Article available online at http://www.idealibrary.com on Microbial Pathogenesis 2002; 32: 149–163 PATHOGENESIS MICROBIAL doi:10.1006/mpat.2001.0490 Early cell signaling by the cytotoxic enterotoxin of Aeromonas hydrophila in macrophages D. A. Ribardo, K. R. Kuhl, I. Boldogh, J. W. Peterson, C. W. Houston & A. K. Chopra* Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1070, U.S.A. (Received October 5, 2001; accepted in revised form January 2, 2002) 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. PGE 2 ) and tumor necrosis factor alpha (TNF) production was demonstrated in macrophages using a cell-permeable calcium chelator BAPTA-AM, which also down-regulated activation of transcription factor NF-B. We showed that Act’s capacity to increase PGE 2 and TNF 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-B leading to PGE 2 and TNF 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 PGE 2 and TNF production, as well as NF-B 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.  2002 Elsevier Science Ltd. All rights reserved Key words: Aeromonas hydrophila, cytotoxic enterotoxin, prostaglandin E 2 , tumor necrosis factor alpha, calcium mobilization, transcription factor NF-B, reactive oxygen species. are being recovered with increasing frequency Introduction from patients with traveler’s diarrhea [1, 2]. An increase in the immunocompromized popu- Aeromonas species (spp.) have been isolated from lation [3], and Aeromonas’ ability to complicate a wide variety of food and water sources and many soft tissue infections have increased the prevalence of this organism in hospital settings [4]. Aeromonas spp. produce a large number of * Author for correspondence. E-mail: achopra@utmb.edu 0882–4010/02/$-See front matter  2002 Elsevier Science Ltd. All rights reserved