Recombinant Flagellar assembly factor FliW 2 (fliW2)

What is Flagellar assembly factor FliW2 and how does it function in bacterial systems?

FliW2 is a protein that functions as a flagellar assembly factor in bacteria, particularly well-characterized in Helicobacter pylori. It acts as a protein antagonist that directly interacts with the global posttranscriptional regulator CsrA. Through this interaction, FliW2 prevents CsrA from binding to target mRNAs such as flagellin transcripts (including flaA mRNA), effectively de-repressing flagellar motility. The mechanism involves FliW2 binding to CsrA to form heterodimers, which prevents CsrA from forming homodimers that would otherwise bind to and repress target transcripts .

How does FliW2 differ from FliW1 in bacterial flagellar systems?

While both FliW1 and FliW2 are flagellar assembly factors, they exhibit distinct structural characteristics and potentially different regulatory functions. In H. pylori, FliW2 (UniProt ID: Q9ZJL5) has been demonstrated to interact directly with CsrA and influence flagellar motility. Comparative sequence analysis and structural predictions using tools like ColabFold with AlphaFold2 algorithm reveal differences in their protein structures. FliW2 contains specific β-barrel regions that form a cleft predicted to interact with CsrA, which may not be present or functionally equivalent in FliW1 (UniProt ID: Q9ZK60). This structural divergence suggests distinct evolutionary roles in flagellar regulation systems across bacterial species .

What experimental evidence demonstrates the role of FliW2 in flagellar motility?

Experimental evidence for FliW2's role in flagellar motility comes from multiple approaches:

• Knockout studies: ΔfliW2 mutant strains demonstrate reduced expression of major flagellin FlaA, impaired flagellar filaments, and attenuated motility compared to wild-type bacteria.

• Protein interaction assays: Direct interaction between FliW2 and CsrA has been demonstrated through bacterial two-hybrid analysis and in vitro pull-down assays.

• Structural predictions and validations: AlphaFold2 predictions identified specific interacting regions between FliW2 and CsrA, which were subsequently validated through truncation experiments.

• Functional regulation assessment: Studies show that FliW2 allosterically antagonizes CsrA activity, preventing it from binding to flaA mRNAs, thus indirectly promoting flagellin expression and flagellar assembly .

What are the recommended approaches for creating recombinant FliW2 expression systems?

To create effective recombinant FliW2 expression systems, researchers should consider the following methodological approach:

• Gene selection and optimization: Identify the complete fliW2 gene sequence from your bacterial species of interest (e.g., H. pylori strain J99, genome accession number: NC_000921.1).

• Expression vector design: Select an appropriate expression vector with:

  • Inducible promoter (e.g., T7 or tac)

  • Affinity tag (His6, GST, or MBP) for purification

  • Appropriate antibiotic resistance markers

  • Consideration of N- or C-terminal tag placement based on predicted structural domains

• Expression conditions optimization:

  • Test multiple expression strains (BL21(DE3), Rosetta, Arctic Express)

  • Vary induction parameters (temperature: 16-37°C; IPTG concentration: 0.1-1.0 mM)

  • Consider auto-induction media for high-density cultures

• Protein solubility enhancement:

  • Co-expression with chaperones

  • Fusion with solubility enhancers (SUMO, thioredoxin)

  • Addition of stabilizing agents (glycerol, arginine) to lysis buffers

• Purification strategy:

  • Implement a multi-step purification approach

  • Include tag removal by specific proteases

  • Validate protein identity by mass spectrometry

  • Assess protein folding by circular dichroism

This approach ensures production of functional recombinant FliW2 suitable for downstream interaction studies and structural analysis .

How can researchers effectively design in-frame deletion mutations of fliW2 for functional studies?

