L-Rhamnosylation of Listeria monocytogenes Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane
Fig 5
WTA l-rhamnosylation delays AMP interaction with the Lm plasma membrane.
(A) Depolarization rate of Lm strains in response to CRAMP. Mid-exponential-phase bacteria pre-stained (15 min) with 30 μM DiOC2(3) were challenged with 50 μg/ml CRAMP and changes in the membrane potential, expressed as the ratio of CRAMP-treated versus untreated samples, were monitored during 30 min. Data represent the mean±SD of three independent experiments. (B) SYTOX Green uptake kinetics of Lm strains in response to CRAMP-mediated membrane permeabilization. Exponential-phase bacteria were incubated (37°C) with PBS (white symbols) or 50 μg/ml CRAMP (black symbols), in the presence of 1 μM SYTOX Green, and the increase in green fluorescence emission was recorded over time. (C and D) Transmission electron microscopy analysis of the subcellular distribution of CRAMP in immunogold-labeled sections of mid-exponential-phase wild type and ΔrmlACBD Lm strains treated with 50 μg/ml CRAMP (15 min, 37°C). (C) Representative images of contrasted sections of Lm cells showing CRAMP-specific gold labeling (10-nm black dots). Scale bar: 0.2 μm. (D) Quantification of the subcellular partition of CRAMP labeling in wild type and ΔrmlACBD Lm strains, for two independent assays. The percentages of cell envelope- and cytoplasm-associated gold dots per bacterium were quantified (at least 90 cells per strain) and the results expressed for each strain as mean±SD. (E and F) Western blot analysis of levels of CRAMP bound to purified cell wall of different Lm strains. Purified cell wall (100 μg) was incubated with CRAMP (5 min), washed and digested overnight with mutanolysin. (E) Supernatants from mutanolysin-treated samples were resolved in 16% Tris-tricine SDS-PAGE and immunoblotted for CRAMP. The Lm cell wall-anchored protein InlA was used as loading control. (F) Quantification of the relative CRAMP levels represented as the mean±SD of four independent blots. *, p≤0.05; **, p≤0.01.