Aims: To understand the effects of Trp residues in linear antimicrobial peptides with a-helical conformations on cell permeation ability and membrane transduction efficacy. Methods and Results: A series of L-K6 analogues were designed and synthesized by replacing Ile or Leu with Trp at different positions on the hydrophobic face of L-K6. The antimicrobial and haemolytic activity and secondary structure of the designed Trp-containing peptides were assessed. In addition, the role of Trp in membrane disruption for these designed peptides was investigated. I1W, I4W and L5W demonstrated stronger activity than the other peptides against both Gram-positive and Gram-negative bacteria. All of the tested peptides preferentially interacted with negatively charged vesicles composed of phosphatidylglycerol (PG)/cardiolipin (CL) or PG/CL/phosphatidylethanolamine, and, to a lesser extent, with zwitterionic vesicles. I1W, I4W and L5W caused calcein release at 2Á5 lmol l À1 . Conclusions:The position of Trp, rather than the number of Trp residues, in these peptides was an important factor in the antimicrobial activity. Trp residues were deeply inserted into negatively charged membranes but were largely exposed in aqueous buffer solution.Significance and Impact of the Study: These Trp-containing peptides may represent good candidates for new antibiotic agents and for use in new therapeutic approaches.

Tryptophan (Trp) residues reportedly exhibit a strong membrane-disruptive activity, and this property endows Trp-containing antimicrobial peptides (AMPs) with a unique ability to interact with the surface of bacterial cell membranes, possibly improving antimicrobial properties. In this study, we investigated the influence of Trp residues engineered to have a distinct preference for the interface region of lipid bilayers on antimicrobial activity. We designed two Trp-substituted AMPs (I1WL5W and I4WL5W) by replacing Ile or Leu residues with two Trp residues at different positions in the L-K6 peptide, and determined their antimicrobial activity and mechanism of membrane action. Both I1WL5W and I4WL5W exhibited significantly higher antimicrobial activity and lower cytotoxicity against Gram-negative and Gram-positive bacteria compared with L-K6. The Trp-substituted peptides had a disordered structure in aqueous solution and adopted an α-helical structure in solutions of 50% trifluoroethanol/water and 30 mM SDS. I1WL5W and I4WL5W caused a significant leakage of calcein from liposomes containing membranes that mimicked those of Escherichia coli and Staphylococcus aureus. Scanning electron microscopy analysis suggested that I1WL5W and I4WL5W killed bacteria by disrupting bacterial cell membranes. Furthermore, fluorescence and quenching data from a variety of liposomes, which mimic different cell membranes, indicated that the Trp-substituted peptides could insert into the lipid bilayers and induce blue shifts in the emission spectra of the Trp residues. I1WL5W and I4WL5W were also less susceptible to acrylamide or KI quenchers. The current work may be important for designing novel Trp-containing peptides exhibiting strong antimicrobial abilities by penetrating bacterial membranes.

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Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in Saccharolobus islandicus, and this revealed that Cmr4α RNase-dead (dCmr4α) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4α in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4α inhibition. Expression of dCmr4α also strongly inhibited plasmid transformation with Cmr2αHD and Cmr2αPalm mutants, but the inhibition was diminished in Cmr2αHD,Palm. Since dCmr4α-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-α system in the S. islandicus cells expressing dCmr4α. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems.