Immune mediators of sea-cucumber Holothuria tubulosa (Echinodermata) as source of novel antimicrobial and anti-staphylococcal biofilm agents

Abstract

The present study aims to investigate coelomocytes, immune mediators cells in the echinoderm Holothuria tubulosa, as an unusual source of antimicrobial and antibiofilm agents. The activity of the 5kDa peptide fraction of the cytosol from H. tubulosa coelomocytes (5-HCC) was tested against a reference group of Gram-negative and Gram-positive human pathogens. Minimal inhibitory concentrations (MICs) ranging from 125 to 500 mg/ml were determined against tested strains. The observed biological activity of 5-HCC could be due to two novel peptides, identified by capillary RP-HPLC/nESI-MS/MS, which present the common chemical-physical characteristics of antimicrobial peptides. Such peptides were chemically synthesized and their antimicrobial activity was tested. The synthetic peptides showed broad-spectrum activity at 12.5 mg/ml against the majority of the tested Gram-positive and Gram-negative strains, and they were also able to inhibit biofilm formation in a significant percentage at a concentration of 3.1 mg/ml against staphylococcal and Pseudomonas aeruginosa strains.The immune mediators in H. tubulosa are a source of novel antimicrobial peptides for the development of new agents against biofilm bacterial communities that are often intrinsically resistant to conventional antibiotics.

Figure 1

Edmundson wheel projection of (A) Holothuroidin 1 and (B) Holothuroidin 2 calculated by the Helical Wheel Projection web tool. Residues are schematically represented by geometric figures. Hydrophilic residues = circles, hydrophobic residues = diamonds, potentially negatively charged = triangles, and potentially positively charged = pentagons. In the centre mean relative hydrophobic moment and hydrophobic arc (@) are indicated.

Figure 2

A ribbon representation of Holoturoidin 1 (A) and Holoturoidin 2 (B). The amphipathic nature of the peptide is shown in this representation with the hydrophilic side above and the hydrophobic side below the polypeptide backbone. The potential surface is superimposed. Color code: acidic residues in red, basic residues in blue, and hydrophobic residues in white.

Figure 3

Inhibition activity of Holothuroidin 1 and 2 on S. aureus ATCC 25923, S. epidermidis ATCC 35984 (light gray square symbol) and P. aeruginosa ATCC 15442 (dark gray square symbol) biofilms. The data are expressed as the mean of three different experiments ± standard deviation. * = p < 0.05; ** = p < 0.01.

Figure 4

Hemolytic activity of H1 and H2 peptides from Holothuria tubulosa hemocytes against rabbit blood cells at different concentrations: white square symbol = 1.5 mg/ml; Black square symbol = 3.2; gray square symbol = 6.2; dark gray square symbol = 50 mg/ml. Data are the mean value of three separate experiments and expressed as percentage of hemolysis ± SD.