We present direct visualization of pores formed by alamethicin (Alm) in a matrix of phospholipids using electrochemical scanning tunneling microscopy (EC-STM). High-resolution EC-STM images show individual peptide molecules forming channels. The channels are not dispersed randomly in the monolayer but agglomerate forming 2D nanocrystals with a hexagonal lattice in which the average channel–channel distance is 1.90 ± 0.1 nm. The STM images suggest that each Alm is shared between the two adjacent channels. Every channel consists of six Alm molecules. Three or four of these molecules have the hydrophilic group oriented toward the center of the channel allowing for water column formation inside the channel. The dimensions of the central pore in the images are consistent with the dimension of the water column in a model of hexameric pore proposed in the literature. The images obtained in this work validate the barrel-stave model of the pore formed in phospholipid membranes by amphiphatic peptides. They also provide direct evidence for cluster formation by such pores.

Staphylococcus pseudintermedius is a common commensal bacterium colonizing the skin and mucosal surfaces of household animals. However, it has recently emerged as a dangerous opportunistic pathogen, comparable to S. aureus for humans. The epidemiological situation is further complicated by the increasing number of methicillin-resistant S. pseudintermedius infections and evidence of gene transmission driving antibiotic resistance between staphylococci colonizing human and zoonotic hosts. In the present study, we describe a unique peptide, BacSp222, that possesses features characteristic of both bacteriocins and virulence factors. BacSp222 is secreted in high quantities by S. pseudintermedius strain 222 isolated from dog skin lesions. This linear, fifty-amino-acid highly cationic peptide is plasmid-encoded and does not exhibit significant sequence similarities to any other known peptides or proteins. BacSp222 kills gram-positive bacteria (at doses ranging from 0.1 to several micromol/l) but also demonstrates significant cytotoxic activities towards eukaryotic cells at slightly higher concentrations. Moreover, at nanomolar concentrations, the peptide also possesses modulatory properties, efficiently enhancing interferon gamma-induced nitric oxide release in murine macrophage-like cell lines. BacSp222 appears to be one of the first examples of multifunctional peptides that breaks the convention of splitting bacteriocins and virulence factors into two unrelated groups.