WTA l-rhamnosylation delays AMP interaction with the <i>Lm</i> plasma membrane.

<p>(<b>A</b>) Depolarization rate of <i>Lm</i> 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>B</b>) SYTOX Green uptake kinetics of <i>Lm</i> 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. (<b>C and D</b>) Transmission electron microscopy analysis of the subcellular distribution of CRAMP in immunogold-labeled sections of mid-exponential-phase wild type and Δ<i>rmlACBD Lm</i> strains treated with 50 μg/ml CRAMP (15 min, 37°C). (C) Representative images of contrasted sections of <i>Lm</i> 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 Δ<i>rmlACBD Lm</i> 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. (<b>E and F</b>) Western blot analysis of levels of CRAMP bound to purified cell wall of different <i>Lm</i> 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 <i>Lm</i> cell wall-anchored protein InlA was used as loading control. (<b>F</b>) Quantification of the relative CRAMP levels represented as the mean±SD of four independent blots. *, <i>p</i>≤0.05; **, <i>p</i>≤0.01.</p

Genes encoding the l-rhamnose biosynthesis pathway are distributed in listeriae and other bacterial species.

<p>Comparison of the genomic organization of the l-rhamnose pathway genes in the genus <i>Listeria</i> and other bacteria. The corresponding species and strains are indicated on the left (<i>Lmo</i>, <i>Listeria monocytogenes</i>; <i>Lin</i>, <i>Listeria innocua</i>; <i>Lse</i>, <i>Listeria seeligeri</i>; <i>Liv</i>, <i>Listeria ivanovii</i>; <i>Lwe</i>, <i>Listeria welshimeri</i>; <i>Smu</i>, <i>Streptococcus mutans</i>; <i>Mtu</i>, <i>Mycobacterium tuberculosis</i>; <i>Sen</i>, <i>Salmonella enterica</i> serovar Typhimurium; <i>Sfl</i>, <i>Shigella flexneri</i>; <i>Pae</i>, <i>Pseudomonas aeruginosa</i>) and listerial serotypes are indicated on the right. Genes are represented by boxed arrows and their names are provided for strain EGD-e. Operons are underlined by dashed arrows and homologs of the <i>rml</i> genes are shown with identical colors. Numbered gaps indicate the genetic distance (Mb, mega base pairs) between <i>rml</i> genes located far apart in the chromosome. Bacterial genomic sequences were obtained from NCBI database and chromosomal alignments assembled using Microbial Genomic context Viewer and Adobe Illustrator.</p

Plasmids and bacterial strains.

<p>Plasmids and bacterial strains.</p

L-Rhamnosylation of <i>Listeria monocytogenes</i> Wall Teichoic Acids Promotes Resistance to Antimicrobial Peptides by Delaying Interaction with the Membrane

<div><p><i>Listeria monocytogenes</i> is an opportunistic Gram-positive bacterial pathogen responsible for listeriosis, a human foodborne disease. Its cell wall is densely decorated with wall teichoic acids (WTAs), a class of anionic glycopolymers that play key roles in bacterial physiology, including protection against the activity of antimicrobial peptides (AMPs). In other Gram-positive pathogens, WTA modification by amine-containing groups such as D-alanine was largely correlated with resistance to AMPs. However, in <i>L</i>. <i>monocytogenes</i>, where WTA modification is achieved solely <i>via</i> glycosylation, WTA-associated mechanisms of AMP resistance were unknown. Here, we show that the L-rhamnosylation of <i>L</i>. <i>monocytogenes</i> WTAs relies not only on the <i>rmlACBD</i> locus, which encodes the biosynthetic pathway for L-rhamnose, but also on <i>rmlT</i> encoding a putative rhamnosyltransferase. We demonstrate that this WTA tailoring mechanism promotes resistance to AMPs, unveiling a novel link between WTA glycosylation and bacterial resistance to host defense peptides. Using <i>in vitro</i> binding assays, fluorescence-based techniques and electron microscopy, we show that the presence of L-rhamnosylated WTAs at the surface of <i>L</i>. <i>monocytogenes</i> delays the crossing of the cell wall by AMPs and postpones their contact with the listerial membrane. We propose that WTA L-rhamnosylation promotes <i>L</i>. <i>monocytogenes</i> survival by decreasing the cell wall permeability to AMPs, thus hindering their access and detrimental interaction with the plasma membrane. Strikingly, we reveal a key contribution of WTA L-rhamnosylation for <i>L</i>. <i>monocytogenes</i> virulence in a mouse model of infection.</p></div

WTA l-rhamnosylation promotes <i>Lm</i> resistance against AMPs.

