Supporting figures and tables.

S1 Fig Proteins involved in Lm WTA l-rhamnosylation. (A) Schematic diagram of the l-rhamnose biosynthesis pathway (adapted from [31, 35]). Each of the RmlACBD proteins catalyzes one of the four reaction steps that convert glucose-1-phosphate into nucleotide-linked l-rhamnose. dTTP, thymidine triphosphate; PPi, pyrophosphate; NADP, nicotinamide adenine dinucleotide phosphate. (B) Alignment of the amino acid sequences of B. subtilis 168 GgaB (GenBank: AAA73513.1) and Lm RmlT (GenBank: NP_464605.1). Boxed sequences correspond to the GT-A glycosyltransferase fold domain, as predicted by the NCBI Conserved Domain Search. The GT-A family signature DxD motif is highlighted in dark gray. The numbers indicate the position of the last amino acid in each line. Protein sequence alignments were obtained with ClustalW2 and edited with UCSF Chimera. S2 Fig Genetic characterization of Lm strains used in this study. (A) Genotypes and gene expression of the constructed Lm strains were confirmed by PCR and RT-PCR. (B) Comparison of the rmlACBD transcription levels in ΔrmlT versus wild type Lm strains by quantitative real-time PCR. Data represent the mean±SD of three independent analyses. *, p≤0.05. S3 Fig HPLC analyses of the cell wall sugar and muropeptide composition from Lm strains. (A) HPAEC-PAD analysis of the sugar composition of cell wall purified from Lm 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. (B) Reverse-phase HPLC analysis of the muropeptide composition from different Lm strains, following overnight digestion of purified peptidoglycan samples with mutanolysin and reduction with NaBH4. Muropeptide species (monomeric, dimeric, trimeric, etc.) were eluted with a 5–30% methanol gradient and detected by UV absorption at 206 nm. S4 Fig Dose-dependent survival response of Lm strains to different AMPs. Quantification of viable bacteria after treatment of mid-exponential-phase Lm strains (2 h, 37°C) with increasing concentrations of gallidermin, CRAMP or LL-37. The average replicate values from AMP-treated samples were expressed as percentage of surviving bacteria relative to the values of the respective untreated control samples (set at 100). Data represent mean±SD of three independent experiments. Asterisks indicate statistical significance between wild type and mutant strains (*, p≤0.05; ***, p≤0.001), while hashes indicate statistical significance between mutant and respective complemented strains (#, p≤0.05; ###, p≤0.001). S5 Fig Zeta potential profile of wild type and WTA
l
-rhamnosylation mutant Lm strains. S6 Fig Determination of the Lm membrane potential magnitude by flow cytometry. The membrane potential of untreated and sodium azide (1.5 mM)-treated suspensions of DiOC2(3)-stained wild type EGD-e suspensions was analyzed (see Materials and Methods) to determine the red/green fluorescence ratio values corresponding, respectively, to a basal (100%) and null (0%) membrane potential. S7 Fig 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 115 min. S8 Fig Growth of Lm strains in broth and inside eukaryotic host cells. (A) Stationary-phase cultures were diluted 100-fold in BHI broth and incubated at 37°C in aerobic and shaking conditions. Optical density values at 600 nm (OD600) from each culture were measured every hour. (B) Intracellular multiplication in J774A.1 murine macrophages. Cells (2×105/well) were infected (45 min) with Lm, treated with 20 μg/ml gentamicin (75 min) and lysed at 2, 5, 7 and 20 h post-infection for quantification of intracellular viable bacteria in BHI agar. S1 Table. Homology between the RmlACBD proteins of Lm EGD-e and other strains and species. S2 Table. Primers.
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