Recombinant Pseudomonas aeruginosa B-type flagellin (fliC)

What are the key structural differences between A-type and B-type flagellin in P. aeruginosa?

B-type flagellin from P. aeruginosa (strain PAO1) differs significantly from A-type flagellin (strain PAK) in its posttranslational modifications and genetic basis. B-type flagellin is posttranslationally modified with an excess mass of up to 700 Da, with modifications occurring at two serine residues (positions 191 and 195). Each site contains a deoxyhexose linked to a unique 209 Da modification containing a phosphate moiety .

The genetic differences are equally notable. While A-type strains possess an extensive flagellar glycosylation island of 14 genes, B-type strains have a significantly reduced locus comprising only four genes (PA1088 to PA1091) . Research indicates minimal polymorphisms among the glycosylation islands of different B-type strains.

Methodological approaches to investigate these differences include:

• Mass spectrometry to characterize posttranslational modifications

• PCR analysis and sequencing of flagellar glycosylation island genes

• Mutational analysis of PA1088-PA1091 to determine specific functions in B-type flagellin modification

How does the unique D2 domain structure of FliC influence its immunological properties?

The D2 domain of P. aeruginosa FliC exhibits a unique structure consisting of two β-sheets and one α-helix that has not been found in other flagellins . In silico modeling of the flagellar filament suggests that this D2 domain is exposed to solution, making it particularly important for immunogenicity .

To investigate the immunological significance of the D2 domain:

• Generate recombinant constructs with modified D2 domains while preserving the D1 domain

• Assess TLR5 activation using reporter cell assays to confirm preservation of this function

• Measure antibody responses against wild-type and modified proteins

• Perform epitope mapping studies to identify immunodominant regions within the D2 domain

• Evaluate cross-protection between A-type and B-type flagellin immunization models

What strategies can optimize expression and purification of recombinant B-type flagellin?

Optimized protocols for recombinant B-type flagellin production should account for its unique structural features and modifications:

• Expression systems selection:

  • E. coli systems for high yield but unmodified protein

  • P. aeruginosa expression for native posttranslational modifications

  • Consider codon optimization for the target expression system

• Purification strategy:

  • Affinity tags (His6, GST) positioned to avoid interference with folding

  • Ion exchange chromatography exploiting the charged nature of modifications

  • Size exclusion chromatography as a final polishing step

• Quality control measures:

  • Mass spectrometry to confirm posttranslational modifications

  • Circular dichroism to verify secondary structure

  • Functional assays including TLR5 binding and polymerization capacity

  • Endotoxin removal crucial for immunological studies

How does B-type flagellin contribute to P. aeruginosa virulence mechanisms?

While specific data on B-type flagellin's role in virulence is limited in the provided search results, research on flagellin in related systems suggests multiple virulence-associated functions:

To investigate B-type flagellin's specific contributions to P. aeruginosa virulence:

• Generate clean deletion mutants of fliC in B-type strains

• Compare phenotypes of wild-type and mutant strains in:

  • Motility assays (swimming, swarming, twitching)

  • Biofilm formation assays

  • Host cell adhesion and invasion models

  • Secretion of TTSS effectors

• Conduct in vivo virulence studies in appropriate animal models

• Assess transcriptional changes in the mutant to identify compensatory mechanisms

What methods effectively assess the impact of flagellin glycosylation on bacterial motility?

The unique glycosylation pattern of B-type flagellin likely influences flagellar assembly and function. To assess this relationship:

• Comparative motility analysis:

  • Generate glycosylation-deficient mutants (targeting PA1088-PA1091)

  • Perform swimming motility assays in semi-solid agar

  • Conduct swarming assays on appropriate media

  • Utilize high-speed video microscopy to analyze swimming patterns and velocity

• Structural assessment:

  • Electron microscopy to analyze flagellar filament morphology and integrity

  • Atomic force microscopy to determine mechanical properties of flagella

  • In vitro polymerization assays with purified glycosylated and non-glycosylated flagellin

• Functional correlations:

  • Biofilm formation capacity

  • Surface attachment efficiency

  • Virulence in infection models

Such studies would illuminate how the specific modifications at serine residues 191 and 195 contribute to flagellar function and bacterial fitness.

How does the interaction between B-type flagellin and TLR5 differ from that of A-type flagellin?

Methodological approaches to investigate this question:

• Direct binding studies:

  • Gel filtration and native PAGE analysis to demonstrate direct interaction

  • Surface plasmon resonance to determine binding kinetics and affinity

  • ELISA-based binding assays with recombinant TLR5

• Structural approaches:

  • In silico modeling of B-type FliC-TLR5 complex

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Site-directed mutagenesis of predicted interface residues

• Functional analysis:

  • TLR5 reporter cell assays comparing A-type and B-type flagellin

  • Cytokine production profiles in TLR5-expressing cells

  • NF-κB activation kinetics and dose-response relationships

These comparative analyses would reveal how structural differences between flagellin types translate to functional differences in immune activation.

What strategies enhance the immunogenicity of B-type flagellin-based vaccines?

Recent advances in flagellin-based vaccine development offer several approaches to enhance B-type flagellin immunogenicity:

• Nanoparticle delivery systems:
  A promising approach involves genetic fusion of flagellin to self-assembled nanocarriers like ferritin. This strategy has proven effective for A-type flagellin, forming homogenous nanoparticles and inducing a Th1-predominant immune response that differs significantly from recombinant FliC alone . The ferritin-flagellin fusion (reFliC-FN) provided enhanced protection against both A-type and B-type flagellin-expressing P. aeruginosa strains .

