| Aeromonas hydrophila, a gram-negative bacterium that causes gastroenteritis, wound infections, septicemia, and other diseases in humans, produces a cytotoxic enterotoxin (Act). Act is a potent virulence factor that possesses several biological activities, including hemolysis, cytotoxicity, enterotoxicity, and lethality. Previous studies indicated that Act induced inflammatory signaling in murine macrophages and caused host cell death. The induction of key inflammatory mediators seen in Act-treated macrophages may result from involvement of Act in one or more signal transduction pathways, which might occur via interaction of Act with a host cell membrane receptor or via internalization and subsequent interaction of Act with intracellular signal transduction molecules. The purpose of this study was to identify specific host cell signaling pathways activated by Act and determine the mechanism of Act-induced host cell death. Based on microarray and proteomics analyses of Act-treated murine macrophages and human intestinal epithelial cells, Act significantly altered host transcriptional profiles, indicating which signaling pathways might be activated by Act, and directly led to the discovery that apoptosis was the mechanism of Act-induced cell death. Act-mediated apoptosis was caspase-dependent, involved release of cytochrome c and AIF from mitochondria, and required the presence of the death-associated receptor for TNF-alpha (TNFR-1). An investigation of host signaling pathways revealed that Act activated all three major mitogen-activated protein kinase pathways (p38, JNK, and ERK1/2), culminating in the activation of C/EBP-beta and AP-1 transcription factors in murine macrophages and human intestinal epithelial cells. Act-mediated inflammatory cell signaling was also apparent in murine peritoneal macrophages and polarized epithelial cells when treated on the apical side, which strengthened the biological relevance of the results. In order to determine how Act might mediate its effects on host cells, human protein microarrays were utilized to demonstrate that Act could bind 9 human proteins, including galectin-3, and SNAP23. Knockdown of galectin-3 and SNAP23 using siRNA technology greatly abrogated Act-induced apoptosis in murine macrophages and human intestinal epithelial cells, indicating their involvement in toxin-mediated cell death. Considered together, these new data demonstrated that Act induced extensive host cell signaling and classical apoptosis and shed light on the mechanism of action of Act. |
