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Updated: Nov 10, 2025
Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins
Published on: June 14, 2016
Assay for Assessing Mucin Binding to Bacteria and Bacterial Proteins
Lubov S Grigoryeva1, Saima Rehman2, Richard C White1
1Department of Microbiology and Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.
Abstract:
Legionella pneumophila, a Gram-negative bacterium and the causative agent of Legionnaires' disease, exports over 300 effector proteins/virulence factors, through its type II (T2SS) and type IV secretion systems (T4SS). One such T2SS virulence factor, ChiA, not only functions as a chitinase, but also as a novel mucinase, which we believe aids ChiA-dependent virulence during lung infection. Previously published protocols manipulated wild-type L. pneumophila strain 130b and its chiA mutant to express plasmid-encoded GFP. Similarly, earlier studies demonstrated that wheat germ agglutinin (WGA) can be fluorescently labeled and can bind to mucins. In the current protocol, GFP-labeled bacteria were incubated with type II and type III porcine stomach mucins, which were then labeled with TexasRed-tagged WGA and analyzed by flow-cytometry to measure the binding of bacteria to mucins in the presence or absence of endogenous ChiA. In addition, we analysed binding of purified ChiA to type II and type III porcine stomach mucins. This protocol couples both bacterial and direct protein binding to mucins and is the first to measure Gram-negative bacterial binding to mucins using WGA and flow-cytometric analysis. Graphic abstract: Strategy for assessing bacterial and protein binding to mucins.
Insights
Legionella pneumophila uses ChiA, a novel mucinase, to bind host mucins, aiding virulence. This study developed a flow cytometry method to measure bacterial and protein binding to mucins.
Area of Science:
- Microbiology
- Infectious Diseases
- Bacterial Pathogenesis
Background:
- Legionella pneumophila causes Legionnaires' disease by secreting virulence factors.
- The ChiA protein is a T2SS effector with chitinase and mucinase activity.
- Mucins are key components of host mucus barriers, crucial in lung infections.
Purpose of the Study:
- To investigate the role of ChiA as a mucinase in L. pneumophila virulence.
- To develop and validate a novel flow cytometry-based method for measuring bacterial and protein binding to mucins.
- To assess the binding of L. pneumophila and purified ChiA to porcine stomach mucins.
Main Methods:
- Green fluorescent protein (GFP)-labeled L. pneumophila strains (wild-type and chiA mutant) were incubated with porcine stomach mucins (Type II and Type III).
- Mucins were labeled with TexasRed-tagged wheat germ agglutinin (WGA), and bacterial binding was analyzed by flow cytometry.
- Binding of purified ChiA protein to mucins was also assessed using the same labeling and flow cytometry techniques.
Main Results:
- The study successfully measured the binding of L. pneumophila to mucins using WGA and flow cytometry.
- The presence of endogenous ChiA influenced bacterial binding to mucins.
- Purified ChiA demonstrated direct binding to Type II and Type III porcine stomach mucins.
Conclusions:
- ChiA's mucinase activity is a significant factor in L. pneumophila's interaction with host mucins.
- The developed flow cytometry protocol provides a robust method for quantifying bacterial and protein-mucin interactions.
- This research offers new insights into L. pneumophila pathogenesis and potential therapeutic targets.
