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EBioMedicine
2020 May 01;55:102775. doi: 10.1016/j.ebiom.2020.102775.
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Antimicrobial and antibiofilm activity of the EeCentrocin 1 derived peptide EC1-17KV via membrane disruption.
Ma L
,
Ye X
,
Sun P
,
Xu P
,
Wang L
,
Liu Z
,
Huang X
,
Bai Z
,
Zhou C
.
Abstract
BACKGROUND: The antibiotic resistance and biofilm formation of pathogenic microbes exacerbate the difficulties of anti-infection therapy in the clinic. The structural modification of antimicrobial peptides (AMP) is an effective strategy to develop novel anti-infective agents.
METHOD: Seventeen amino acids (AA) in the longer chain of EeCentrocin 1 (from the edible sea-urchin Echinus esculentus) were truncated and underwent further modification. To produce lead peptides with low toxicity and high efficacy, the antimicrobial activity or cytotoxicity of peptides was evaluated against various multidrug-resistant bacteria/fungi or mammalian cells in vivo/ in vitro. In addition, the stability and modes of action of the lead peptide were investigated.
FINDINGS: EC1-17KV displayed potent activity and an expanded antimicrobial spectrum, especially against drug-resistant gram-negative bacteria and fungi, attributable to its enhanced amphiphilicity and net charge. In addition, it exhibits bactericidal/fungicidal activity and effectively increased the animal survival rate and mitigated the histopathological damage induced by multidrug-resistant P. aeruginosa or C. albicans in infected mice or G. mellonella. Moreover, EC1-17KV had a poor ability to induce resistance in bacteria and fungi and exhibited desirable high-salt/high-temperature tolerance properties. In bacteria, EC1-17KV promoted divalent cation release to damage bacterial membrane integrity. In fungi, it changed C. albicans membrane fluidity to increase membrane permeabilization or reduced hyphal formation to suppress biofilm formation.
INTERPRETATION: EC1-17KV is a promising lead peptide for the development of antimicrobial agents against antibiotic resistant bacteria and fungi.
FUNDING: This work was funded by the National Natural Science Foundation of China (No. 81673483, 81803591); National Science and Technology Major Project Foundation of China (2019ZX09721001-004-005); National Key Research and Development Program of China (2018YFA0902000); "Double First-Class" University project (CPU2018GF/GY16); Natural Science Foundation of Jiangsu Province of China (No. BK20180563); and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.
Fig. 1. Structure of the designed peptides. (a) Amino acid sequences and key physicochemical properties of different peptide mutants. μH: hydrophobic moment; GRAVY: grand average of hydropathy; pI: isoelectric point. (b) The conformation of peptides in 500 μM SUVs (DPPG/CL/DPPE at a mass ratio of 2:1:7) was analysed by circular dichroism. (c) CD assay for EC1-17KV secondary structure in different solutions.
Fig. 2. Antimicrobial activity and toxicities of peptides. (a) Disk diffusion antibacterial assay. Poly B: polymyxin B; CAZ: ceftazidime; AMB: amphotericin B; FLU: fluconazole. (b) Haemolytic activity against SRBCs. 0.1% Triton X-100 is positive control, *p<0.05; ⁎⁎p<0.01 vs EC1-17RV at the same concentration [Student t-test]. The cytotoxicity of peptides to murine spleen cells (c) and other eukaryotic cells (d) was measured by MTT assay. (e) Drug resistance study for EC1-17KV in bacteria or fungi after 24 subcultures following the initial exposure. (f) The high-salt and high temperature tolerance of EC1-17KV was determined by HPLC and microdilution assays.
Fig. 3. Time-kill curves of EC1-17KV against drug-resistant bacteria or fungi. (a) MDR P. aeruginosa, (b) ceftazidime-resistant E. coli, (c) MDR Enterococcus faecalis, (d) MDR Mlicrococcus scarlatinae, (e) fluconazole-resistant C. albicans, (f) fluconazole-resistant C. neoformans. Control: treated with normal saline.
Fig. 4. EC1-17KV protects mice against MDR P. aeruginosa-induced acute systemic infection. (a) The survival rate of mice during the observation period of 7 consecutive days. (b) Bacterial counts in the lungs and blood of infected mice. (c) ELISA for the content of TNF-α, IL-6, IL-1β and IL-18 in mouse serum. (d) Western blot for the expression of NLRP3, IL-1β, cleaved IL-1β, caspase 1 and cleaved caspase-1 to measure the activation of the NLRP3 inflammasome. (e) H&E staining of mouse lungs. The total lung lesion scores were generated in a blinded manner by a certified pathologist according to a semi-quantitative scoring rank (right). Scale bars, 100 μm. *p<0.05; ⁎⁎p<0.01 vs model group [One-Way ANOVA Tukey test].
