mutants in Treponema hyodysenteriae demonstrates that these structures are essential for bacterial motility and virulence . Similar dependencies on flagellar function for pathogenesis have been observed in other spirochetes, suggesting functional conservation across species.

Evolutionary considerations:
The presence of multiple related FlaB proteins (FlaB1, FlaB2, FlaB3) suggests gene duplication events followed by functional diversification. This pattern of multiple flagellar core proteins appears to be conserved across various spirochete species, indicating its evolutionary importance for these bacteria.

What insights can comparative analysis of flaB1, flaB2, and flaB3 provide regarding the evolution of flagellar structure in spirochetes?

Comparative analysis of the three FlaB proteins offers valuable insights into flagellar evolution:

Evolutionary implications from structural analysis:

• Shared characteristics: All three FlaB proteins (FlaB1, FlaB2, and flaB3) in Treponema hyodysenteriae undergo O-linked glycosylation and share extensive immunological and N-terminal sequence similarities , suggesting common ancestry through gene duplication events

• Structural diversification: Despite their similarities, these proteins have distinct molecular weights - with flaB2 characterized as a 34 kDa protein and FlaB1 as a 38-kDa protein - indicating evolutionary divergence after duplication

• Functional specialization: The observation that individual flagellar protein mutants display altered but not abolished motility suggests functional specialization of each protein, with partial but incomplete compensation by the remaining proteins

Research approaches for evolutionary analysis:

• Comparative genomic analysis of flagellar gene clusters across spirochete species

• Phylogenetic analysis of FlaB protein sequences to establish evolutionary relationships

• Structural comparison of FlaB proteins to identify conserved and divergent domains

• Functional complementation studies to assess the degree of functional equivalence between proteins

Selective pressures:
The conservation of multiple FlaB proteins across spirochetes suggests that maintaining this diversity provides selective advantages, potentially including:

• Enhanced structural stability of the flagellar filament

• Improved motility characteristics in different environmental conditions

• Immune evasion through antigenic variation