After appropriate time intervals the crude solutions were analyzed by HPLC and mass spectroscopy. Unlike M33 incubated without enzymes (Figs. 3a, 3b, 3c and 3d), M33-L was degraded within 1h by staphylococcal aureolysin (Fig. 3e), through hydrolysis at R6-L7 and S8-A9 peptide bonds (Fig. 3f). Conversely, M33-D was completely stable to proteolysis by this metalloprotease, remaining unaltered after 24 h of incubation (Figs. 3g and 3h). Incubation of M33-L with P. aeruginosa elastase showed moderate peptide stability after 5 h (a peak corresponding to a retention time of 23 min is still present in Fig. 3i), and again the cleavage sites were R6-L7 and S8-A9 peptide bonds (Fig. 3j). In contrast, the M33-D peptide resisted degradation by elastase for 24 h (Figs. 3k and 3l). The cleavage sites of both peptides are illustrated in Fig. 3m and the MS peaks are assigned to the fragments. Altogether, these results suggest that the 1326631 increased stability of M33-D to staphylococcal aureolysin could be at least partly responsible for the increased activity exhibited by this isomer 
against S. aureus. The same phenomenon could also explain theBinding of M33-L and M33-D to Lipopolysaccharide (LPS) and Lipoteichoic Acid (LTA)In a previous report [13] we hypothesized that LPS was the first bacterial structure to interact with M33-L. In order to evaluate possible differential binding of M33-L and M33-D to Gramnegative LPS and to Gram-positive LTA, we therefore analyzed the interactions of both peptides with LPS and LTA by surface plasmon resonance. LTA from S. aureus and Streptococcus faecalis, and LPS from E. coli, P. aeruginosa and K. pneumoniae were injected at a concentration of 10 mg/ml over immobilized M33-L or M33D peptides. No significant difference in binding or kinetic rates that could explain such dissimilar antimicrobial activity of the two peptides was observed (Fig. 1).Interaction of M33 with Liposomes Mimicking Bacterial CellsTo investigate interaction of peptides M33-D and M33-L with the bacterial membrane, including possible perturbation,Antimicrobial Activity of M33 Peptide D-IsomerTable 1. MICs of M33-L and M33-D for different bacteria species and strains.Relevant featuresa Reference inhibitor strain, wild type FQr AGr ESCr NEMr (MBL/IMP-13) Reference strain, wild type FQr AGr ESCr NEMr (MBL/VIM-1) Reference strain, wild type FQr AGr ESCr (ESBL/CTX-M-15) Reference strain, wild type MR MR VANiSpecies and Autophagy strains P.aeruginosa 
ATCC 27853 P.aeruginosa AV 65 K.pneumoniae ATCC 13833 K.pneumoniae 7086042 E.coli ATCC 25922 E.coli W03BG0025 S.aureus ATCC 29213 S.aureus USA 300 S.aureus 3851 S.epidermidis ATCC 14990 S.epidermidisaM33-L (mM) 1.5 3 1.5 3 3 0.7 6 6 12 1.5M33-D (mM) 1.5 3 3 6 3 3 1.5 1.5 0.7 0.4 0.Reference strain, wild type MRM33 antimicrobial activity was evaluated on reference strains and clinical isolates (mostly with an MDR phenotype). Relevant resistance phenotypes and resistance determinants are indicated. Resistance phenotypes: FQr, resistant to fluoroquinolones; AGr, resistant to aminoglycosides (gentamycin, amikacin, and/or tobramycin); ESCr, resistant to expanded-spectrum cephalosporins; NEMr, resistant to carbapenems (imipenem and/or meropenem); MRr, methicillin-resistant; VANi, vancomycinintermediate. Resistance determinants: ESBL, extended spectrum b-lactamase; MBL, metallo-b-lactamase. doi:10.1371/journal.pone.0046259.tincreased activity of M33-D against S. epidermidis (Table 1), which produces an ortholog of aureolysin (the metallo-.After appropriate time intervals the crude solutions were analyzed by HPLC and mass spectroscopy. Unlike M33 incubated without enzymes (Figs. 3a, 3b, 3c and 3d), M33-L was degraded within 1h by staphylococcal aureolysin (Fig. 3e), through hydrolysis at R6-L7 and S8-A9 peptide bonds (Fig. 3f). Conversely, M33-D was completely stable to proteolysis by this metalloprotease, remaining unaltered after 24 h of incubation (Figs. 3g and 3h). Incubation of M33-L with P. aeruginosa elastase showed moderate peptide stability after 5 h (a peak corresponding to a retention time of 23 min is still present in Fig. 3i), and again the cleavage sites were R6-L7 and S8-A9 peptide bonds (Fig. 3j). In contrast, the M33-D peptide resisted degradation by elastase for 24 h (Figs. 3k and 3l). The cleavage sites of both peptides are illustrated in Fig. 3m and the MS peaks are assigned to the fragments. Altogether, these results suggest that the 1326631 increased stability of M33-D to staphylococcal aureolysin could be at least partly responsible for the increased activity exhibited by this isomer against S. aureus. The same phenomenon could also explain theBinding of M33-L and M33-D to Lipopolysaccharide (LPS) and Lipoteichoic Acid (LTA)In a previous report [13] we hypothesized that LPS was the first bacterial structure to interact with M33-L. In order to evaluate possible differential binding of M33-L and M33-D to Gramnegative LPS and to Gram-positive LTA, we therefore analyzed the interactions of both peptides with LPS and LTA by surface plasmon resonance. LTA from S. aureus and Streptococcus faecalis, and LPS from E. coli, P. aeruginosa and K. pneumoniae were injected at a concentration of 10 mg/ml over immobilized M33-L or M33D peptides. No significant difference in binding or kinetic rates that could explain such dissimilar antimicrobial activity of the two peptides was observed (Fig. 1).Interaction of M33 with Liposomes Mimicking Bacterial CellsTo investigate interaction of peptides M33-D and M33-L with the bacterial membrane, including possible perturbation,Antimicrobial Activity of M33 Peptide D-IsomerTable 1. MICs of M33-L and M33-D for different bacteria species and strains.Relevant featuresa Reference strain, wild type FQr AGr ESCr NEMr (MBL/IMP-13) Reference strain, wild type FQr AGr ESCr NEMr (MBL/VIM-1) Reference strain, wild type FQr AGr ESCr (ESBL/CTX-M-15) Reference strain, wild type MR MR VANiSpecies and strains P.aeruginosa ATCC 27853 P.aeruginosa AV 65 K.pneumoniae ATCC 13833 K.pneumoniae 7086042 E.coli ATCC 25922 E.coli W03BG0025 S.aureus ATCC 29213 S.aureus USA 300 S.aureus 3851 S.epidermidis ATCC 14990 S.epidermidisaM33-L (mM) 1.5 3 1.5 3 3 0.7 6 6 12 1.5M33-D (mM) 1.5 3 3 6 3 3 1.5 1.5 0.7 0.4 0.Reference strain, wild type MRM33 antimicrobial activity was evaluated on reference strains and clinical isolates (mostly with an MDR phenotype). Relevant resistance phenotypes and resistance determinants are indicated. Resistance phenotypes: FQr, resistant to fluoroquinolones; AGr, resistant to aminoglycosides (gentamycin, amikacin, and/or tobramycin); ESCr, resistant to expanded-spectrum cephalosporins; NEMr, resistant to carbapenems (imipenem and/or meropenem); MRr, methicillin-resistant; VANi, vancomycinintermediate. Resistance determinants: ESBL, extended spectrum b-lactamase; MBL, metallo-b-lactamase. doi:10.1371/journal.pone.0046259.tincreased activity of M33-D against S. epidermidis (Table 1), which produces an ortholog of aureolysin (the metallo-.