Designing effective in-frame deletion mutations of fliW2 requires a carefully planned approach:

• Strategic deletion design:

  • Map functional domains using protein prediction tools

  • Design deletions that preserve reading frame of downstream genes

  • Retain 5-10 codons at N- and C-termini to minimize polar effects

• Primer design for mutagenesis:

  • For inverse PCR approach: Design complementary primers that flank but exclude the region to be deleted

  • Include restriction sites for marker cassette insertion if needed

  • Verify primer specificity using in silico PCR tools

• Construction procedure:

  • Amplify ~1.5 kb fragment containing fliW2 and flanking sequences (~750 bp upstream and downstream)

  • Clone into appropriate vector (e.g., pGEMTeasy)

  • Perform inverse PCR to create the deletion

  • Insert selectable marker (e.g., chloramphenicol resistance cassette)

• Transformation and selection:

  • Transform constructed deletion vector into target bacteria via natural transformation or electroporation

  • Select transformants using appropriate antibiotics

  • Confirm recombination by PCR, Southern blotting, and DNA sequencing

• Validation of non-polar effects:

  • Perform RT-qPCR of flanking genes to verify absence of polar effects

  • Complement the mutation with wild-type gene on separate vector

  • Compare phenotypes of wild-type, mutant, and complemented strains

This systematic approach minimizes unintended consequences while enabling clear interpretation of FliW2 function in flagellar assembly .

What are the key variables to control when studying FliW2-CsrA interactions?

When designing experiments to study FliW2-CsrA interactions, researchers must carefully control these critical variables:

Controlling these variables ensures reproducible and interpretable results when investigating the molecular mechanisms of FliW2-CsrA interaction .

How does the C-terminal extension of CsrA influence its interaction with FliW2?

The C-terminal extension of CsrA plays a critical role in mediating its interaction with FliW2, as revealed through both computational predictions and experimental validation:

AlphaFold2 structural predictions identified that the C-terminal extension helix–loop–helix (residues 55–76) of CsrA forms a specific interaction interface with the β-barrel region of FliW2. This interaction domain is distinct from CsrA's N-terminal RNA binding domain, suggesting an allosteric regulatory mechanism.

Systematic truncation experiments provide compelling evidence for the importance of this C-terminal region. Using bacterial two-hybrid analysis with T25-fused FliW2 and T18-fused CsrA truncation variants, researchers demonstrated that:

• Full-length CsrA and variants with minor C-terminal truncations (CsrA 1-72, CsrA 1-63) maintained binding capacity with FliW2.

• More extensive C-terminal truncations (CsrA 1-58, CsrA 1-54) eliminated FliW2 binding, as evidenced by significantly reduced β-galactosidase activity compared to negative controls.

This region-specific interaction creates a regulatory mechanism where FliW2 binding does not directly compete with RNA for the same binding site on CsrA, but instead causes allosteric changes that prevent CsrA from binding to target mRNAs like flaA. This allosteric antagonism represents a sophisticated control mechanism for bacterial flagellar biosynthesis .

What computational approaches are most effective for predicting FliW2-protein interactions?

For predicting FliW2-protein interactions, a multi-layered computational approach incorporating the following methods has proven most effective:

• Deep learning structural prediction:

  • AlphaFold2 implementation through ColabFold has demonstrated high accuracy in predicting FliW2-CsrA interactions

  • The procedure employs MMseqs2 for homology searches against UniRef30, PDB70, and environmental databases

  • This approach successfully identified the β-barrel region of FliW2 as forming a cleft that interacts with CsrA's C-terminal extension

• Multiple sequence alignment analysis:

  • Using tools like CLC Genomic Workbench to align FliW homologs across bacterial species

  • Identifying conserved residues that may represent functional interaction sites

  • Comparing alignment patterns between FliW1 and FliW2 to distinguish unique binding interfaces

• Molecular dynamics simulations:

  • Validating predicted interaction interfaces through energy minimization

  • Assessing stability of proposed protein-protein complexes

  • Identifying potential conformational changes upon binding

• RNA structure prediction:

  • Tools like UNAFold Web Server for RNA secondary structure prediction

  • Essential for understanding how FliW2-CsrA interaction affects CsrA binding to mRNA targets

  • Particularly relevant for analyzing the 5'UTR of flaA mRNA and CsrA binding motifs

Integration of these computational approaches provides researchers with testable hypotheses about critical residues and conformational changes involved in FliW2-protein interactions, which can then be experimentally validated through site-directed mutagenesis and interaction assays .

What mechanisms explain how FliW2 allosterically antagonizes CsrA activity?

The allosteric antagonism of CsrA by FliW2 involves a sophisticated molecular mechanism:

• Binding-induced conformational changes:

  • FliW2 binds to the C-terminal extension (residues 55-76) of CsrA through its β-barrel region

  • This interaction induces conformational changes in CsrA's structure

  • Although the binding does not directly block CsrA's N-terminal RNA binding domain, it alters its configuration

• Disruption of CsrA dimerization:

  • Native CsrA functions as a homodimer to effectively bind target mRNAs

  • FliW2 binding promotes formation of FliW2-CsrA heterodimers

  • These heterodimers prevent formation of functional CsrA homodimers needed for RNA binding

• Altered RNA binding affinity:

  • The allosteric changes induced by FliW2 reduce CsrA's affinity for target mRNAs

  • This prevents CsrA from binding to critical sequences like the 5'UTR of flaA mRNA

  • As a result, ribosomes can access the ribosome binding sites that would otherwise be blocked by CsrA

• Competitive binding equilibrium:

  • A dynamic equilibrium exists between FliW2-CsrA complexes and CsrA-mRNA complexes

  • This creates a responsive regulatory system that can adapt to changing cellular conditions

  • When FliW2 levels increase, CsrA activity is inhibited, promoting flagellar gene expression

This allosteric mechanism represents an elegant regulatory strategy that allows bacteria to fine-tune flagellar gene expression without directly competing for the same binding sites on the global regulator CsrA .

How can researchers overcome challenges in detecting weak FliW2-CsrA interactions?

Detecting weak or transient FliW2-CsrA interactions requires specialized approaches to enhance sensitivity and specificity:

• Crosslinking strategies:

  • Implement chemical crosslinking with optimized bifunctional reagents (DSS, formaldehyde)

  • Use photo-activatable crosslinkers for site-specific interaction analysis

  • Apply crosslinking prior to cell lysis to capture transient interactions

  • Analyze crosslinked products by immunoblotting or mass spectrometry

• Enhanced two-hybrid systems:

  • Optimize the Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) system by:

    • Testing both N- and C-terminal fusion orientations

    • Using split-reporter systems with enhanced sensitivity

    • Implementing low-temperature incubation (25-30°C) to stabilize interactions

    • Adjusting inducer concentrations to optimize protein expression levels

• Advanced biophysical methods:

  • Surface Plasmon Resonance (SPR) with regeneration optimization

  • Microscale Thermophoresis (MST) for solution-based interaction analysis

  • Bio-Layer Interferometry (BLI) with extended association phases

  • Isothermal Titration Calorimetry (ITC) with concentrated protein samples

• Proximity-based detection methods:

  • Bioluminescence Resonance Energy Transfer (BRET)

  • Fluorescence Resonance Energy Transfer (FRET)

  • Split luciferase complementation assays

  • Proximity ligation assays for detecting interactions in situ

• Native detection systems:

  • Co-immunoprecipitation with optimized gentle lysis conditions

  • Sequential epitope tag purification for higher specificity

  • Mass spectrometry analysis with targeted multiple reaction monitoring

By implementing these advanced methodologies, researchers can overcome sensitivity limitations and successfully detect weak or transient interactions between FliW2 and CsrA proteins that might be missed by standard techniques .

What strategies can address issues with recombinant FliW2 solubility and stability?

When facing challenges with recombinant FliW2 solubility and stability, researchers should implement this systematic troubleshooting approach:

• Expression optimization:

  • Reduce induction temperature (16-20°C) to slow protein folding

  • Decrease inducer concentration (0.1-0.5 mM IPTG) to reduce expression rate

  • Test auto-induction media for gradual protein expression

  • Evaluate specialized expression strains (Arctic Express, Rosetta-gami)

• Buffer optimization:

  • Screen various pH conditions (pH 6.0-8.5) to find stability optima

  • Test stabilizing additives:

    • Osmolytes: glycerol (5-20%), sucrose (5-10%)

    • Amino acids: arginine, proline (50-200 mM)

    • Detergents: non-ionic (0.01-0.1% Triton X-100, NP-40)

    • Salt concentration variations (100-500 mM NaCl)

• Fusion tag strategies:

  • Implement solubility-enhancing fusion partners:

    • MBP (Maltose-Binding Protein)

    • SUMO (Small Ubiquitin-like Modifier)

    • Thioredoxin (TrxA)

    • NusA (N-utilization substance protein A)

  • Test both N-terminal and C-terminal tag positions

  • Include flexible linkers between tag and FliW2

• Co-expression approaches:

