LecA (PA-IL) from Pseudomonas aeruginosa linked to Fluorescein

LecA (PA-IL) from Pseudomonas aeruginosa linked to Fluorescein

LecA (PA-IL) from Pseudomonas aeruginosa linked to Fluorescein is a fluorescently labeled lectin used to study bacterial adhesion mechanisms, biofilm formation, and host-pathogen interactions. This conjugate enables real-time visualization of galactose-specific binding events in experimental systems.

Key Characteristics of LecA (PA-IL):

• Function:
  LecA is a tetrameric, calcium-dependent lectin that binds α-galactose residues on host glycosphingolipids. It facilitates bacterial adhesion to epithelial cells, promotes biofilm formation, and contributes to cytotoxicity during infections.

• Disrupts lung epithelial barrier function, increasing permeability and bacterial dissemination.
• Acts as a virulence factor in chronic infections like cystic fibrosis.

• Structural Features:

• Molecular weight: ~12.8 kDa (monomer).
• Contains a carbohydrate-binding site with conserved tryptophan residues (e.g., W42) critical for galactose recognition.
• Requires Ca²⁺ ions for structural stability and ligand binding.

• Fluorescein Conjugation:
• The fluorescein tag allows quantitative tracking of LecA interactions with galactose-containing substrates.
• Applications include:
• • • Fluorescence polarization assays for ligand affinity measurements.
    • Imaging bacterial adhesion to host tissues.
    • High-throughput screening of glycomimetic inhibitors.

Applications in Research:

1. Biofilm Studies:
   LecA stabilizes biofilms by crosslinking extracellular polysaccharides (e.g., Psl). Fluorescein-labeled LecA helps monitor biofilm architecture and inhibitor efficacy.
2. Drug Discovery:
   Used to identify non-carbohydrate inhibitors via NMR-based methods (e.g., protein-observed ¹⁹F NMR). Glycoclusters with β-d-galactoside ligands show anti-adhesion potential by blocking LecA-mediated binding.
3. Pathogenesis Analysis:
   In vivo studies demonstrate that LecA inhibition with α-methyl-galactoside reduces lung injury and bacterial load.

Production and Validation:

• Recombinantly expressed in E. coli with affinity purification.
• Functional activity post-labeling confirmed via:

• Calcium-dependent binding assays.
• Structural integrity checks using X-ray crystallography.

This tool is vital for advancing therapeutic strategies targeting LecA-mediated infections, particularly in antibiotic-resistant P. aeruginosa strains.

Citations:

1. https://pubmed.ncbi.nlm.nih.gov/32573695/
2. https://experiments.springernature.com/articles/10.1007/978-1-0716-0430-4_25
3. https://pmc.ncbi.nlm.nih.gov/articles/PMC7874386/
4. https://pmc.ncbi.nlm.nih.gov/articles/PMC6920806/
5. https://journals.asm.org/doi/full/10.1128/iai.01204-08
6. https://pmc.ncbi.nlm.nih.gov/articles/PMC2681743/