• Strategic modification of glycosylation:
  Given the importance of glycosylation in B-type flagellin, targeted enhancement or modification of these patterns could potentially improve immunogenicity. This approach would require detailed understanding of the natural modifications at serine residues 191 and 195 .

• Adjuvant combinations:
  Flagellin itself acts as an adjuvant through TLR5 activation, but additional adjuvants targeting complementary pathways could synergistically enhance responses. When designing such combinations, researchers should carefully assess safety profiles through preliminary biocompatibility assays .

How can cross-protection between A-type and B-type flagellin strains be measured?

Designing studies to evaluate cross-protection requires comprehensive methodological approaches:

• Immunization protocols:

  • Vaccinate with either A-type or B-type recombinant flagellin

  • Include ferritin-flagellin nanoparticles shown to enhance cross-protection

  • Test various dosing schedules and routes of administration

• Immune response assessment:

  • Measure serum-specific antibody levels via indirect ELISA

  • Determine antibody subtypes (IgG1, IgG2a, IgA) to characterize response type

  • Analyze splenocyte proliferation upon restimulation with homologous and heterologous flagellins

  • Quantify cytokine-producing cells via ELISpot (IFN-γ, IL-4)

• Challenge studies:

  • Challenge with virulent strains expressing both A-type and B-type flagellin

  • Determine LD50 values compared to control groups

  • Measure bacterial clearance from tissues

  • Evaluate survival rates and durations

  • Perform histopathological examination of infected tissues

This comprehensive approach would provide clear data on the degree of cross-protection and identify immune correlates associated with protection.

How does flagellin glycosylation affect its adjuvant properties?

The unique glycosylation pattern of B-type flagellin may influence its adjuvant capacity through several mechanisms:

• TLR5 activation modulation:

  • Glycosylation could affect protein folding and stability

  • Modified residues might influence receptor binding kinetics

  • Changes in signaling threshold or duration could result

• Interaction with other pattern recognition receptors:

  • Glycan structures might enable recognition by lectins and C-type lectin receptors

  • This could provide additional activation signals to antigen-presenting cells

  • Synergistic activation of multiple pathways could enhance adjuvant effects

• Experimental approaches:

  • Compare TLR5 activation by glycosylated and enzymatically deglycosylated flagellin

  • Measure dendritic cell maturation markers (CD80, CD86, MHCII)

  • Assess cytokine profiles induced by different flagellin variants

  • Evaluate adjuvant potency when co-administered with model antigens

Understanding these relationships would enable rational design of flagellin-based adjuvants with optimized properties for specific applications.

How can structure-based design improve B-type flagellin as a vaccine antigen?

Crystal structure determination of FliC reveals important features that can guide rational design efforts:

The crystal structure of P. aeruginosa FliC at 2.1 Å resolution shows a conserved D1 domain folded into a rod-shaped structure responsible for TLR5 binding, and a unique D2 domain with two β-sheets and one α-helix not found in other flagellins . In silico modeling suggests the D2 domain is exposed in the flagellar filament, making it particularly relevant for immunogenicity .

Strategic approaches for structure-based optimization include:

• Preserving the TLR5-binding D1 domain while enhancing D2 domain immunogenicity

• Targeting modifications to exposed residues identified through structural modeling

• Stabilizing conformational epitopes through strategic disulfide bond engineering

• Creating chimeric constructs incorporating protective epitopes from other P. aeruginosa antigens

• Designing flagellin variants with enhanced stability but retained immunogenicity

These approaches should be guided by the structural model of the FliC-TLR5 complex to ensure that modifications preserve this critical interaction .

What methods can differentiate between cellular responses to A-type versus B-type flagellin?

Understanding differential cellular responses to flagellin variants requires sophisticated immunological techniques:

• Transcriptomic profiling:

  • RNA-seq of macrophages or dendritic cells exposed to each flagellin type

  • Pathway analysis to identify differentially activated signaling networks

  • Validation of key findings through targeted qPCR

• Single-cell analysis:

  • CyTOF or multi-parameter flow cytometry to identify responding cell populations

  • Single-cell RNA-seq to characterize heterogeneity in cellular responses

  • Imaging mass cytometry to assess spatial relationships in tissue responses

• Functional assessments:

  • T cell polarization assays (Th1, Th2, Th17)

  • Macrophage activation phenotypes (M1/M2 polarization)

  • Dendritic cell maturation and antigen presentation capacity

  • Neutrophil activation and NETosis induction

These approaches would reveal how structural differences between flagellin types translate to functional immunological differences, informing vaccine design and therapeutic applications.

How might synthetic biology approaches utilize B-type flagellin structures for biotechnology applications?

The self-assembling properties and unique structure of B-type flagellin offer exciting possibilities for biotechnology:

• Engineered vaccine delivery systems:

  • Building on the successful ferritin-flagellin fusion approach

  • Creating multivalent display platforms for multiple antigens

  • Designing controlled release systems responsive to specific environmental triggers

• Biosensing applications:

  • Exploiting the TLR5-binding capability for inflammation detection

  • Engineering the variable regions to bind specific analytes

  • Creating signal amplification systems based on flagellar polymerization

• Biomaterial development:

  • Utilizing self-assembly properties to create nanostructured materials

  • Engineering flagellin with novel functional domains

  • Developing stimulus-responsive materials based on conformational changes

The implementation of these applications requires:

• Precise structural knowledge of B-type flagellin

• Understanding the rules governing self-assembly

• Identification of modification sites that preserve structure and function

• Development of high-throughput screening systems to evaluate engineered variants