Early cell signaling by the cytotoxic enterotoxin of Aeromonas hydrophila in macrophages

A cytotoxic enterotoxin (Act) of Aeromonas hydrophila is an important virulence factor with hemolytic, cytotoxic and enterotoxic activities. In this report, we demonstrated Act rapidly mobilized calcium from intracellular stores and evoked influx of calcium from the extracellular milieu in macrophages. A direct role of calcium in Act-induced prostaglandin (e.g. PGE₂) and tumor necrosis factor alpha (TNFα) production was demonstrated in macrophages using a cell-permeable calcium chelator BAPTA-AM, which also down-regulated activation of transcription factor NF-κB. We showed that Act's capacity to increase PGE₂ and TNFα production could be blocked by inhibitors of tyrosine kinases and protein kinase A. In addition, Act caused up-regulation of the DNA repair enzyme redox factor-1 (Ref-1), which potentially could promote DNA binding of the transcription factors allowing modulation of various genes involved in the inflammatory response. Taken together, a link between Act-induced calcium release, regulation of downstream kinase cascades and Ref-1, and activation of NF-κB leading to PGE₂ and TNFα production was established. Since Act also caused extensive tissue damage, we showed that Act increased reactive oxygen species, and the antioxidant N-acetyl cysteine, blocked Act-induced PGE₂ and TNFα production, as well as NF-κB nuclear translocation in macrophages. We have demonstrated for the first time early cell signaling initiated in eukaryotic cells by Act, which leads to various biological effects associated with this toxin.