  • Co-express with protein partners (e.g., truncated CsrA domains)

  • Co-express with molecular chaperones (GroEL/ES, DnaK/J/GrpE)

  • Implement dual-plasmid systems with tunable expression ratios

• Refolding strategies (if inclusion bodies form):

  • Optimize solubilization in chaotropic agents (6-8 M urea or 4-6 M guanidine HCl)

  • Implement step-wise dialysis for controlled refolding

  • Use on-column refolding during affinity purification

  • Add redox pairs (GSH/GSSG) to facilitate disulfide bond formation

• Storage stabilization:

  • Flash-freeze in liquid nitrogen with cryoprotectants

  • Test lyophilization with appropriate excipients

  • Determine optimal storage buffer composition

  • Evaluate protein stability at different temperatures (-80°C, -20°C, 4°C)

This comprehensive approach addresses the multifaceted challenges in producing stable, soluble recombinant FliW2 for functional and structural studies .

How might understanding FliW2 function contribute to new antimicrobial development strategies?

Understanding FliW2 function opens several promising avenues for antimicrobial development:

• Targeting bacterial motility:

  • Flagellar motility is essential for colonization by many pathogens, including H. pylori

  • Compounds disrupting FliW2 function could impair bacterial motility and reduce virulence

  • This approach may be particularly valuable against pathogens with increasing antibiotic resistance

• Protein-protein interaction inhibitors:

  • Small molecules designed to interfere with FliW2-CsrA interaction

  • Peptide mimetics of the FliW2 binding region could competitively inhibit the interaction

  • Structure-based drug design targeting the β-barrel region of FliW2 or the C-terminal extension of CsrA

• Posttranscriptional regulation disruption:

  • Manipulating CsrA activity through FliW2 targeting could dysregulate multiple virulence factors

  • This represents a multi-target approach that might reduce the likelihood of resistance development

  • RNA-protein interaction modulators could complement direct protein-protein interaction inhibitors

• Adjuvant therapy potential:

  • FliW2-targeting compounds might enhance conventional antibiotic efficacy

  • Immobilizing bacteria through flagellar disruption could increase susceptibility to host immune defenses

  • Combination therapies targeting both growth and motility pathways may provide synergistic effects

• Host-pathogen interaction disruption:

  • Understanding how flagellar regulation affects host immune response recognition

  • Development of compounds that enhance pathogen detection by the host immune system

  • Potential for immunomodulatory approaches based on flagellar protein expression

This research direction offers particular promise for developing narrow-spectrum antimicrobials with reduced selection for resistance compared to conventional antibiotics targeting essential cellular processes .

What are the critical knowledge gaps in understanding FliW2 regulation across bacterial species?

Despite progress in characterizing FliW2 function, several critical knowledge gaps remain:

• Evolutionary conservation and divergence:

  • Limited understanding of FliW2 functional conservation across diverse bacterial phyla

  • Unclear evolutionary relationship between FliW1 and FliW2 homologs

  • Insufficient data on species-specific adaptations in FliW2 structure and function

• Regulatory network integration:

  • Incomplete characterization of how FliW2 activity integrates with other flagellar regulatory systems

  • Limited understanding of environmental signals that modulate FliW2 expression or activity

  • Gaps in knowledge about potential post-translational modifications affecting FliW2 function

• Structural mechanisms:

  • While AlphaFold2 predictions provide insights, high-resolution crystal structures of FliW2-CsrA complexes are lacking

  • Conformational dynamics during binding events remain poorly characterized

  • Molecular basis for allosteric regulation needs further elucidation

• Temporal regulation:

  • Limited understanding of the dynamics of FliW2-CsrA interactions during flagellar assembly

  • Unclear how FliW2 contributes to the sequential assembly of flagellar components

  • Insufficient data on the temporal coordination with flagellar gene expression

• Additional protein partners:

  • Potential for FliW2 to interact with proteins beyond CsrA remains largely unexplored

  • Comprehensive interactome studies are needed across different bacterial species

  • Functional significance of potential additional interactions requires investigation

• In vivo relevance:

  • Limited in vivo studies connecting molecular mechanisms to bacterial behavior

  • Insufficient data on the importance of FliW2 during host colonization and infection

  • Unclear how FliW2 function affects bacterial adaptation to different environments

Addressing these knowledge gaps will require integrated approaches combining structural biology, systems biology, and in vivo models to fully understand FliW2's role in bacterial physiology and pathogenesis .

