Reduction Impairs the Antibacterial Activity but Benefits the LPS Neutralization Ability of Human Enteric Defensin 5

Oxidized human defensin 5 (HD5(OX)), a Paneth cell-secreted antibacterial peptide with three characteristic disulfide bonds, protects the host from invasion by morbigenous microbes in the small intestine. HD5(OX) can be reduced by thioredoxin (Trx) in vitro, while the biochemical properties of the reduced linear peptide, HD5(RED), remain unclear. Here, we first confirm that HD5(RED) does exist in vivo. Furthermore, we reveal that the recruitment of HD5(RED) to the outer membrane of Gram-negative bacteria and to the anionic lipid A is lower than that of HD5(OX), and HD5(RED) is less efficient in penetrating bacterial outer and inner membranes and inducing membrane depolarization, which confers an attenuated antibacterial activity to HD5(RED). However, due to its higher structural flexibility, the binding of HD5(RED) to bacterial lipopolysaccharide (LPS) is markedly stronger than that of HD5(OX). Consequently, HD5(RED) is more effective in suppressing the production of the pro-inflammatory cytokine TNF-α in LPS-stimulated macrophages by blocking the interaction between LPS and LPS-binding protein, thus suggesting that HD5(RED) might act as a scavenger to neutralize LPS in the gut. This study provides insights into the antibacterial and immunoregulatory effects of HD5(RED) and expands the known repertoire of the enteric defensins.