A Novel Peptide with Antifungal Activity from Red Swamp Crayfish Procambarus clarkii

Abstract

The defense system of freshwater crayfish Procambarus clarkii as a diversified source of bioactive molecules with antimicrobial properties was studied. Antimicrobial activity of two polypeptide-enriched extracts obtained from hemocytes and hemolymph of P. clarkii were assessed against Gram positive (Staphylococcus aureus, Enterococcus faecalis) and Gram negative (Pseudomonas aeruginosa, Escherichia coli) bacteria and toward the yeast Candida albicans. The two peptide fractions showed interesting MIC values (ranging from 11 to 700 μg/mL) against all tested pathogens. Polypeptide-enriched extracts were further investigated using a high-resolution mass spectrometry and database search and 14 novel peptides were identified. Some peptides and their derivatives were chemically synthesized and tested in vitro against the bacterial and yeast pathogens. The analysis identified a synthetic derivative peptide, which showed an interesting antifungal (MIC and MFC equal to 31.2 μg/mL and 62.5 μg/mL, respectively) and antibiofilm (BIC₅₀ equal to 23.2 μg/mL) activities against Candida albicans and a low toxicity in human cells.

Keywords: Candida albicans; antibiofilm activity; antibiotic resistant strains; crustacean antimicrobial peptides; high-resolution mass spectrometry.

Figure 1

Inhibition of biofilm formation of the derivative peptide #14d against C. albicans ATCC 10231.

Figure 2

SEM images showing C. albicans ATCC 10231 biofilm after 24 h of treatment with synthetic derivative peptide #14d at sub-MIC concentration of 25 μg/mL. (A), and non-treated control (B). Bars: 10 μm.

Figure 3

Characterization of membrane damages caused by peptide #14d in C. albicans ATCC 10231 analyzed by fluorescence microscope (40×) using propidium iodide (PI). Cells were treated with increasing concentrations of peptide #14d (15–25 μg/mL) and incubated for 24 h.; 25 μg/mL (A), 20 μg/mL (B), and 15 μg/mL (C). Growth control treated of C. albicans ATCC 10231 in Sabouroud medium (D). Bars: 20 μm.

Figure 4

Fluorescence images illustrating ROS overproduction on inhibition of biofilm of C. albicans ATCC 10231 cells. ROS production was detected using 2′,7′ dichlorofluorescein diacetate (DCFH-DA). (A,B) C. albicans ATCC 10231 cells treated with peptide #14d at 25 μg/mL; (C,D) C. albicans ATCC 10231 cells treated with peptide #14d at 20 μg/mL. Bars: 100 μm.

Figure 5

Viability of HCT-116 in presence of peptide #14d after 24 h and 48 h of incubation.

Figure 6

(A) Cartoon representation of the structure of the most representative cluster of peptides #14 (blue) and #14d (red), obtained from MD simulations. (B) Ramachandran plots showing the values of the psi and phi angles assumed by each residue. (C) Electrostatic potential (red and blue for negative and positive values, respectively) for peptides structures, shown as surfaces.