Selectivity for and destruction of Salmonella typhimurium via a membrane damage mechanism of a cell-penetrating peptide ppTG20 analogue.

P7, an analogue of the cell-penetrating peptide (CPP) ppTG20, was derived by replacing Phe and Trp with Arg based on the structure-activity relationships of CPPs and antimicrobial peptides (AMPs). P7 showed antimicrobial activity against Salmonella typhimurium at 4 μM and possessed broad antimicrobial activity greater than its parent peptide. P7 displayed good selectivity, with low haemolysis below its minimum inhibitory concentration range, but displayed cytotoxic activity against the HT29 and MDA-MB231 mammalian cell lines. Studies of calcein leakage from egg yolk L-α-phosphatidylcholine/egg yolk L-α-phosphatidyl-DL-glycerol (EYPC/EYPG) (bacterial membrane mimic) and EYPC/cholesterol (eukaryotic membrane mimic) vesicles also demonstrated that P7 exhibited high selectivity and caused pore formation in the bacterial membrane. Circular dichroism experiments suggested that the conformation of P7 underwent transitions from a random coil in sodium phosphate buffer to an α-helical conformation in bacterial model membranes. P7 induced influx of the membrane fluorescent probe 1-N-phenylnaphthylamine (NPN) and the nucleic acid fluorescent probe SYTOX(®) Green by increasing live S. typhimurium cell outer membrane and plasma membrane permeability, respectively. P7 also induced ion channel formation in the cell plasma membrane causing leakage of potassium ions. Flow cytometric analysis demonstrated that S. typhimurium cell membrane integrity was destroyed following incubation with P7. These results indicated that P7 exhibited good bacterial selectivity and exerted its antibacterial activity by a membrane damage mechanism. Furthermore, these results suggested that CPPs may represent a source of templates for AMP design.