# Breaking disulfide bonds in a weakly bactericidal α-defensin unleashes a potent antimicrobial peptide with an altered conformation

## TL;DR

Modifying a weak antimicrobial peptide by breaking disulfide bonds makes it highly effective against bacteria, offering a new approach to combat antibiotic resistance.

## Contribution

Breaking disulfide bonds in cryptdin 1 transforms it into a potent antimicrobial peptide with a new conformation and improved activity.

## Key findings

- Disulfide-devoid cryptdin 1 (L-Crp1) disintegrates bacterial membranes effectively.
- L-Crp1 adopts a helix-loop-helix conformation, enhancing its antimicrobial activity.
- L-Crp11-25, a shortened version, rescues mice from sepsis by reducing bacterial burden and inflammation.

## Abstract

Harnessing antimicrobial peptides as bactericidal agents affords an attractive approach to developing new anti-infective therapies. We found that abolishing disulfide bonding in mouse cryptdin 1 (Crp1), a weakly bactericidal α-defensin of 35 residues, turned it into a potent antimicrobial peptide against Gram-negative bacteria. Here we report that Crp1 in its natively folded β-sheet structure forms high-ordered nanonets to cloak, but not kill, Escherichia coli, whereas its disulfide-devoid linear counterpart (L-Crp1) readily disintegrates the bacterial membrane as monomers. L-Crp1 adopts a helix-loop-helix conformation in molecular dynamics simulations, likely conducive to productive peptide-membrane interactions detrimental to bacteria. A truncated peptide spanning the helix-loop-helix, L-Crp11-25, maintains the same conformation as and similar membranolytic and bactericidal activities to L-Crp1. Remarkably, intraperitoneally administered L-Crp11-25 rescues E. coli-challenged mice from lethality in a sepsis model by effectively reducing bacterial burden, inflammation and tissue damage. Our studies cultivate additional mechanistic insights into the mode of action of defensins and shed new light on how to harness these host factors for potential therapeutic use.

A recent epidemiological study estimates that antibiotic-resistant bacterial infections directly and indirectly killed 4.71 million patients worldwide in 2021 and will cause 8.22 million annual deaths by 2050. Searching new classes of antibiotics refractory to the existing resistance mechanisms for antibacterial therapy is imperative for global public health and economic growth. Toward this end, cationic antimicrobial peptides are particularly attractive drug candidates as they primarily target the anionic microbial membrane by a mechanism that deters the rise of resistance. However, obstacles abound in their clinical translation, including, but not limited to, subpar bactericidal activity and safety concerns. In studying a weakly bactericidal mouse α-defensin, cryptdin 1 (Crp1), we made a surprising finding that breaking its three structurally stabilizing disulfide bonds through Cys-to-Ala mutations unleashed an antimicrobial peptide against Gram-negative bacteria with an altered conformation but significantly improved potency and selectivity. A further shortened peptide of 25 amino acid residues, L-Crp11-25, was safe and highly efficacious in rescuing E. coli-infected mice from lethality by reducing bacterial burden, inflammation and tissue damage. Our study demonstrates how to harness naturally occurring antimicrobial peptides as novel anti-infective agents to combat increasingly prevalent antibiotic resistance.