Recombinant Listeria monocytogenes serotype 4b Flagellar hook-basal body complex protein FliE (fliE)

What is the role of FliE in Listeria monocytogenes flagellar assembly?

FliE functions as an essential adaptor protein within the flagellar basal body complex of L. monocytogenes. Similar to other flagellar basal body proteins like FlhB, FliM, and FliY, FliE likely mediates the connection between the MS-ring and the rod structure, serving as a crucial structural component for proper flagellar assembly. Studies on related flagellar proteins have demonstrated that these components not only facilitate bacterial motility but also actively regulate flagellar synthesis . Methodologically, this has been established through constructing deletion mutants using homologous recombination strategies, followed by comparative analysis of motility capabilities, flagellar morphology, and protein expression profiles between wild-type and mutant strains.

What molecular techniques are recommended for initial characterization of FliE function?

For initial characterization of FliE function in L. monocytogenes, researchers should employ a systematic approach combining:

• Gene deletion using homologous recombination to create a ΔfliE mutant

• Complementation studies to verify phenotype restoration

• Motility assays in semi-solid media (e.g., tryptic soy agar) at 30°C

• Transcriptional analysis of flagellar-related genes using qRT-PCR

• Protein expression analysis via Western blotting

• Electron microscopy to visualize flagellar structures

This methodological approach parallels successful strategies used for characterizing other flagellar proteins such as FlhB, FliM, and FliY, where gene deletion resulted in complete abolishment of motility and flagella synthesis, with these phenotypes being fully restored in complemented strains . Researchers should monitor growth kinetics at both 30°C and 37°C to ensure that any observed phenotypic changes are not due to general growth defects.

What is the optimal Design of Experiments (DoE) approach for studying FliE interactions with other flagellar proteins?

When investigating FliE interactions with other flagellar proteins, researchers should implement a systematic Design of Experiments (DoE) approach. This methodology optimizes experimental efficiency by systematically varying experimental factors while monitoring responses:

Factors to consider:

• Temperature conditions (levels: 25°C, 30°C, 37°C)

• Growth media composition (levels: BHI, minimal media, modified media)

• Genetic background (levels: wild-type, different mutant strains)

• Expression induction parameters (if using recombinant systems)

Responses to measure:

• Protein-protein interaction strength (co-immunoprecipitation results)

• Flagellar assembly completion

• Bacterial motility

• Transcription levels of related genes

A factorial design would be most appropriate, allowing researchers to identify not only main effects but also interaction effects between factors . This approach enables efficient identification of key influencing factors with fewer experimental runs than would be required by varying one factor at a time. For initial screening, researchers might employ a fractional factorial design to identify the most significant factors before proceeding to optimization experiments using response surface methodology .

How can recombinant FliE be effectively produced and purified for structural studies?

Production and purification of recombinant L. monocytogenes FliE for structural studies requires careful optimization of expression systems and purification protocols:

• Expression system selection: While E. coli is commonly used for heterologous protein expression, researchers should consider testing multiple expression systems, including L. innocua (a non-pathogenic Listeria species) which may better maintain native protein folding and modifications .

• Expression vector design: Include a fusion tag (His-tag or GST) for purification, but ensure placement (N- or C-terminal) doesn't interfere with protein folding. Include a TEV protease cleavage site for tag removal.

• Induction parameters: Optimize temperature (typically lower temperatures like 18°C reduce inclusion body formation), inducer concentration, and induction duration through factorial experimental design .

• Purification protocol:

  • Initial capture: Affinity chromatography (IMAC for His-tagged proteins)

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography

• Quality control:

  • SDS-PAGE for purity assessment

  • Western blotting for identity confirmation

  • Circular dichroism for secondary structure verification

  • Dynamic light scattering for aggregation analysis

This methodological approach parallels strategies used for other flagellar proteins, where protein purity and structural integrity are essential for downstream applications such as crystallography, NMR, or cryo-EM structural determination.

What techniques are most effective for visualizing FliE localization within the bacterial cell?

