Type of presentation: Poster

LS-11-P-1590 Investigation of the membrane effects and mode of action of the synthetic peptides Os and Os-C

Taute H.¹, Bester M. J.¹, Gaspar A. R.², Neitz A. W.²

¹Department of Anatomy, Faculty of Health Sciences, University of Pretoria, South Africa, ²Department of Biochemistry, Faculty of Natural and Agricultural Sciences, University of Pretoria, South Africa

helena.taute@up.ac.za

Antimicrobial peptides (AMPs) are found in all forms of life, and form part of the innate immune system. Strategies for the development of AMPs for clinical application involves the isolation and characterisation of these AMPs, then to use these peptides as templates for the synthesis of shorter peptides with greater stability, lower cost of production and greater antimicrobial activity.

The peptide Os is a synthetic defensin C-terminal derived peptide from the tick Ornithodoros savignyi. Both Os and its analogue, in which three cysteine residues were omitted (Os-C), were previously found to be bactericidal to Gram-positive and Gram-negative bacteria. Peptides investigated for clinical application must not be toxic to mammalian cells. Cell culture based studies have shown that both peptides were not cytotoxic (Prinsloo et al. 2013).

In this study the morphological effects and mode of killing of Os and Os-C was further investigated using SEM and confocal fluorescence microscopy (triple staining with DAPI - stain all cells, CTC - viable cells and FITC - cells with permeabilised membranes). Possible cytotoxicity was further evaluated using human erythrocytes which represent a typical eukaryotic membrane.

SEM of melittin (Mel), a known lytic peptide, showed distinct blebbing of the cell surfaces of E. coli and B. subtilis at 25 µM. In contrast both Os (0.77 µM) and Os-C (1.74 µM), at their minimum bactericidal concentrations (MBCs), caused leakage of intracellular content that led to a flattened morphology (fig 1).

Triple fluorescence staining showed that E. coli exposed to 2.5 µM Mel had undergone membrane permeabilisation while no viable bacteria remained. Likewise at the MBC of Os and Os-C membranes were damaged with few viable cells. At concentrations ten times lower than the MBCs, Os and Os-C caused membrane permeabilisation, however the bacteria remain viable. This suggests a dual mechanism of action that includes both the membrane and intracellular targets.

At 25 µM Mel caused 100% haemolysis with the formation of echinocytes at concentrations as low as 2.5 µM. Both Os and Os-C did not cause haemolysis up to 100 µM and SEM showed normal membrane morphology and biconcave cells (fig 2).

In conclusion, preliminary indications are that these peptides have a different mechanism of action than Mel, possibly affecting multiple targets including membranes and intracellular components. The peptides show no toxicity towards human erythrocytes. Further investigation will be done by fluorescently labelling the peptide and tracking with confocal microscopy. Cytotoxicity towards human leukocytes will also be investigated.

Thank you to the personnel of the Micrscopy Unit at the University of Pretoria for all your assistance during this project.

Fig. 1: SEM of B. subtilis treated with 25 µM Mel showing membrane blebbing (A), and 0.77 µM Os showing flattened cell morphology (B). Scale bars = 1 µm

Fig. 2: SEM of human erythrocytes exposed to 2.5 µM Mel showing the formation of echinocytes (A) and 100 µM Os showing normal morphology (B). Scale bars = 1 µm