Transmembrane Pores Formed by Human Antimicrobial Peptide LL-37

Abstract

Human LL-37 is a multifunctional cathelicidin peptide that has shown a wide spectrum of antimicrobial activity by permeabilizing microbial membranes similar to other antimicrobial peptides; however, its molecular mechanism has not been clarified. Two independent experiments revealed LL-37 bound to membranes in the α-helical form with the axis lying in the plane of membrane. This led to the conclusion that membrane permeabilization by LL-37 is a nonpore carpet-like mechanism of action. Here we report the detection of transmembrane pores induced by LL-37. The pore formation coincided with LL-37 helices aligning approximately normal to the plane of the membrane. We observed an unusual phenomenon of LL-37 embedded in stacked membranes, which are commonly used in peptide orientation studies. The membrane-bound LL-37 was found in the normal orientation only when the membrane spacing in the multilayers exceeded its fully hydrated value. This was achieved by swelling the stacked membranes with excessive water to a swollen state. The transmembrane pores were detected and investigated in swollen states by means of oriented circular dichroism, neutron in-plane scattering, and x-ray lamellar diffraction. The results are consistent with the effect of LL-37 on giant unilamellar vesicles. The detected pores had a water channel of radius 23-33 Å. The molecular mechanism of pore formation by LL-37 is consistent with the two-state model exhibited by magainin and other small pore-forming peptides. The discovery that peptide-membrane interactions in swollen states are different from those in less hydrated states may have implications for other large membrane-active peptides and proteins studied in stacked membranes.

Membrane Core-Specific Antimicrobial Action of Cathelicidin LL-37 Peptide Switches Between Pore and Nanofibre Formation

“…With DMPC the α-helicity of LL-37 did not change after 2 hours; the helicity was increased in the presence of DOPC, however, it also did not change after 2 hours. Accordingly, LL-37 is bound to the membrane in α-helical form, consistent with earlier literature reports1224. Hence the formation of fibres does not involve the refolding of the peptide and the fibres are not amyloid-like since no significant β-structure is observed that would be characteristic of an amyloidogenic peptide44.…”

“…Hence, it was proposed that LL-37 is a nonspecific, albeit highly effective, cell killer that acts via the carpet mechanism2122. However, it was shown that LL-37 disrupts the lipid bilayer without breaking the membrane into small fragments, and fluorescence measurements also suggested a pore forming mechanism2324. The activity against mammalian cell membranes is also ambiguous: it was proposed that LL-37 could act, at least in part, by decreasing the fluidity and hence lowering the permeability of epithelial cell membranes, making it harder for certain bacteria to attack25.…”

High-quality 3D structures shine light on antibacterial, anti-biofilm and antiviral activities of human cathelicidin LL-37 and its fragments

“…Based on spectroscopic evidence, it was suggested that either a carpet or toroidal pore mechanism was possible for LL-37 [108]. Diffraction measurements, albeit at high peptide concentrations, show a trans-bilayer orientation of the peptide, consistent with pore formation [109]. Time-lapse fluorescence microscopy was suggested to be more consistent with a pore model [110].…”

Functional Linkage between Genes That Regulate Osmotic Stress Responses and Multidrug Resistance Transporters: Challenges and Opportunities for Antibiotic Discovery

“…TolC works synergistically in the multicomponent transporters of the resistance-nodulation division (RND) superfamily, the major facilitator superfamily (MFS), and the ATP-binding cassette (ABC) superfamily of MDR efflux pumps (21). Of note, exposure of E. coli to pore-forming cationic antimicrobial peptides (CAMPs) (22,23) led to the upregulation of the marRAB operon (24). This effect requires the presence of Rob, which may act as a signal of osmotic stress caused by membrane disruption (24).…”