For effective visualization of FliE localization within L. monocytogenes cells, researchers should employ complementary imaging techniques:

• Immunofluorescence microscopy:

  • Fix bacterial cells with 4% paraformaldehyde

  • Permeabilize cell walls (crucial for Gram-positive bacteria) using lysozyme treatment

  • Use primary antibodies specific to FliE and fluorophore-conjugated secondary antibodies

  • Counter-stain with DAPI to visualize bacterial DNA

• Fluorescent protein fusions:

  • Create genomic fliE-GFP or fliE-mCherry fusions

  • Verify functionality through motility assays

  • Examine localization patterns at different growth stages

  • Use time-lapse microscopy to track dynamic localization

• Immunogold electron microscopy:

  • Use gold-conjugated antibodies against FliE

  • Examine the precise localization within the flagellar apparatus

  • Compare localization patterns in wild-type versus mutant strains

• Super-resolution microscopy:

  • Techniques like STORM or PALM provide nanometer-scale resolution

  • Useful for determining spatial relationships between FliE and other flagellar components

These visualization approaches should be implemented at both 30°C and 37°C to observe temperature-dependent differences in localization patterns, as flagellar gene expression in L. monocytogenes is known to be temperature-regulated .

How does FliE contribute to Listeria monocytogenes virulence and host cell invasion?

The contribution of FliE to L. monocytogenes virulence involves complex interactions between flagellar structure, motility, and regulatory networks:

Experimental approach for investigating FliE's role in virulence:

• In vitro invasion assays:

  • Compare invasion efficiency between wild-type, ΔfliE mutant, and complemented strains

  • Use different cell lines (e.g., Caco-2, HepG2) to assess tissue-specific effects

  • Quantify bacterial adhesion, internalization, and intracellular replication

• Animal infection models:

  • Mouse models of listeriosis to assess systemic spread

  • Oral infection models to evaluate gastrointestinal colonization

  • Quantify bacterial burden in various organs (liver, spleen, brain)

• Transcriptomic analysis:

  • Compare gene expression profiles between wild-type and ΔfliE strains

  • Focus on virulence-associated genes and potential compensatory mechanisms

  • Analyze at both 30°C and 37°C to identify temperature-dependent effects

Similar to other flagellar proteins, FliE likely influences virulence through multiple mechanisms beyond motility alone. Research on related flagellar proteins has shown that these components can affect the expression of various virulence factors through regulatory cross-talk . Different L. monocytogenes clones exhibit varying levels of virulence, with hypervirulent clones (e.g., CC1) showing better intestinal colonization and invasion capabilities compared to hypovirulent clones (e.g., CC9, CC121) . The specific contribution of FliE to these clone-dependent virulence differences represents an important research question.

What bioinformatic approaches are recommended for analyzing evolutionary conservation of FliE across Listeria species?

Advanced bioinformatic analysis of FliE evolutionary conservation requires a comprehensive approach:

• Sequence retrieval and alignment:

  • Collect FliE sequences from all available Listeria species and strains

  • Include sequences from related bacterial genera for outgroup comparison

  • Use MUSCLE or MAFFT for multiple sequence alignment

  • Refine alignments manually to address potential misalignments

• Phylogenetic analysis:

  • Construct maximum likelihood trees using RAxML or IQ-TREE

  • Implement appropriate evolutionary models (LG+G, WAG+F+G)

  • Perform bootstrap analysis (≥1000 replicates) to assess branch support

  • Compare FliE phylogeny with species phylogeny to identify potential horizontal gene transfer events

• Structural conservation analysis:

  • Predict secondary and tertiary structures using AlphaFold or I-TASSER

  • Map conservation patterns onto structural models

  • Identify functionally important domains based on conservation patterns

• Selection pressure analysis:

  • Calculate dN/dS ratios to identify sites under positive or purifying selection

  • Use PAML or HyPhy for codon-based analyses

  • Correlate selection patterns with structural features and functional domains

This bioinformatic approach can reveal evolutionary relationships between pathogenic and non-pathogenic Listeria species. For example, research on L. innocua (generally considered non-pathogenic) has identified atypical hemolytic isolates that challenge this classification . Comparative analysis of flagellar proteins between these species could provide insights into the evolution of virulence capabilities.

