Effect of Sp-LECin on morphological and structural changes of E. faecium, A. baumannii, P. aeruginosa, P. stutzer, A. niger and F. oxysporum observed by a scanning electron microscopy (SEM).

The bactericidal kinetics of Sp-LECin against E. faecium (A), A. baumannii (B), P. aeruginosa (C) and P. stutzeri (D) at 1×, and 2× MBC. Data represent mean ± standard error of mean from three independent biological replicates.

Effect of Sp-LECin on the membrane permeability of P. aeruginosa and A. baumannii. (A,B) Outer membrane permeability after Sp-LECin treatment was measured by N-phenyl-1-naphthylamine (NPN) uptake assay. The NPN fluorescence was recorded at the excitation and emission wavelengths of 350 and 420 nm, respectively. (C,D) Inner membrane permeability after Sp-LECin treatment was measured by SYTOX Green uptake assay. The SYTOX Green fluorescence was recorded at the excitation and emission wavelengths of 485 and 530 nm, respectively. The bars indicate the mean ± standard error of mean (n = 3). (E,F) Confocal laser-scanning microscope (CLSM) images representing the cell membrane permeability by SYTO 9 and propidium iodide (PI) staining in P. aeruginosa and A. baumannii cells treated with Sp-LECin.

The binding property of Sp-LECin to LPS. (A) The Effect of exogenous LPS (0, 6, 12, 24 and 48 μg/mL) on the antibacterial activity of Sp-LECin (24 μM) was assessed by measuring the growth curve of P. aeruginosa. The bars indicate the mean ± standard error of mean (n = 3). (B) Determination of the binding property of Sp-LECin to LPS by the LAL assay. The bars indicate the mean ± standard error of mean (n = 5).

Effect of Sp-LECin on ROS production in P. aeruginosa. Intracellular ROS was monitored using 2,7-dichlorofuorescin diacetate (DCFH-DA) probe after treatment with different concentrations of Sp-LECin (0, 12, 24 and 48 μM) for 0.5 (A) and 1 h (B), respectively. LL-37 was used as a positive control. Significant difference between control group and AMP treatment group was indicated by asterisks as *** p < 0.001. The bars indicate the mean ± standard error of mean (n = 5).

Anti-biofilm activity of Sp-LECin against P. aeruginosa. (A) The inhibitory effect of Sp-LECin on the formation of P. aeruginosa biofilm. The biofilm mass was quantified by crystal violet (CV) and the absorbance at 595 nm was measured. (B) The inhibitory effect of Sp-LECin against the preformed biofilm of P. aeruginosa. The amount of reduced resazurin and the residual amount of oxidized resazurin was determined by measuring at the absorbance at 560 nm and 620 nm, respectively. The corrected A560 value (AR560) was calculated using the following formula: AR560 = A560 − (A620 × RO) and RO = AO560/AO620, where A560 and A620 are sample absorbance and AO560 and AO620 are the absorbance of medium containing 0.1 mM resazurin. Significant difference between control group and Sp-LECin treatment group was indicated by asterisks as * p < 0.05, ** p < 0.01 and *** p < 0.001. The bars indicate the mean ± standard error of mean (n = 5).

In vitro cytotoxicity and hemolytic activity and the in vivo effectiveness of Sp-LECin. The cytotoxicity of Sp-LECin on crab hemocytes (A), human embryonic kidney cells (HEK-293T) (B), human hepatic cells (L02) (C), and mouse embryonic fibroblast (3T3) (D), were determined by the MTS-PMS assay. (E) Hemolytic activity of Sp-LECin against mouse red blood cells. Significant difference between the control group and the Sp-LECin treatment group was indicated by asterisks as * p < 0.05 and ** p < 0.01. The bars indicated the mean ± standard error of mean (n = 3). (F) In vivo effectiveness of Sp-LECin in P. aeruginosa-challenged zebrafish (n = 20/group). The survival curve of each group was analyzed using the log-rank test.