Assay for assessing mucin binding to bacteria and bacterial proteins

Lubov S. Grigoryeva, Saima Rehman, Richard C. White, James A. Garnett*, Nicholas P. Cianciotto

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Article numbere3933
JournalBio-protocol
Volume11
Issue number5
DOIs
StatePublished - Mar 5 2021

Keywords

  • Bacterial flow-cytometry
  • ELISA
  • Legionella pneumophila
  • Mucin
  • Mucin-bacteria binding
  • Mucin-binding proteins

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Plant Science

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