Naturally occurring cationic antimicrobial peptides (AMPs)
mostly
adopt α-helical structures in bacterial membrane mimetic environments.
To explore the design of novel β-sheet AMPs, we identified two
short cationic amphipathic β-strand segments from the crystal
structure of the innate immune protein, MyD88. Interestingly, of these,
the 10-residue arginine–valine-rich synthetic MyD88-segment,
KRCRRMVVVV (M3), exhibited β-sheet structure when bound to the
outer membrane Gram-negative bacterial component, LPS. Isothermal
titration calorimetric data showed that M3 bound to LPS with high
affinity, and the interaction was hydrophobic in nature. Supporting
these observations, computational studies indicated strong interactions
of multiple and consecutive valine residues of M3 with the acyl chain
of LPS. Moreover, M3 adopted nanosheet and nanofibrillar structure
in 25% acetonitrile/water and isopropanol, respectively. M3 showed
substantial antibacterial activities against both Gram-positive and
Gram-negative bacteria which it appreciably retained in the presence
of human serum and physiological salts. M3 was non-hemolytic against
human red blood cells and non-cytotoxic to 3T3 cells up to 200 μM
and to mice in vivo at a dose of 40 mg/kg. Furthermore, M3 neutralized
LPS-induced pro-inflammatory responses in THP-1 cells and rat bone
marrow-derived macrophages. Consequently, M3 attenuated LPS-mediated
lung inflammation in mice and rescued them (80% survival at 10 mg/kg
dose) against a lethal dose of LPS. The results demonstrate the identification
of a 10-mer LPS-interacting, β-sheet peptide from MyD88 with
the ability to form nanostructures and in vivo activity against LPS
challenge in mice. The identified M3-template provides scope for designing
novel bioactive peptides with β-sheet structures and self-assembling
properties.
Many antibiotics are ineffective in killing Gram-negative bacteria due to the permeability barrier of the outer-membrane LPS. Infections caused by multi-drug-resistant Gram-negative pathogens require new antibiotics, which are often difficult to develop. Antibiotic potentiators disrupt outer-membrane LPS and can assist the entry of large-scaffold antibiotics to the bacterial targets. In this work, we designed a backbone-cyclized ultra-short, six-amino-acid-long (WKRKRY) peptide, termed cWY6 from LPS binding motif of β-boomerang bactericidal peptides. The cWY6 peptide does not exhibit any antimicrobial activity; however, it is able to permeabilize the LPS outer membrane. Our results demonstrate the antibiotic potentiator activity in the designed cWY6 peptide for several conventional antibiotics (vancomycin, rifampicin, erythromycin, novobiocin and azithromycin). Remarkably, the short cWY6 peptide exhibits wound-healing activity in in vitro assays. NMR, computational docking and biophysical studies describe the atomic-resolution structure of the peptide in complex with LPS and mode of action in disrupting the outer membrane. The dual activities of cWY6 peptide hold high promise for further translation to therapeutics.
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