Abstract
Melatonin, an endogenous hormone molecule, has a variety of biological functions, but a functional role of melatonin in the infection of Gram-negative bacterium Vibrio vulnificus has yet to be described. In this study, we investigated the molecular mechanism of melatonin in the apoptosis of human intestinal epithelial (HCT116) cells induced by the hemolysin (VvhA) produced by V. vulnificus. Melatonin (1 μM) significantly inhibited apoptosis induced by the recombinant protein (r) VvhA, which had been inhibited by the knockdown of MT2. The rVvhA recruited caveolin-1, NCF-1, and Rac1 into lipid rafts to facilitate the production of ROS responsible for the phosphorylation of PKC and JNK. Interestingly, melatonin recruited NCF-1 into non-lipid rafts to prevent ROS production via MT2 coupling with Gαq. Melatonin inhibited the JNK-mediated phosphorylation of c-Jun responsible for Bax expression, the release of mitochondrial cytochrome c, and caspase-3/-9 activation during its promotion of rVvhA-induced apoptotic cell death. In addition, melatonin inhibited JNK-mediated phosphorylation of Bcl-2 responsible for the release of Beclin-1 and Atg5 expression during its promotion of rVvhA-induced autophagic cell death. These results demonstrate that melatonin signaling via MT2 triggers recruitment of NCF-1 into non-lipid rafts to block ROS production and JNK-mediated apoptotic and autophagic cell deaths induced by rVvhA in intestinal epithelial cells.
Highlights
Introduction of cells infected with aVvhA mutant, VvhA, a pore-Vibrio vulnificus is a halophilic Gram-negative bacter- forming toxin, is known for its cytolytic activity against ium frequently found in warm coastal waters
Melatonin prevents apoptosis induced by VvhA To confirm that the VvhA produced by V. vulnificus used in this study induced cytotoxicity in HCT116 cells, the cells were exposed to various concentrations (0–200 pg/mL) of recombinant protein of VvhA (rVvhA) for 24 h. rVvhA treatments from 50 to 200 pg/mL significantly induced cytotoxicity of HCT116 cells (Fig. 1a)
These results indicate that the protective effect of melatonin on V. vulnificus infection is related to blockage of apoptosis caused by rVvhA, and that melatonin triggers the MT2 receptor-mediated signaling pathway
Summary
Introduction of cells infected with aVvhA mutant, VvhA, a pore-Vibrio vulnificus is a halophilic Gram-negative bacter- forming toxin, is known for its cytolytic activity against ium frequently found in warm coastal waters. The extent erythrocytes and host cells[5]. It has been suggested of V. vulnificus-related disease can range from gastro- that the balance of intracellular ROS in host cells during enteritis to primary sepsis (vibriosis) in high-risk individuals[1]. Infection of V. vulnificus is cytotoxic to host cells, and its virulence is mediated by secreted cytotoxins and enzymes, such as VvhA, MARTX, and Vvp[2,3,4]. Recent studies have suggested that rVvhA increases ROS provirulence effects of VvhA were controversial in an in vivo duction through the lipid raft-dependent c-Src/NOX signaling pathway[8].
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