Recombinant Legionella pneumophila subsp. pneumophila Flagellar L-ring protein (flgH)

What is the Flagellar L-ring protein (FlgH) in Legionella pneumophila?

FlgH is a critical structural protein that forms the L-ring component of the flagellar basal body in L. pneumophila. Similar to its counterpart in Salmonella typhimurium, L. pneumophila FlgH functions as a lipoprotein that anchors the flagellar structure to the outer membrane of the bacterial cell wall. The protein is synthesized as a precursor with a signal peptide that undergoes processing to form the mature protein with an approximate molecular mass of 25 kDa. The N-terminal region of FlgH contains a consensus sequence (LTG C) for lipoylation and signal peptide cleavage, which is essential for its proper localization and function .

How does the flagellar system contribute to L. pneumophila pathogenicity?

The flagellum of L. pneumophila significantly enhances the bacterium's pathogenicity by facilitating:

• Host cell encounter - The flagellum enables bacterial motility, increasing the probability of contact with host cells

• Invasion efficiency - Flagellated L. pneumophila demonstrates higher invasion rates in both amoebae and human HL-60 cells compared to non-flagellated mutants

• Initial infection establishment - Studies with flagellar mutants indicate that while adhesion and intracellular replication remain unaffected, the invasion efficiency is severely reduced, particularly in human cells

Unlike many virulence factors that are regulated by temperature above 37°C, flagellar expression in L. pneumophila is coordinatively regulated with other virulence-associated traits, including cell envelope modifications, osmotic resistance, and evasion of macrophage lysosomes .

What is the genetic organization and regulation of flgH in L. pneumophila?

The flgH gene in L. pneumophila is part of a complex regulatory network that controls flagellar assembly and expression. The flagellar system expression is regulated at multiple levels:

• Transcriptional regulation - The alternative sigma factor FliA (σ28) controls the expression of flagellar genes, including flgH

• Temperature-dependent expression - Flagellar genes are repressed at temperatures above 37°C

• Coordinate regulation - Expression occurs in concert with other virulence-associated traits

• Regulatory proteins - FlaR, a regulator of the LysR family, likely contributes to the regulation of flagellar genes

The flgH gene likely follows a similar regulatory pattern to flaA, which has been extensively studied in L. pneumophila.

What are the structural and functional homologies between FlgH in L. pneumophila and other bacterial species?

FlgH proteins share conserved structural features across bacterial species despite sequence variations. Based on studies in Salmonella typhimurium, we can infer the following comparative features for L. pneumophila FlgH:

The N-terminus of FlgH is responsible for anchoring the basal body in the outer membrane, while the C-terminus likely interacts with the P-ring to form the L,P-ring complex that serves as a bushing for the rotating flagellar rod .

How do post-translational modifications affect FlgH functionality?

Post-translational modifications, particularly lipoylation, are critical for FlgH functionality. In Salmonella, the FlgH protein undergoes lipoylation at the N-terminal cysteine residue, which is essential for proper membrane anchoring and function. Research findings indicate:

• Inhibition of lipoylation (using globomycin) causes accumulation of precursor forms of FlgH

• The mature FlgH incorporates [³H]palmitate, confirming its nature as a lipoprotein

• Purified hook-basal body complexes contain radiolabeled FlgH, demonstrating that the lipid modification is retained in assembled flagellar structures

• Mutation of the N-terminal cysteine significantly impairs function, though can be partially compensated by overexpression

• Alternative acylation (possibly at the N-terminal alpha-amino group) may occur at low levels even in the absence of the primary lipoylation site

These findings suggest that under normal physiological conditions, lipoyl modification is necessary for FlgH to function properly as the L-ring protein of the flagellar basal body in L. pneumophila as well.

What are the implications of FlgH in L. pneumophila vaccine development?

Although no direct studies on FlgH-based vaccines for L. pneumophila are reported in the provided search results, research on other flagellar proteins suggests potential applications:

• Recombinant flagellin A (FlaA) from L. pneumophila elicits strong innate and adaptive immune responses in mice

• FlaA immunization provides 60% survival against lethal challenge with L. pneumophila

• A fusion protein of FlaA and peptidoglycan-associated lipoprotein (PAL) shows enhanced protective efficacy (100% survival) against lethal challenge

As a structural component of the flagellum, FlgH might similarly serve as a vaccine candidate or as part of a multi-component vaccine. Its outer membrane localization makes it potentially accessible to antibodies, which could enhance bacterial clearance or neutralization.

How can the functionality of recombinant FlgH be assessed?

The functionality of purified recombinant FlgH can be assessed through multiple complementary approaches:

Structural Integrity Assessment:

• Circular dichroism (CD) spectroscopy to evaluate secondary structure

• Thermal shift assays to determine protein stability

• Limited proteolysis to detect properly folded domains

Membrane Association Tests:

• Liposome binding assays using fluorescently labeled protein

• Flotation gradient ultracentrifugation to detect membrane association

• Analysis of [³H]palmitate incorporation to confirm lipoylation

Functional Complementation:

• Transform flgH-deficient L. pneumophila mutants with a plasmid expressing recombinant FlgH

• Assess restoration of motility on soft agar plates (0.3% agar)

• Electron microscopy to confirm flagellar assembly

• In vitro host cell invasion assays to evaluate functional flagella formation

Protein-Protein Interaction Studies:

• Pull-down assays with other flagellar components, particularly P-ring proteins

• Surface plasmon resonance (SPR) to measure binding kinetics

• Bacterial two-hybrid system to detect interactions in vivo

What approaches are effective for studying FlgH interactions with other flagellar proteins?

Investigating the interactions between FlgH and other flagellar components requires specialized techniques:

In Vitro Interaction Studies:

• Co-immunoprecipitation using anti-FlgH antibodies or antibodies against His-tagged recombinant FlgH

• GST pull-down assays with GST-tagged FlgH and other flagellar proteins

• Isothermal titration calorimetry (ITC) to determine binding affinities and thermodynamic parameters

• Microscale thermophoresis (MST) for quantitative interaction analysis in solution

In Vivo Interaction Mapping:

• Bacterial two-hybrid system using complementary fragments of adenylate cyclase fused to potential interacting partners

• Förster resonance energy transfer (FRET) using fluorescently tagged proteins expressed in L. pneumophila

• Cross-linking followed by mass spectrometry (XL-MS) to identify interaction interfaces

Structural Studies:

• X-ray crystallography of FlgH alone or in complex with interaction partners

• Cryo-electron microscopy of purified flagellar basal bodies to visualize FlgH in its native context

• NMR spectroscopy for mapping interaction interfaces using isotopically labeled proteins

Data Analysis Matrix for Interaction Studies:

How can recombinant FlgH contribute to developing novel anti-Legionella therapeutics?

Recombinant FlgH offers several potential applications in therapeutic development:

Vaccine Development:

• As a structural protein essential for flagellar function, FlgH represents a potential vaccine antigen

• Its surface exposure facilitates antibody binding

• Combination with established immunogenic proteins like FlaA could enhance protective efficacy

Targeted Antivirulence Strategies:

• Small molecule inhibitors targeting FlgH could disrupt flagellar assembly, reducing bacterial motility and invasion

• Peptide inhibitors designed to interfere with FlgH-FlgI (P-ring) interactions might prevent functional flagella formation

Diagnostic Applications:

• Anti-FlgH antibodies could serve as diagnostic tools for detecting L. pneumophila

• Recombinant FlgH could be used for antibody screening in serological assays

Research with other flagellar proteins has demonstrated the potential of this approach, with recombinant FlaA providing 60% protection and a FlaA-PAL fusion protein offering 100% protection against lethal challenge with L. pneumophila in mouse models .

What are the key challenges in structural studies of FlgH and how can they be addressed?

Structural characterization of FlgH presents several challenges:

Challenges and Solutions:

• Membrane Association:

  • Challenge: As a lipoprotein, FlgH has hydrophobic regions that complicate expression and purification

  • Solutions:

    • Express truncated constructs lacking the lipid modification site

    • Use mild detergents during purification

    • Apply membrane mimetics (nanodiscs, amphipols) for structural studies

• Conformational Heterogeneity:

  • Challenge: FlgH likely adopts different conformations in isolation versus assembled in the L-ring

  • Solutions:

    • Use chemical cross-linking to stabilize specific conformations

    • Co-express with interaction partners to promote native folding

    • Apply cryo-EM for visualizing conformational ensembles

• Crystallization Difficulties:

  • Challenge: Membrane proteins often resist crystallization

  • Solutions:

    • Screen extensive crystallization conditions with varying detergents

    • Consider lipidic cubic phase crystallization

    • Use fusion proteins (T4 lysozyme, BRIL) to increase soluble surface area

• Expression Yields:

  • Challenge: Membrane proteins typically express at lower levels

  • Solutions:

    • Optimize codon usage for expression host

    • Test different promoter strengths and induction conditions

    • Consider specialized expression strains (C41, C43) designed for membrane proteins

How does the study of L. pneumophila FlgH contribute to understanding bacterial co-infection mechanisms?

The study of FlgH and other flagellar proteins in L. pneumophila provides insights into bacterial co-infection mechanisms and host-pathogen interactions:

• Studies of L. pneumophila flagellar structures contribute to understanding bacterial motility, which is critical during co-infections when multiple pathogens compete for the same ecological niche

• Understanding the immune response to flagellar proteins like FlgH may help explain how prior or concurrent infections affect susceptibility to L. pneumophila

• Recent research has shown co-infection possibilities with different variants of pathogens, such as the co-infection of Delta and Beta variants of SARS-CoV-2, which can lead to recombination events

• The mechanisms by which structural proteins like FlgH contribute to bacterial survival in multi-species biofilms may inform strategies to combat complex infections

• Flagellar proteins serve as pathogen-associated molecular patterns (PAMPs) that trigger host immune responses, potentially altering the course of co-infections

The lessons learned from studying L. pneumophila FlgH may be applicable to other bacterial pathogens that utilize similar structural proteins for virulence and survival in host environments.

How can researchers verify the correct folding and functionality of purified recombinant FlgH?

Ensuring proper folding and functionality of recombinant FlgH is crucial for meaningful experimental results. Multiple complementary approaches can be employed:

Biophysical Characterization:

• Circular dichroism (CD) spectroscopy to assess secondary structure content

• Differential scanning fluorimetry (DSF) to determine thermal stability

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to verify oligomeric state

• Intrinsic tryptophan fluorescence to monitor tertiary structure

Biochemical Verification:

• Limited proteolysis to identify stable domains (properly folded regions resist digestion)

• Binding assays with known interaction partners (particularly P-ring components)

• Lipid binding assays to confirm membrane interaction capability

• Antibody recognition using conformation-specific antibodies

Functional Validation:

• Complementation assays in flgH-deficient bacterial strains

• Motility restoration in mutant L. pneumophila

• In vitro assembly assays with other flagellar components

• Electron microscopy to visualize incorporation into flagellar structures

Quality Assessment Criteria for Recombinant FlgH: