The Multifaceted Antibacterial Mechanisms of the Pioneering Peptide Antibiotics Tyrocidine and Gramicidin S

Cyclic β-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used Antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a Bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins. Interestingly, they also cause DNA damage and interfere with DNA-binding proteins. Despite sharing 50% sequence identity with tyrocidines, gramicidin S causes only mild lipid demixing with minor effects on membrane fluidity and permeability. Gramicidin S delocalizes peripheral membrane proteins involved in cell division and cell envelope synthesis but does not affect integral membrane proteins or DNA. Our results shed a new light on the multifaceted Antibacterial mechanisms of these Antibiotics and explain why resistance to them is virtually nonexistent.IMPORTANCE Cyclic β-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first Antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the Bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established Antibiotics kill bacteria. Our results shed new light on the Antibacterial mechanism of β-sheet peptide Antibiotics and explain why they are still so effective and why there is so little resistance to them.