Structure-activity relationships of the antimicrobial peptide arasin 1 - and mode of action studies of the N terminal, proline-rich region

Arasin 1 is a 37 amino acid long proline-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. In this work the active region of arasin 1 was identified through structure-activity studies using different peptide fragments derived from the arasin 1 sequence. The pharmacophore was found to be located in the proline/arginine-rich NH2 terminus of the peptide and the fragment arasin 1(1–23) was almost equally active to the full length peptide. Arasin 1 and its active fragment arasin 1(1–23) were shown to be non-toxic to human red blood cells and arasin 1(1–23) was able to bind chitin, a component of fungal cell walls and the crustacean shell. The mode of action of the fully active N-terminal arasin 1(1–23) was explored through killing kinetic and membrane permeabilization studies. At the minimal inhibitory concentration (MIC), arasin 1(1–23) was not bactericidal and had no membrane disruptive effect. In contrast, at concentrations of 5×MIC and above it was bactericidal and interfered with membrane integrity. We conclude that arasin 1(1–23) has a different mode of action than lytic peptides, like cecropin P1. Thus, we suggest a dual mode of action for arasin 1(1–23) involving membrane disruption at peptide concentrations above MIC, and an alternative mechanism of action, possibly involving intracellular targets, at MIC

Antibacterial activities in various tissues of the horse mussel, Modiolus modiolus

A search for antibacterial activity in different organs/tissues of the horse mussel, Modiolus modiolus, was conducted. Dried samples were extracted with 60% (v/v) acetonitrile, containing 0.1% (v/v) trifluoroacetic acid. Due to high salt content, two liquid phases were obtained; an acetonitrile-rich phase (ACN extract) and an aqueous phase. The aqueous phase was further subjected to solid phase extraction (SPE). Eluates from SPE and ACN extracts were tested for antibacterial, lysozyme, and toxic activity. Antibacterial activity was demonstrated in extracts from several tissues, including plasma, haemocytes, labial palps, byssus, mantle, and gills. Some of the extracts were sensitive to proteinase K treatment, indicating antibacterial peptides and/or proteins. Lysozyme-like activity and toxic activity against Artemia salina nauplii was detected in fractions from the gills, mantle, muscle, and haemocytes. Results from this study indicate that M. modiolus is a promising source for identifying novel drug lead compounds

Screening for antibacterial and antifungal activities in marine benthic invertebrates from northern Norway

Benthic marine invertebrates collected from sub-Arctic regions of northern Norway, were found to be a promising source of novel bioactive compounds against human and fish pathogenic bacteria and fungi. Lyophilized material from seven species of ascidians, six sponges and one soft alcyonid coral were extracted with 60% acidified acetonitrile (ACN). After separation into an ACN-rich phase (ACN extract) and an aqueous phase, and subsequent solid phase extraction of the aqueous phase, fractions differing in polarity were obtained and screened for antibacterial and antifungal activities, along with the more lipophilic ACN-extracts. Antimicrobial activity was determined against two Gram-negative, two Gram-positive bacteria, and two strains of fungi. Notably, all the invertebrate species in the study showed activity against all four strains of bacteria and the two strains of fungi. In general, the aqueous fractions displayed highest antimicrobial activity, and the most potent extracts were obtained from the colonial ascidian Synoicum pulmonaria which displayed activity against bacteria and fungi at a concentration of 0.02 mg/ml; the lowest concentration tested

Effect of arasin 1(1–23), PR-39(1–26) and cecropin P1 on <i>E. coli</i> viability.

<p>Relative light units (rlu) produced by <i>E.coli</i> HB101, constitutively expressing the <i>luxCDABE</i> operon are shown, after AMP treatment at different concentrations. Light emission is shown relative to the untreated control for the 4 selected incubation times. The mean of three independent measurements is indicated ± S.D. MIC against <i>E. coli</i> HB101 is 4, 1 and 1 µM for arasin 1(1–23), PR-39(1–26) and cecropin P1, respectively.</p

Overview of synthetic arasin 1 and its deletion peptides.

<p>Shaded letters indicate characteristic residues for arasin (Cys: black) and for proline-rich AMPs (Pro, Arg: grey). Acm = Acetamidomethyl. Predicted disulphide bridges are indicated for arasin 1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053326#pone.0053326-Stensvag1" target="_blank">[27]</a>.</p

Chitin binding capacity of (A) arasin 1(1–23) and (B) arasin 1(20–37)-Acm.

<p>Fifty micrograms of peptides were incubated with 40 mg chitin, and subsequently washed with 0.1 M, 1 M NaCl, and hot (95°C) 10% acetic acid. The obtained supernatants were subjected to RP-HPLC on a C₁₈ column. The resulting chromatograms show unbound material and peptides released from chitin by the different washing solutions, respectively.</p

Killing kinetics of <i>E. coli</i> cells treated with arasin 1(1–23) or cecropin P1.

<p>Colony forming units (CFU) of <i>E. coli</i> HB101 subjected to peptide treatment or water (negative control) are shown. Sample aliquots were withdrawn and washed in high salt solution at the time points indicated and subsequently plated for colony counts. Data are expressed as the average number of colony forming cells ± S.D. for three independent experiments.</p

Bacterial membrane integrity after treatment with arasin 1(1–23) or cecropin P1.

<p>Percentage of fluorescent cells (PI-positive) measured by flow cytometry after incubation of <i>E. coli</i> HB101 cells with 4, 20, and 40 µM arasin 1(1–23) or 4 µM cecropin P1 is shown. The background level of permeabilized cells, obtained using untreated samples, was always lower than 3% and was subtracted to the corresponding peptide-treated sample. Data are expressed as the average of % PI-positive cells ± S.D. for four independent experiments.</p

CD spectra of arasin 1(1–23) (A) and arasin 1 (B).

<p>Peptides were analyzed at a final concentration of 20 µM in 10 mM sodium phosphate buffer pH 7.0 (solid line) or with the addition of 10 mM SDS (dotted lines). Spectra are the smoothed means of at least two measurements, each composed of three different scans.</p