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Fig. 3. ZY4 exhibits biofilm inhibition and eradication activities and kills persister cells. Inhibitory effects of ZY4 on P. aeruginosa (A) and A. baumannii (B biofilm formation. Effects of ZY4 on established biofilm eradication of P. aeruginosa CICC21625 (C) and A. baumannii 22933 (D). Bactericidal activities of ZY¢ against persister cells of P. aeruginosa CICC21625 (E) and A. baumannii 22933 (F). Data represent means + SD of 3 individual experiments. *P < 0.05 **P < 0.01.

Figure 3 ZY4 exhibits biofilm inhibition and eradication activities and kills persister cells. Inhibitory effects of ZY4 on P. aeruginosa (A) and A. baumannii (B biofilm formation. Effects of ZY4 on established biofilm eradication of P. aeruginosa CICC21625 (C) and A. baumannii 22933 (D). Bactericidal activities of ZY¢ against persister cells of P. aeruginosa CICC21625 (E) and A. baumannii 22933 (F). Data represent means + SD of 3 individual experiments. *P < 0.05 **P < 0.01.

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Fig. 1. Primary sequence and structure prediction of ZY4 and its analogs. (A) Sequence and physicochemical properties of the designed peptides. (B) The 3- dimensional structures represent the last frames (t = 10 ns) of cathelicidin-BF15-a3 and cathelicidin-BF15-a4. The different colors represent various secondary structure types: green, turn; red, helix; white, coil. The yellow lines in cathelicidin-BF15-a4 indicate the disulfide bond. (C) The cathelicidin-BF15-a4 with lower and steady rmsd levels indicates that the structure of ZY4 is stabler than the structure of cathelicidin-BF15-a3. (D) The lower and steady Rg values of cath- elicidin-BF15-a4 indicate that after introducing the disulfide bond, cathelicidin-BF15-a4 was stabler than cathelicidin-BF15-a3.
Fig. 1. Primary sequence and structure prediction of ZY4 and its analogs. (A) Sequence and physicochemical properties of the designed peptides. (B) The 3- dimensional structures represent the last frames (t = 10 ns) of cathelicidin-BF15-a3 and cathelicidin-BF15-a4. The different colors represent various secondary structure types: green, turn; red, helix; white, coil. The yellow lines in cathelicidin-BF15-a4 indicate the disulfide bond. (C) The cathelicidin-BF15-a4 with lower and steady rmsd levels indicates that the structure of ZY4 is stabler than the structure of cathelicidin-BF15-a3. (D) The lower and steady Rg values of cath- elicidin-BF15-a4 indicate that after introducing the disulfide bond, cathelicidin-BF15-a4 was stabler than cathelicidin-BF15-a3.
Fig. 2. ZY4kills bacteria by permeabilizing the cell membrane. After treatment with or without ZY4 for 30 min, the membrane morphology of P. aeruginosa CICC21625 (A) and A. baumannii 22933 (B) was determined by SEM. The arrows indicate the typical damage to the plasma membranes of the bacteria. Killing kinetics of ZY4 against P. aeruginosa (C) and A. baumannii (D). Data represent means + SD of 3 individual experiments.
Fig. 2. ZY4kills bacteria by permeabilizing the cell membrane. After treatment with or without ZY4 for 30 min, the membrane morphology of P. aeruginosa CICC21625 (A) and A. baumannii 22933 (B) was determined by SEM. The arrows indicate the typical damage to the plasma membranes of the bacteria. Killing kinetics of ZY4 against P. aeruginosa (C) and A. baumannii (D). Data represent means + SD of 3 individual experiments.
Fig. 4. ZY4 inhibits P. aeruginosa lung infection and inflammation in vivo. (A) Following intranasal inoculation of mice (n = 8) with P. aeruginosa C1 and subsequent i.v. administration of ZY4 or colistin (CS) to examine their therapeutic effects, bacteria load in lung homogenate is shown. (B) Representative his- topathological images of processed lung section with hematoxylin and eosin (H&E) staining. Images are presented at a magnification of 40x. (C) The number of counted infiltrated inflammatory cells within processed lung sections from B. Data represent the mean + SD of 8 individual experiments. *P < 0.05, **P < 0.01.
Fig. 4. ZY4 inhibits P. aeruginosa lung infection and inflammation in vivo. (A) Following intranasal inoculation of mice (n = 8) with P. aeruginosa C1 and subsequent i.v. administration of ZY4 or colistin (CS) to examine their therapeutic effects, bacteria load in lung homogenate is shown. (B) Representative his- topathological images of processed lung section with hematoxylin and eosin (H&E) staining. Images are presented at a magnification of 40x. (C) The number of counted infiltrated inflammatory cells within processed lung sections from B. Data represent the mean + SD of 8 individual experiments. *P < 0.05, **P < 0.01.
Fig. 5. ZY4 suppresses dissemination of bacteria to target organs. (A-D) ZY4 suppresses dissemination of P. aeruginosa to target organs. Mice (n = 10) were i.p. injected with P. aeruginosa C1, followed by i.p injection of ZY4 (10 mg/kg), colistin (10 mg/kg), levofloxacin (100 mg/kg), or vehicle (PBS). Bacterial burder was determined in the blood (A), liver (B), spleen (C), and kidney (D). (E-/) ZY4 inhibits cytokine release in LPS shock mice model. Mice (n = 8) were i.p. injectec with LPS (15 mg/kg) or PBS (control), followed by i.p. injection of ZY4 (10 mg/kg) or PBS 30 min after LPS injection. Levels of IL-10 (E), IFN-y (F), TNF-a (G), MCP-1 (H), and IL-6 (/) in plasma were determined 6 and 18 h after LPS injection, respectively. U-M) ZY4 suppresses dissemination of A. baumannii to target organs. A baumannii was i.p. injected into female C57BL/6 mice (n = 8); 1 h after the injection, ZY4 (1, 2, 4 mg/kg), colistin (1 mg/kg), and vehicle (PBS) were i.p. applied Bacterial burdens in blood (J), lung (kK), liver (L), and spleen (M) are shown. NC, normal healthy mice. Data represent the mean + SD of 8 individual experiments *P < 0.05, **P < 0.01.
Fig. 5. ZY4 suppresses dissemination of bacteria to target organs. (A-D) ZY4 suppresses dissemination of P. aeruginosa to target organs. Mice (n = 10) were i.p. injected with P. aeruginosa C1, followed by i.p injection of ZY4 (10 mg/kg), colistin (10 mg/kg), levofloxacin (100 mg/kg), or vehicle (PBS). Bacterial burder was determined in the blood (A), liver (B), spleen (C), and kidney (D). (E-/) ZY4 inhibits cytokine release in LPS shock mice model. Mice (n = 8) were i.p. injectec with LPS (15 mg/kg) or PBS (control), followed by i.p. injection of ZY4 (10 mg/kg) or PBS 30 min after LPS injection. Levels of IL-10 (E), IFN-y (F), TNF-a (G), MCP-1 (H), and IL-6 (/) in plasma were determined 6 and 18 h after LPS injection, respectively. U-M) ZY4 suppresses dissemination of A. baumannii to target organs. A baumannii was i.p. injected into female C57BL/6 mice (n = 8); 1 h after the injection, ZY4 (1, 2, 4 mg/kg), colistin (1 mg/kg), and vehicle (PBS) were i.p. applied Bacterial burdens in blood (J), lung (kK), liver (L), and spleen (M) are shown. NC, normal healthy mice. Data represent the mean + SD of 8 individual experiments *P < 0.05, **P < 0.01.
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