Fig. 5. EC1-17KV damages bacterial membrane integrity by competitively replacing Ca2+ to bind to LPS. (a) NPN uptake and (b) SYTO9/PI staining for outer and inner membrane permeability detection, the data are representative of three experiments. The increased fluorescence of NPN indicates increased outer membrane permeability, whereas a decreased SYTO (green)/PI (red) ratio means increased inner membrane permeability. (c) TEM assay of EC1-17KV-induced P. aeruginosa membrane disruption. Images in the dotted boxes are enlarged in the leftmost column. Red arrow: vacuoles or discontinuous cell membrane. (d) Leakage of entrapped calcein from EC1-17KV-treated liposomes. Left: artificial human erythrocyte cell membrane; Right: artificial P. aeruginosa membrane. (e) Ca2+ concentration and (f) LPS release in supernatant from peptide-treated P. aeruginosa. The effects of external Ca2+ on EC1-17KV-induced LPS release (g) and on the fold change of the peptide MIC (h). (i) The effect of EDTA on the fold change of the peptide MIC with external Ca2+ (4.0 mM). Scale bars: c (left): 200 nm; c (right): 1 μm. ⁎⁎p<0.01 vs non-Ca2+ group [One-Way ANOVA Tukey test].
Fig. 6. EC1-17KV has potent antibiofilm activity against P. aeruginosa. (a) Biofilm formation or (b) preformed-biofilm dispersion in the presence or absence of EC1-17KV was measured by crystal violet staining. (c) Bacterial spread into the subcutaneous tissue or sessile bacteria attached to the catheter surface were counted by the plate dilution method. (d) H&E staining of mouse subcutaneous tissue infected by the implanted biofilm. Scale bars, 50 μm. *p<0.05; ⁎⁎p<0.01 vs model group [One-Way ANOVA Tukey test].
Fig. 7. Effective antifungal activity of EC1-17KV in C. albicans-infected G. mellonella. (a) Survival plot of G. mellonella after inoculation with C. albicans and treatment with a single dose of EC1-17KV or fluconazole over 72 h postinfection. (b) Plate dilution method for CFU counting in the larval homogenate after 24 h of infection. (c) Visual appearance of larvae and cocoon formation of G. mellonella. Red arrow: complete and thick cocoon; yellow arrow: incomplete and thin cocoon. (d) Melanization of G. mellonella after C. albicans injection was measured by the optical density (OD) of the haemolymph. (e) H&E staining of larvae in the presence or absence of EC1-17KV treatment. Black arrow: hyperemia and inflammatory cell infiltration; Control: non-infected larvae; Model: infected larvae with saline treatment; FLU: 10 μg fluconazole. Scale bars, 100 μm. *p<0.05; ⁎⁎p<0.01 vs model group [One-Way ANOVA Tukey test].
Fig. 8. EC1-17KV exerts fungicidal effects by disrupting fungal membrane integrity. (a) Zeta potential on the surface of C. albicans. (b) Flow cytometry and (c) fluorescence microscopy were employed to detect the binding activity of EC1-17KV to the surface of C. albicans (yeast and mycelial forms). Green: FITC-labelled EC1-17KV. (d) TEM assay for the ultrastructure of C. albicans. Images in the dotted boxes are enlarged in the rightmost column. (e-f) PI uptake and PI staining assay for the permeability of fungal cell membrane in the presence of EC1-17KV. (g) The leakage of intracellular K+ from C. albicans exposed to different concentrations of EC1-17KV. (h) The expression changes of ergosterol synthesis-related genes were measured by qRT-PCR. (i) Membrane fluidity after EC1-17KV treatment was determined using a TMA-DPH probe. A decrease in DPH fluorescence anisotropy reflects an increase in membrane fluidity. Scale bars: c: 50 μm; d (left): 1 μm; d (right): 200 nm; e: 25 μm. *p<0.05; ⁎⁎p<0.01 vs control group [One-Way ANOVA Tukey test].
Fig. 9. EC1-17KV exerts antifungal activity by inhibiting biofilm formation and damaging preformed biofilms. The adhesion of C. albicans to (a) HBECs or (b) poly-L-lysine-coated polystyrene cell culture plate was detected by Wright staining or XTT assay. (c) The cell surface hydrophobicity (CSH) of EC1-17KV-treated C. albicans was detected. (d) Effects of EC1-17KV on hyphal formation in RPMI 1640 medium. (e) qRT-PCR analysis of the expression of filamentation-related genes (left); EC1-17KV hindered the switch between yeast, pseudohyphae and hyphae (right). The biofilm formation (f) or preformed biofilm dispersion (g) of C. albicans treated with EC1-17KV was detected by FDA staining and crystal violet staining. (h) EPS component reduction in preformed biofilms after EC1-17KV treatment. Carbohydrate: FITC-ConA, green; Nucleic acids: DAPI, blue; protein: SYPRO orange, red. Scale bars: a, d (right) and h: 50 μm; d (left): 25 μm; f and g: 100 μm. *p<0.05; ⁎⁎p<0.01 vs control group [Student t-test].
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