The Human Antimicrobial Peptide LL-37 Transfers Extracellular DNA Plasmid to the Nuclear Compartment of Mammalian Cells via Lipid Rafts and Proteoglycan-dependent Endocytosis

Abstract

Antimicrobial peptides, such as LL-37, are found both in nonvertebrates and vertebrates, where they represent important components of innate immunity. Bacterial infections at epithelial surfaces are associated with substantial induction of LL-37 expression, which allows efficient lysis of the invading microbes. Peptide-mediated lysis results in the release of bacterial nucleic acids with potential pathobiological activity in the host. Here, we demonstrate that LL-37 targets extracellular DNA plasmid to the nuclear compartment of mammalian cells, where it is expressed. DNA transfer occurred at physiological LL-37 concentrations that killed bacterial cells, whereas virtually no cytotoxic or growth-inhibitory effects were observed in mammalian cells. Furthermore, LL-37 protected DNA from serum nuclease degradation. LL-37⅐DNA complex uptake was a saturable time-and temperature-dependent process and was sensitive to cholesterol-depleting agents that are known to disrupt lipid rafts and caveolae, as shown by flow cytometry. Confocal fluorescence microscopy studies showed localization of internalized DNA to compartments stained by cholera toxin B, a marker of lipid rafts, but failed to demonstrate any co-localization of internalized DNA with caveolin-positive endocytotic vesicles. Moreover, LL-37-mediated plasmid uptake and reporter gene expression were strictly dependent on cell surface proteoglycans. We conclude that the human antimicrobial peptide LL-37 binds to, protects, and efficiently targets DNA plasmid to the nuclei of mammalian cells through caveolae-independent membrane raft endocytosis and cell surface proteoglycans.

Cationic antimicrobial peptides in psoriatic skin cooperate to break innate tolerance to self‐DNA

“…Thus, internalization is largely independent of the binding of the peptide to a specific cell surface receptor, although some specificity may be conferred by different subtypes of proteoglycans due to the structure of the complexes [36]. The process of proteoglycan-mediated endocytosis has also been described for LL37 [37] and is in accordance with the fact that LL37 enantiomers or LL37 mutants, which both retain the cationic structure of the peptide but not its specific amino acid sequence, are still able to condense DNA and trigger endosomal TLR9 activation in pDCs (Supporting Information Fig. 3) [24].…”

Cathelicidin LL-37 Increases Lung Epithelial Cell Stiffness, Decreases Transepithelial Permeability, and Prevents Epithelial Invasion by Pseudomonas aeruginosa

“…Our findings support a model in which P. aeruginosa internalization can infect epithelial cells through more than one pathway (47), including targeting molecules associated with lipid rafts (48,49), such as Lyn (50) and caveolin-2 (51). LL-37's ability to interact with lipid rafts is suggested by its high surface activity (52), the caveolae-independent membrane raft-dependent endocytosis of an LL-37/DNA plasmid complex (53), and LL-37-mediated/raft-dependent LPS internalization (54). Because LL-37 activates multiple plasma receptors, and the cellular mechanical response to agonists depends on the receptor engaged (55), it is also possible that the LL-37-mediated increase in F-actin concentration results in the formation of distinct cytoskeletal networks with mechanical properties that affect bacterial invasion.…”

Serum amyloid P component facilitates DNA clearance and inhibits plasmid transfection: implications for human DNA vaccine

“…Recent studies indicate that several extracellular DNA-binding proteins, such as high-mobility group B 1, anti-microbial peptide LL37 and heparan sulfate proteoglycan, may be important for DNA uptake under physiological conditions. [17][18][19]30 Therefore, we used two DNA-binding peptides, TAT and LL37, to simulate physiological transfection agents, as reported previously. Using the luciferase-expressing plasmid pGL4.17-CMV and two non-phagocytic cell lines, we found that the transfection efficiency of the plasmid significantly increased after a pre-incubation of LL37 or TAT.…”

“…30 For CL treatment, 100 mg ml À1 CL was administered in cell culture for 30 min and then removed by phosphate-buffered saline washing. For intracellular DNA analysis, cells were washed with phosphatebuffered saline containing dextran sulfate (100 mg ml À1 ) for three times to remove extracellular DNA.…”