How do environmental factors beyond temperature influence FliE expression and function?

The expression and function of flagellar proteins in L. monocytogenes are influenced by multiple environmental factors beyond temperature. To investigate these influences on FliE:

These environmental factors often interact with each other and with the temperature-dependent regulatory system. For example, the MogR/GmaR regulatory system that controls flagellar gene expression in response to temperature may also integrate signals from other environmental cues. Advanced experimental designs using DoE principles should include interaction terms to capture these complex relationships between environmental factors.

What are common challenges in generating viable fliE deletion mutants and how can they be addressed?

Creating viable fliE deletion mutants in L. monocytogenes presents several challenges that researchers must address:

• Potential essentiality issues:

  • If complete deletion is lethal, consider creating conditional mutants

  • Use inducible systems (e.g., IPTG-inducible promoters) to control expression

  • Create partial deletions that maintain essential functions

• Polar effects on downstream genes:

  • Design deletions that maintain reading frame

  • Use non-polar cassettes that don't affect downstream gene expression

  • Verify transcription of adjacent genes in the mutant strain

• Complementation difficulties:

  • Use site-specific integration vectors to ensure stable complementation

  • Test multiple promoters to achieve native-like expression levels

  • Consider both in trans (plasmid-based) and in cis (chromosome-integrated) complementation

• Phenotypic verification:

  • Compare growth curves at different temperatures (similar to Figure 1 in source )

  • Verify motility using semi-solid agar assays

  • Examine flagellar structures using electron microscopy

• Genetic verification:

  • PCR verification of deletion and insertion sites

  • Whole-genome sequencing to identify potential compensatory mutations

  • Transcriptomic analysis to verify expected changes in gene expression

These strategies are based on successful approaches used for other flagellar proteins in L. monocytogenes, where deletion mutants for flhB, fliM, and fliY were successfully generated despite their crucial roles in flagellar assembly and motility .

How can researchers resolve contradictory data regarding FliE function in different experimental systems?

When confronted with contradictory data regarding FliE function across different experimental systems, researchers should implement a systematic troubleshooting approach:

• Standardize experimental conditions:

  • Ensure consistent growth media composition across experiments

  • Standardize temperature, pH, and growth phase for sample collection

  • Use identical genetic backgrounds for all comparisons

• Validate methodological approaches:

  • Perform side-by-side comparisons of different assay methods

  • Include appropriate positive and negative controls in all experiments

  • Blind experimenters to sample identity when possible

• Consider strain-specific variations:

  • Different L. monocytogenes strains or serotypes may exhibit different phenotypes

  • Sequence the fliE gene and regulatory regions from all strains used

  • Test the same deletion in multiple strain backgrounds

• Evaluate environmental context:

  • Contradictions may reflect genuine biological responses to different conditions

  • Systematically vary environmental factors using DoE principles

  • Develop mathematical models to explain context-dependent behaviors

• Consult with collaborators:

  • Arrange for independent replication in different laboratories

  • Share detailed protocols to identify subtle methodological differences

  • Consider joint experiments with standardized materials

This systematic approach recognizes that contradictory data often reflects biological complexity rather than experimental error. For example, studies on L. monocytogenes virulence have shown that different clonal complexes exhibit varying levels of pathogenicity and environmental adaptation , which could influence the phenotypic consequences of fliE deletion.

What are the key considerations for designing cross-linking experiments to identify FliE protein interaction partners?

Designing effective cross-linking experiments to identify FliE protein interaction partners requires careful optimization:

• Cross-linker selection:

  • Use membrane-permeable cross-linkers for intact cells (e.g., formaldehyde, DSP)

  • Consider spacer arm length (2-15Å) based on expected proximity of interaction partners

  • Select chemistry compatible with intended analysis (e.g., reversible for certain MS approaches)

• Cross-linking conditions optimization:

  • Titrate cross-linker concentration to avoid non-specific aggregation

  • Optimize reaction time to capture transient interactions

  • Control temperature to maintain native protein conformations

• Sample preparation:

  • For membrane-associated proteins like FliE, use appropriate detergents for solubilization

  • Consider membrane fractionation before or after cross-linking

  • Use appropriate protease inhibitors to prevent degradation

• Analysis methods:

  • Immunoprecipitation followed by western blotting for known candidates

  • Mass spectrometry for unbiased identification of novel partners

  • Proximity labeling approaches (BioID, APEX) as complementary methods

• Control experiments:

  • Include non-cross-linked samples

  • Use irrelevant proteins as negative controls

  • Validate interactions through reciprocal pull-downs

  • Confirm biological relevance through mutational analysis

This methodological approach has been successfully applied to study protein-protein interactions in bacterial flagellar systems, revealing complex interaction networks that govern flagellar assembly and regulation .