What is the optimal experimental design for studying FliW2 function in bacterial pathogenesis?

An optimal experimental design for studying FliW2 function in bacterial pathogenesis requires a multi-faceted approach that integrates molecular, cellular, and in vivo methodologies:

• Genetic manipulation strategy:

  • Create a comprehensive mutation panel:

    • Complete gene deletion (ΔfliW2)

    • Point mutations in key functional residues (based on AlphaFold2 predictions)

    • Domain-specific deletions

    • Regulated expression systems (inducible/repressible)

  • Include complementation constructs to confirm phenotype specificity

  • Develop fluorescently tagged FliW2 variants for localization studies

• Molecular characterization:

  • Assess impact on flagellar gene expression:

    • Transcriptome analysis (RNA-seq) comparing wild-type and mutants

    • Targeted qRT-PCR of flagellar gene expression

    • Protein expression analysis of key flagellar components

  • Evaluate protein-protein interactions using multiple methods:

    • Bacterial two-hybrid analysis

    • Co-immunoprecipitation

    • Pull-down assays

    • In situ proximity ligation assays

• Cellular phenotype assessment:

  • Quantitative motility assays:

    • Soft-agar motility assessment

    • Video microscopy with automated tracking

    • Microfluidic-based migration assays

  • Flagellar structure analysis:

    • Transmission electron microscopy

    • Cryo-electron microscopy

    • Immunofluorescence microscopy

  • Biofilm formation evaluation

• Host interaction studies:

  • Adhesion assays with relevant cell lines

  • Invasion assays (if applicable to the pathogen)

  • Co-culture systems with immune cells

  • Transepithelial migration assessment

• In vivo infection models:

  • Animal colonization studies comparing wild-type and FliW2 mutants

  • Competition assays between strains

  • Tissue-specific bacterial localization

  • Immune response characterization

• Control variables and validation:

  • Include multiple bacterial strains to confirm generalizability

  • Use appropriate statistical methods for each experiment type

  • Perform biological and technical replicates

  • Include relevant control mutations in other flagellar genes

This comprehensive experimental design enables researchers to connect molecular mechanisms of FliW2 function to bacterial pathogenesis while controlling for confounding variables and ensuring reproducibility .

How can researchers effectively integrate computational and experimental approaches in FliW2 studies?

Effective integration of computational and experimental approaches for FliW2 research follows this iterative workflow:

• Initial sequence-based analysis:

  • Perform comprehensive sequence alignment of FliW2 across bacterial species

  • Identify conserved domains and residues

  • Predict secondary structure elements

  • Generate testable hypotheses about functional regions

• Structural prediction and modeling:

  • Apply AlphaFold2 through ColabFold for high-confidence structure prediction

  • Perform molecular dynamics simulations to assess structural stability

  • Identify potential protein-protein interaction interfaces

  • Model FliW2-CsrA complexes using docking algorithms

• Experimental validation of structural features:

  • Design targeted mutations based on computational predictions

  • Express and purify recombinant proteins with specific mutations

  • Assess structural integrity using circular dichroism or limited proteolysis

  • Validate interaction interfaces using hydrogen-deuterium exchange mass spectrometry

• Functional correlation studies:

  • Test mutation effects on protein-protein interactions using bacterial two-hybrid analysis

  • Assess impact on flagellar gene expression with reporter constructs

  • Quantify motility phenotypes with automated tracking systems

  • Correlate structural perturbations with functional outcomes

• Refinement through iterative cycles:

  • Use experimental data to refine computational models

  • Generate new predictions based on experimental outcomes

  • Design next-generation mutations with increased specificity

  • Develop more sophisticated interaction models

• Systems-level integration:

  • Incorporate FliW2 function into broader flagellar regulatory networks

  • Develop mathematical models of CsrA-FliW2-RNA interactions

  • Predict system-wide effects of perturbations

  • Design experiments to test network-level hypotheses

This integrated approach leverages the strengths of both computational prediction and experimental validation, creating a powerful methodology for understanding FliW2 function at multiple levels of biological organization. The workflow enables researchers to rapidly generate and test hypotheses while maintaining biological relevance and experimental feasibility .