<p>(<b>A</b>) Growth of <i>Lm</i> strains in BHI broth supplemented with 5% NaCl. A growth curve of wild type EGD-e in the absence of 5% NaCl was included as a control for optimal growth. (<b>B</b>) Growth of mid-exponential-phase <i>Lm</i> strains untreated (black symbols) or challenged with 50 μg/ml (gray symbols) or 1 mg/ml (white symbols) of lysozyme. Optical density of the shaking cultures was monitored spectrophotometrically at 600 nm. (<b>C</b>) Quantification of viable bacteria after treatment of mid-exponential-phase <i>Lm</i> strains (2 h, 37°C) with gallidermin (1 μg/ml), CRAMP or LL-37 (5 μg/ml). Averaged replicate values from AMP-treated samples were normalized to untreated control samples and the transformed data expressed as the percentage of surviving bacteria relative to wild type <i>Lm</i> (set at 100). Data represent mean±SD of three independent experiments. *, <i>p</i>≤0.05; ***, <i>p</i>≤0.001.</p

A functional <i>rml</i> operon is required for glycosylation of <i>Lm</i> WTAs with l-rhamnose.

<p>(<b>A</b>) Alcian blue-stained 20% polyacrylamide gel containing WTA extracts from logarithmic-phase cultures of different <i>Lm</i> strains. (<b>B–D</b>) HPAEC-PAD analyses of the sugar composition of the (B) WTA, (C) peptidoglycan and (D) cytoplasmic fractions isolated from the indicated <i>Lm</i> strains. Samples were hydrolyzed in 3 M HCl (2 h, 95°C), diluted with water and lyophilized before injection into the HPLC equipment. Standards for ribitol (Rib), l-rhamnose (Rha), glucosamine (GlcN), and muramic acid (Mur) were eluted under identical conditions to allow peak identification.</p

WTA l-rhamnosylation interferes with the <i>Lm</i> cell wall crossing by AMPs.

<p>(<b>A and B</b>) Flow cytometry analysis of <i>Lm</i> surface-exposed CRAMP levels in mid-exponential-phase <i>Lm</i> strains, following incubation (5 min) in a 5-μg/ml solution of the peptide and immunolabeling with anti-CRAMP and Alexa Fluor 488-conjugated antibodies. (A) Representative experiment showing overlaid histograms of CRAMP-treated (solid line) and untreated (dashed line) samples, with mean fluorescence intensity (MFI) values from treated samples indicated by vertical dashed lines. (B) Mean±SD of the MFI values of CRAMP-treated samples from three independent experiments. (<b>C</b>) Cell surface charge analysis of <i>Lm</i> strains deficient for WTA l-rhamnosylation as determined by cytochrome c binding assays. Mid-exponential-phase bacteria were incubated with equine cytochrome c (0.5 mg/ml), centrifuged and the supernatant was recovered for spectrophotometric quantification of the unbound protein fraction. Values from <i>Lm</i>-containing samples are expressed as the percentage of unbound cytochrome c relative to control samples lacking bacteria. Data represent the mean±SD of three independent experiments. (<b>D and E</b>) Flow cytometry analysis of total <i>Lm</i>-associated CRAMP levels in mid-exponential-phase <i>Lm</i> strains, following incubation (5 min) with a 5-μg/ml solution of fluorescently labeled peptide (5-FAM-CRAMP). (D) Representative experiment showing overlaid histograms of FAM-CRAMP-treated (solid line) and untreated (dashed line) samples, with MFI values from treated samples indicated by vertical dashed lines. (E) Mean±SD of the MFI values of 5-FAM-CRAMP-treated samples from three independent experiments. (<b>F</b>) Fluorometric quantification of the unbound CRAMP fraction in the supernatant of suspensions of mid-exponential-phase <i>Lm</i> strains, following incubation (5 min) with a 5-μg/ml solution of 5-FAM-CRAMP. Data are expressed as the percentage of unbound fluorescent peptide relative to control samples lacking bacteria, and represent the mean±SD of three independent experiments performed in triplicates. ns = not significant, <i>p</i>>0.05; **, <i>p</i>≤0.01; ***, <i>p</i>≤0.001.</p