## 3 Discussion
The notion that Arg- and Trp-containing peptides are more cytotoxic than the commensurate Lys peptides has also been reported previously by other authors and has been observed both in in vitro and in animal model studies (see, for example, [75; 76]). While the antimicrobial and antib Biofilm activity of such Lys-substituted peptides is typically somewhat reduced compared to Arg-containing peptides (see Tables 2 and 4), their better selectivity for bacterial over human cells would make them the preferred choice in clinical settings. Shortening the length of the Lys sidechain [29; 32; 77] could further improve the selectivity [25], although these changes would need to be balanced by maintaining the antibofilm activity. The data depicted in Figs 2 and 6 clearly indicate that substituting Lys with the shorter Dab or Dap unnatural amino acids leads to improved antimicrobial and antibofilm activities. It is important to stress here that Tritrp-Dap has no effect on RBCs or PBMC's [25], which would make AMPs substituted with this analog very suitable for clinical applications. While the sidechain amino groups of Orn and Dab have pKa values comparable to Lys, once incorporated into a peptide, Dap is an unusual amino acid with a sidechain pKa value of 6.3 which can perturb the backbone structure of the peptide [78; 79]. Of note, shortening of the Lys sidechains also makes the peptides resistant to trypsin and other related proteolytic activities [32; 80; 81], which could further enhance their activities in in vivo situations. The antimicrobial mechanism of action of Tritrp has been investigated in some detail. Biophysical studies with vesicles made of model bacterial and mammalian membranes were evaluated by calcein leakage experiments and lipid flip-flop experiments. These studies indicated that Tritrp could act preferentially on the bacterial cytoplasmic membrane, likely forming dynamic toroidal pores that cause membrane leakage [30; 37; 82; 83]. Also, calorimetry studies indicated that Tritrp could bind strongly to vesicles made of _E. coli_polar lipid extract and to other model membranes containing phospholipids with the negatively charged phosphatidylglycerol headgroups [82]. The latter are abundant in bacterial membranes, but they are absent in mammalian membranes [84]. In doing so, it can seriously perturb the regular bilayer structure of such membranes [85]. Additional studies with a living engineered _E. coli_ strain showed that Tritrp and several of its analogs could perturb the inner membrane of this Gram-negative bacterium [30; 32]. However, subsequent studies have also indicated the possibility of intracellular actions of Tritrp, where it could bind to DNA and interfere in intracellular macromolecular synthesis [31]. Tritrp and its analogs could enter the bacterial cell through disproportionation of transient toroidal pores [86]. In a similar manner, it has been shown that the puroinodine A peptide can also cause leakage in the bacterial cytoplasmic membrane, while causing perturbations of intracellular activities as well [41; 42]. Likewise, indolicidin can also perturb cytoplasmic bacterial membranes [87] and it can inhibit the intracellular synthesis of DNA, RNA and proteins [45]. Recent studies with indolicidin indicate that at concentrations below the MIC, this peptide can induce extensive lipid flip-flop in bacterial membrane [88; 89]. Taken together, these data indicate that Tritrp, indolicidin and puroinodine A share several mechanisms through which they can kill bacterial cells. By being able to form membrane pores, they are clearly distinct from the Trp- and Arg-rich hexepeptides, which are too short to form such pores. In our recent study on the effects of the hexapeptides on _S. aureus_[23], we argued that the cationic Trp- and Arg-rich hexapeptides can cause unmixing of the bacterial cytoplasmic membrane by clustering with the headgroups of phosphatidylglycerol phospholipids, leaving behind domains enriched in phosphatidylcholine-ethanolamine.