VP1686, a Vibrio type III secretion protein, induces Toll-like receptor-independent apoptosis in macrophage through NF-κB inhibition

Abstract

Vibrio parahaemolyticus, causative agent of human gastrointestinal diseases, possesses several virulent machineries including thermostable direct hemolysin and type III secretion systems (TTSS1 and -2). In this report, we establish that TTSS1-dependent secretion and translocation of a V. parahaemolyticus effector protein VP1686 into the cytosol induces DNA fragmentation in macrophages. We performed yeast two-hybrid screening to identify the molecules involved in VP1686-mediated cell death pathways and showed that nuclear factor RelA p65/NF-κB physically interacts with VP1686. To understand the impact of this interaction on the NF-κB DNA binding activities in infected macrophages, we analyzed a series of deletion mutants for the TTSS and its secreted proteins. Induction of DNA binding activity of NF-κB was significantly suppressed, and increased macrophage apoptosis has been associated with V. parahaemolyticus strain, which contains both VP1686 and TTSS1. Macrophages lacking Toll-like receptor adaptor molecules MyD88 (myeloid differentiation primary response protein 88) or TRIF (TIR domain-containing adapter-inducing interferon β) showed similar sensitivity to VP1686. As a consequence of NF-κB suppression, microarray analysis has revealed that VP1686 translocation alerted the expression of many genes that have known functions in cellular responses to apoptosis, cell growth, and transcriptional regulation. Our results suggest an important role for Vibrio effector protein VP1686 that activate a conserved apoptotic pathway in macrophages through suppression of NF-κB activation independent of Toll-like receptor signaling. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.