Structure and functional analysis of the Legionella pneumophila chitinase ChiA reveals a novel mechanism of metal-dependent mucin degradation
Fig 4
L. pneumophila surface association of ChiA and mucin binding.
(A) Whole cell ELISA of L. pneumophila wild-type 130b (WT), chiA mutant NU318 (chiA), proA mutant AA200 (proA) and mip mutant NU203 (mip) detected with antiserum specific for either ChiA, ProA, or Mip. (B) Whole cell ELISA of chiA mutant incubated with either recombinant ChiA-FL or subdomains (NT, N1, N2, N3, CTD) and detected with antiserum specific for ChiA. PBS buffer alone was used as a control (-). Multiple comparisons against the control were made using a one-way ANOVA, Holm-Šídák multiple comparisons test; * P < 0.05, *** P < 0.001. (C) ELISA analysis of binding between immobilised type I-S, II or III porcine stomach mucins and His-tagged ChiA-FL, subdomains (NT, N1, N2, N3, CTD) and controls (SslE, NttE) detected with anti-His-tag antibody. BSA-coated wells were used as controls. *** P < 0.001; verses control empty well by two-tailed Student’s test. (D) Mucin binding to GFP-expressing L. pneumophila WT or chiA mutant strains were incubated at 25°C or 37°C with PBS, type I-S, II or III mucins followed by Texas Red-tagged wheat germ agglutinin (WGA). Mucin binding to bacteria was quantified by flow cytometry. * P < 0.05; verses PBS control by two-tailed Student’s test. All data represent the mean and standard deviation for triplicate experiments.