Activity of the novel peptide arminin against multiresistant human pathogens shows the considerable potential of phylogenetically ancient organisms as drug sources

Abstract

The emergence of multidrug-resistant bacteria highlights the need for new antibacterial agents. Arminin 1a is a novel antimicrobial peptide discovered during investigations of the epithelial defense of the ancient metazoan Hydra. Following proteolytic processing, the 31-amino-acid-long positively charged C-terminal part of arminin 1a exhibits potent and broad-spectrum activity against bacteria, including multiresistant human pathogenic strains, such as methicillin-resistant Staphylococcus aureus (MRSA) strains (minimal bactericidal concentration, 0.4 microM to 0.8 microM). Ultrastructural observations indicate that bacteria are killed by disruption of the bacterial cell wall. Remarkably, the antibacterial activity of arminin 1a is not affected under the physiological salt conditions of human blood. In addition, arminin 1a is a selective antibacterial agent that does not affect human erythrocyte membranes. Arminin 1a shows no sequence homology to any known antimicrobial peptide. Because of its high level of activity against multiresistant bacterial strains pathogenic for humans, the peptide arminin 1a is a promising template for a new class of antibiotics. Our data suggest that ancient metazoan organisms such as Hydra hold promise for the detection of novel antimicrobial molecules and the treatment of infections caused by multiresistant bacteria.

FIG. 1.

(a) Schematic representation and size of the domains characterized by the separation of negatively and positively charged amino acid residues of arminin 1a. Underlined sequence, amino acid signal sequence; box with dashed border, putative cleavage site; dashed underline, putative amidation signal; red amino acids, negatively charged residues; blue amino acids, positively charged residues. The whole peptide has a predicted molecular mass of 10.5 kDa, and the functional cationic C-terminal part (c-arminin 1a) has a predicted molecular mass of 3.9 kDa. (b) In situ hybridization with the gene for arminin 1a showed strong blue staining in the entodermal (en) epithelium along the body column in Hydra. ec, ectodermal.

FIG. 2.

Arminin 1a is a member of a gene family containing eight members. Amino acid sequence alignment with the ClustalW algorithm shows a conserved N-terminal part and a variable C-terminal part (gray box). Nearly all positively charged amino acid residues are within the variable part (compare with Fig. 1a). The homology tree illustrates how arminin family members group together according to their sequence identity (the numbers given at the nodes of the tree are the percentage of identical amino acid residues).

FIG. 3.

Activity of c-arminin 1a in the presence of different salt concentrations and on red blood cell membranes. (a) The antimicrobial activity of c-arminin 1a against B. megaterium ATCC 14581 (diamonds), E. coli DH5α (squares), and S. aureus ATCC 12600 (triangles) was tested in the presence of different salt concentrations. Data were obtained from microdilution susceptibility assays performed at pH 7.4 with 0, 75, 150, or 200 mM NaCl. The values are expressed as the mean (including the standard deviation) of the relative activity of two experiments, each of which was performed in duplicate. The activity measured by using buffer without NaCl was set equal to 1. (b) The activity of c-arminin 1a (up to 250 μM) against human red blood cells was tested. The cytotoxic peptide melittin (up to 20 μM) was used as a positive control.

FIG. 4.

Changes in morphology of c-arminin 1a-treated S. aureus ATCC 12600. (a) Transmission electron micrograph of S. aureus (10⁸ cells/ml) incubated with c-arminin 1a (410 μM, i.e., 1,000 times the MBC) for 1.5 h. (b and c) Magnifications of two bacterial cells shown in panel a; the arrowheads point to the detachment of the peripheral cell wall. (d) Transmission electron micrograph of S. aureus (10⁸ cells/ml) incubated in 10 mM sodium phosphate buffer, pH 7.4, for 1.5 h as a negative control (intact cells). Bars, 1 μm.