How does the function of FliE compare with other flagellar basal body proteins like FlhB, FliM, and FliY?

FliE functions as part of the flagellar basal body complex in L. monocytogenes, with both distinct and overlapping roles compared to other basal body proteins:

Comparative functional analysis:

Based on studies of other flagellar proteins, FliE likely shares several characteristics with FlhB, FliM, and FliY:

• Essential role in flagellar assembly and motility

• Involvement in the flagellar type III secretion system (T3SS)

• Potential regulatory function affecting the expression of other flagellar genes

• Temperature-dependent expression pattern

What methodological approaches are most effective for studying the role of FliE in the flagellar type III secretion system?

To effectively study FliE's role in the flagellar type III secretion system (T3SS) of L. monocytogenes, researchers should employ a multi-faceted approach:

• Secretion assays:

  • Measure secretion efficiency of flagellar proteins in wild-type versus ΔfliE strains

  • Use reporter fusion proteins to quantify secretion rates

  • Analyze secretome composition through proteomic approaches

• Structure-function analysis:

  • Generate point mutations in conserved FliE residues

  • Create chimeric proteins with FliE domains from other bacteria

  • Test functionality through complementation of ΔfliE phenotypes

• Protein-protein interaction mapping:

  • Use bacterial two-hybrid systems to identify direct interaction partners

  • Perform co-immunoprecipitation studies with other T3SS components

  • Implement FRET-based approaches to study interactions in living cells

• Real-time secretion visualization:

  • Utilize fluorescent protein fusions to track protein export

  • Apply single-molecule approaches to measure secretion kinetics

  • Develop microfluidic systems for controlled secretion induction

These methodological approaches should consider the temperature-dependent regulation of flagellar genes in L. monocytogenes, testing conditions at both 30°C (permissive for flagellar expression) and 37°C (typically repressive) . Research on other flagellar proteins has demonstrated that components of the flagellar T3SS are involved not only in protein transport but also in regulating flagellar gene expression, suggesting FliE may have similar dual functions .

How can researchers differentiate between structural and regulatory roles of FliE in flagellar assembly?

Differentiating between structural and regulatory roles of FliE in flagellar assembly requires sophisticated experimental design:

• Domain-specific mutational analysis:

  • Create a library of FliE variants with mutations in different domains

  • Evaluate each variant for: (a) protein stability, (b) structural incorporation, and (c) regulatory function

  • Identify separation-of-function mutations that affect regulation without disrupting structure

• Temporal expression studies:

  • Develop inducible expression systems for precise control of FliE production

  • Monitor flagellar gene expression before and after FliE induction

  • Use time-lapse microscopy to track flagellar assembly progression

• Complementation with heterologous proteins:

  • Test if FliE from non-Listeria species can restore structural function without regulatory function

  • Create chimeric proteins combining domains from different species

  • Evaluate restoration of motility versus gene expression separately

• Biochemical approach:

  • Develop in vitro transcription systems with purified components

  • Test direct interaction of FliE with regulatory proteins and DNA

  • Perform chromatin immunoprecipitation to identify potential FliE-associated genomic regions

Based on studies of related flagellar proteins, researchers should consider that FliE may influence gene expression by participating in regulatory feedback loops. For example, in L. monocytogenes, the deletion of flhB, fliM, or fliY resulted in the downregulation of multiple flagellar genes, suggesting these structural proteins also participate in gene regulation . Similar dual functionality might be present in FliE, where its structural role in connecting the MS-ring to the rod might be coupled with regulatory activities affecting the expression of other flagellar components.