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J. Biol. Chem., Vol. 278, Issue 41, 40198-40212, October 10, 2003

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Identification of Aeromonas hydrophila Cytotoxic Enterotoxin-induced Genes in Macrophages Using Microarrays^*

Cristi L. Galindo  , Jian Sha  ¶, Deborah A. Ribardo, Amin A. Fadl, Lakshmi Pillai and Ashok K. Chopra ||

From the Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, Texas 77555-1070

A cytotoxic enterotoxin (Act) of Aeromonas hydrophila possesses several biological activities, and it induces an inflammatory response in the host. In this study, we used microarrays to gain a global and molecular view of the cellular transcriptional responses to Act and to identify important genes up-regulated by this toxin. Total RNA was isolated at 0, 2, and 12 h from Act-treated macrophages and applied to Affymetrix MGU74 arrays, and the data were processed using a multi-analysis approach to identify genes that might be critical in the inflammatory process evoked by Act. Seventy-six genes were significantly and consistently up-regulated. Many of these genes were immune-related, and several were transcription factors, adhesion molecules, and cytokines. Additionally, we identified several apoptosis-associated genes that were significantly up-regulated in Act-treated macrophages. Act-induced apoptosis of macrophages was confirmed by annexin V staining and DNA laddering. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay were used to verify increased expression of some inflammatory and apoptosis-associated genes identified by the microarray analysis. To further confirm Act-induced increases in gene expression, real-time RT-PCR was also used for selected genes. Taken together, the array data provided for the first time a global view of Act-mediated signal transduction and clearly demonstrated an inflammatory response and apoptosis mediated by this toxin in host cells at the molecular level.

Received for publication, June 3, 2003 , and in revised form, June 20, 2003.

^* This work was supported by Grant AI41611 from NIAID, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Predoctoral fellow and recipient of a grant from the National Science Foundation.

These authors contributed equally to the paper.

^¶ Supported in part by a McLaughlin postdoctoral fellowship.

^|| To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Medical Research Bldg., 301 University Blvd., Galveston, TX 77555-1070. Tel.: 409-747-0578; Fax: 409-747-6869; E-mail: achopra{at}utmb.edu.

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