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

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

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 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

Plasmids and bacterial strains.

<p>Plasmids and bacterial strains.</p

Effect of TDZ and DCX on the muropeptide composition of USA300 PGN.

<p>PGN was isolated from cultures grown to exponential phase in the absence or presence of TDZ or DCX and muropeptide compositions were analyzed by HPLC as described in Materials and Methods. Peak numbers are assigned according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064518#pone.0064518-DeJonge1" target="_blank">[30]</a>. The identity of peak 1, 4, 5, 7, and 11 was confirmed by mass spectrometry. Chromatograms are normalized to peak 11.</p

Impact of TDZ on the sensitivity to cell wall targeting antibiotics.

<p>(A-E) Time-kill assays showing the effect of TDZ (16 µg/mL, ¼×MIC) and (A) DCX (0.125 µg/mL, 1×MIC), (B) fosfomycin (FOS, 3 µg/mL), (C) D-cycloserine (CYC, 8 µg/mL), (D) bacitracin (BAC, 32 µg/mL) or (E) vancomycin (VAN, 1.5 µg/mL) on survival of USA300. (F) Susceptibility of post-exponential phase cultures grown in the absence or presence of TDZ (16 µg/mL) to DCX was determined by population analysis.</p

Effect of TDZ on PBPs and autolysis.

<p>(A) Membrane fractions were isolated from USA300 grown to mid-exponential phase in the absence or presence of TDZ (16 µg/mL) and pre-incubated with 16 µg/mL TDZ, 50 µg/mL DCX, or assay buffer. PBPs were labeled with Bocillin-FL, separated by SDS-PAGE, and visualized by fluorography. The PBP profile of MSSA strain Newman is shown for comparison. (B) Zymogram analysis of autolysins extracted from the cell walls of USA300 grown to mid-exponential phase in the absence (1) or presence (2) of 16 µg/mL TDZ. 10 µg or protein extract was separated in a 10% SDS-polyacrylamide gel containing purified cell walls from USA300 grown in BHI. Arrows indicate bands with decreased intensity following TDZ treatment. (C) Unstimulated and Triton X-100 stimulated autolysis of USA300 grown to mid-exponential phase in the absence (squares) and presence (triangles) of 16 µg/mL TDZ. Unstimulated autolysis is represented by open symbols, and Triton X-100 stimulated autolysis by closed symbols.</p