Lipopolysaccharide neutralization by the antibacterial peptide CM4.
Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria. Binding of LPS to the CD14+ murine macrophage cell line RAW264.7 results in pro-inflammatory cytokine secretion. In extreme cases, it leads to septic shock in vivo. Therefore, the pursuit for molecules with antiendotoxin properties is urgent. In this study, we investigated the efficacy of antibacterial peptide CM4 in binding Escherichia coli LPS in vitro. CM4 avidly bound to E. coli LPS, as proven by the limulus amoebocyte lysate assay. Furthermore, the killing activity of CM4 against E. coli was progressively inhibited by increasing concentrations of LPS added to the medium, further confirming the peptide's affinity for endotoxin. Flow cytometric analysis revealed that CM4 inhibited the binding of FITC-conjugated LPS to RAW264.7 cells. Likewise, the inhibition of peptide to LPS-dependent cytokine induction was analyzed. CM4 suppressed LPS-induced TNF-alpha and IL-6 mRNA expression and blocked release of TNF-alpha and NO following LPS challenge in RAW264.7 cells. Together these observations indicate that antibacterial peptide CM4 probably exerts protective actions against endotoxin shock by blocking the binding of LPS to CD14+ cells.
Lin
Qing-Ping
QP
Jiangsu Province Key Laboratory for Molecular and Medical Biotechnology, Nanjing Normal University, Nanjing, China.
Zhou
Liang-Fan
LF
Li
Nan-Nan
NN
Chen
Yu-Qing
YQ
Li
Bao-Cun
BC
Cai
Yu-Feng
YF
Zhang
Shuang-Quan
SQ
D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D048168:Escherichia coli K12 / Q000187:drug effects; D015850:Interleukin-6 / Q000096:biosynthesis / Q000235:genetics; D018950:Lipopolysaccharide Receptors / Q000378:metabolism; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D011485:Protein Binding; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; C534224:CM4 peptide, Bombyx mori; D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide