The mechanism of action of LL-37 revisited : exploring its activity and interaction with the Gram-positive model organism Bacillus subtilis

Abstract

LL-37 is a human antimicrobial peptide produced by neutrophils and epithelial cells, with broad activity against Gram-negative and Gram-positive bacteria. The ability of LL-37 to target bacterial membranes is largely agreed upon, however the mechanism by which it kills bacteria is still somewhat unclear. Using a combination of fluorescence microscopy, growth, and viability assays on the Gram-positive model organism Bacillus subtilis, I show that permeabilisation by LL-37 is not sufficient to trigger bacteriolysis. Instead, LL-37 causes lysis in an indirect, autolysin-dependent manner. Surprisingly, a small subpopulation of cells can resume growth in the presence of LL-37. It emerged this phenomenon is caused by stochasticity in LL-37 binding, which enables these cells to grow and recover the culture. Intriguingly, cultures are then protected from subsequent exposure to LL-37. I found this was due to membrane debris from lysed cells sequestering LL-37, and not DNA from lysed cells as previously suggested by other research groups. Altogether, upon LL-37 treatment, the majority of cells die via membrane disruption which then induces autolysis; however, a small population of cells stochastically survive LL-37 and recover the culture, which is then protected from future LL-37 exposure by membrane debris from lysed sister cells. Finally, I show that, unlike in other bacterial species, membrane headgroup composition, including the presence of lysyl-phosphatidylglycerol, has little impact on the susceptibility of B. subtilis towards first or repeated encounters with LL-37. Instead, only the loss of teichoic acids from the cell envelope increases susceptibility by a substantial degree, due in part to their D-alanylation. Contrary to previous postulations, membrane fluidity also has no impact on LL-37 activity, with both highly fluid and less fluid membranes being equally susceptible. Taken together, these results shed surprising new light on the antibacterial mechanism of action of a major human immune system component.