TY - JOUR
T1 - Assay for Assessing Mucin Binding to Bacteria and Bacterial Proteins
AU - Grigoryeva, Lubov S
AU - Rehman, Saima
AU - White, Richard C
AU - Garnett, James A
AU - Cianciotto, Nicholas P
N1 - Funding Information:
Work in the Cianciotto lab was supported by a National Institutes of Health grant R01AI 043987. LSG and RCW were also partly supported by National Institutes of Health training grants T32 GM08061 and T32 AI0007476, respectively. We thank the Northwestern ImmunoBiology Flow Cytometry Core for the maintenance and use of their equipment. Work in the Garnett lab was supported by MRC grants MR/M009920/1 and MR/R017662/1.
Publisher Copyright:
Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.
Copyright:
Copyright 2021 Elsevier B.V., All rights reserved.
PY - 2021/3/5
Y1 - 2021/3/5
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85116052446&partnerID=8YFLogxK
U2 - 10.21769/BioProtoc.3933
DO - 10.21769/BioProtoc.3933
M3 - Article
C2 - 33796607
SN - 2331-8325
VL - 11
SP - e3933
JO - Bio-protocol LLC
JF - Bio-protocol LLC
IS - 5
M1 - e3933
ER -
