Mechanism of action of membrane-active marine natural product theonellamide A

• Main Authors: Espiritu, Rafael Atillo, Matsumori, Nobuaki, Kinoshita, Masanao, Murata, Michio, Nishimura, Shinichi, Kakeya, Hideaki, Matsunaga, Shigeki, Yoshida, Minoru
• Format: text
• Published: Animo Repository 2013
• Subjects: Sponges—Composition; Membranes (Biology); Surface plasmon resonance; Nuclear magnetic resonance; Chemistry
• Online Access: https://animorepository.dlsu.edu.ph/faculty_research/5115
• Institution: De La Salle University

Theonellamides (TNMs) are members of a distinctive family of antifungal and cytotoxic bicyclic dodecapeptides isolated from the marine sponge Theonella sp. Recently, it was shown that TNMs recognize 3β-hydroxysterol-containing membranes, induce glucan overproduction, and damage cellular membranes. However, to date, the detailed mode of action of TNM at a molecular level has not been elucidated. In this study, to gain insight into its mechanism of action, surface plasmon resonance (SPR) experiments, solid-state nuclear magnetic resonance (2H and 31P NMR) measurements, and microscopic observation were performed on theonellamide A (TNM-A). SPR experiments revealed that TNM-A binding to membranes occurs in two steps, and that the incorporation of cholesterol or ergosterol significantly enhances the affinity of the peptide to the membrane, particularly in the initial binding step. These findings, together with the fact that binding of TNM-A to epicholesterol-containing liposomes and sterol-free liposomes was comparably weak, confirmed the preference of the peptide for the 3β-hydroxysterol-containing membranes. Then, solid-state 2H NMR measurements were conducted using deuterium-labeled cholesterol, ergosterol, or epicholesterol, which verified the direct interaction between TNM-A and 3β-hydroxysterols in lipid bilayers. Next, to evaluate changes in the lamellar structure induced by TNM-A, we performed solid state 31P NMR measurements of liposomes, and found that TNM-A induced partial disruption of the membrane structure. Then, we carried out differential interference microscopy analysis to directly observe membrane morphology. Microscopy images of liposomes showed that TNM-A induced various kinds of fluctuation of membrane surface structure such as deformation, fission, budding and wrinkling, which were observed both in cholesterol-containing and sterol-free liposomes, but more frequently in the former. The membrane fission process observed in liposomes might be related to TNM-induced vacuolar fragmentation in yeasts reported earlier. Altogether, this study demonstrates that the membrane action of TNM-A occurs in two processes; (1) TNM-A binds to membrane surface at the initial step, which is promoted by the direct interaction with sterol, and (2) TNM-A integrated on membrane surface induces membrane deformation in a concentration dependent manner.