Permeabilization of bacterial membranes assay.

<p>NCP-2 and NCP-3-mediated membrane permeabilization of <i>E</i>. <i>coli</i> ML-35 pYC. Inner membrane damage was evaluated at 600 nm through the conversion of ortho-Nitrophenyl-b-galactoside (ONPG) by cytoplasmic β-galactosidase. Outer membrane damage was evaluated at 405 nm through the conversion of the chromogenic cephalosporin CENTA by periplasmic β-lactamase.</p

Propidium iodide (PI) dead-cell stain assay.

<p>Permeabilization of the inner membrane of <i>P</i>. <i>aeruginosa</i> ATCC 27853 as a function of contact time (min) with 12.5 μg/ml of NCP-3 and NCP-2. Viable cells are impermeable to PI and are blue due to counterstaining with 4’,6-diamidino-2-phenylindole (DAPI). Conversely, PI penetrates and stains the DNA of membrane-compromised cells (red emission).</p

NCP-2 predicted structural features.

<p>(A) Three-dimensional structure. (B) Molecular surface colored by electrostatic potential (ranging from 1 to 10 kcal/(mol <i>e</i>)). (C) Distribution of basic (blue) and hydrophobic (red) amino acids. (D) Molecular surface colored by hydrophobicity (using the Kyte-Doolittle scale). Visualized using University of California, San Francisco (UCSF) Chimera.</p

NCP-3 predicted structural features.

<p>(A) Three-dimensional structure. (B) Molecular surface colored by electrostatic potential (ranging from 1 to 10 kcal/(mol <i>e</i>)). (C) Distribution of basic (blue) and hydrophobic (red) amino acids. (D) Molecular surface colored by hydrophobicity (using the Kyte-Doolittle scale). Visualized using University of California, San Francisco (UCSF) Chimera.</p

Antimicrobial activity (inhibition percentages) of NCP-0, NCP-2 and NCP-3 in presence of sodium chloride or MH broth.

<p>Antimicrobial activity (inhibition percentages) of NCP-0, NCP-2 and NCP-3 in presence of sodium chloride or MH broth.</p

Time kill assay for NCP-2 and NCP-3 against Gram-negative and Gram-positive bacteria.

<p>Bacterial suspensions (5x10⁵ CFU/ml) were exposed to peptides at the concentration of 12.5 μg/ml. Percentage of inhibition was evaluated at fixed time intervals by CFU count.</p

Amino acidic sequences of peptides belonging to the NCPs family.

<p>Amino acidic sequences of peptides belonging to the NCPs family.</p

Representative results of the anti-mycobacterial activity of NCP-3 against <i>Mycobacterium fortuitum</i> DSMZ 46621 and <i>Mycobacterium smegmatis</i> DSMZ 43756 evaluated by resazurin assay.

<p>Bacterial suspensions (5x10⁵ CFU/ml) were exposed to NCP-3 at 37°C for 72 hours, then resazurin was added and plates were read after 24 hours at 37°C. Viable cells convert resazurin (blue) to resorufin (pink).</p

MBC values for NCP-0, NCP-2,NCP-3, NCP-3a and NCP-3b against Gram-negative and Gram-positive bacteria.

<p>MBC values for NCP-0, NCP-2,NCP-3, NCP-3a and NCP-3b against Gram-negative and Gram-positive bacteria.</p

Haemolysis and cytotoxicity tests.

<p>(a) Hemolysis assay of NCP-2 and NCP-3 on sheep red blood cells. (b) Cytotoxicity assay of NCP-2 and NCP-3 on Madin-Darby Bovine Kidney (MDBK) cells. Reported percentages are referred to the hemolytic/cytopathic effect scale, where 0% = all intact cells and 100% = hemolytic/cytopathic effect extended to all cells.</p