Microarray and proteomics analyses of human intestinal epithelial cells treated with the Aeromonas hydrophila cytotoxic enterotoxin

C. L. Galindo, A. A. Fadl, Jian Sha, L. Pillai, C. Gutierrez, Ashok Chopra

Research output: Contribution to journalArticle

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Abstract

We performed microarray analyses on RNA from human intestinal epithelial (HT-29) cells treated with the cytotoxic enterotoxin (Act) of Aeromonas hydrophila to examine global cellular transcriptional responses. Based on three independent experiments, Act upregulated the expression of 34 genes involved in cell growth, adhesion, signaling, immune responses (including interleukin-8 [IL-8] production), and apoptosis. We verified the upregulation of 14 genes by real-time reverse transcriptase-PCR and confirmed Act-induced production of IL-8 by enzyme-linked immunosorbent assay on supernatants from nonpolarized and polarized HT-29 cells. Maximal production of IL-8 in response to Act required the presence of intracellular calcium, since chelation of calcium with BAPTA-AM significantly reduced Act-induced IL-8 production in HT-29 cells. We also examined activation of mitogen-activated protein kinases and, as demonstrated by Western blot analysis of apical side-treated polarized HT-29 cells, Act induced phosphorylation of p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase 1/2. In addition, KinetWorks proteomics screening of whole-cell lysates revealed Act-induced phosphorylation of cyclic AMP-response element binding protein (CREB), c-Jun, adducin, protein kinase C, and signal transducer and activator of transcription 3 (STAT3) and decreased phosphorylation of protein kinase Bα, v-raf-1 murine leukemia viral oncogene homolog 1 (i.e., Raf1), and STAT1. We verified activation of CREB and activator protein 1 in polarized cells by gel shift assay. This is the first description of human intestinal epithelial cell transcriptional alterations, phosphorylation or activation of signaling molecules, cytokine production, and calcium mobilization in response to this toxin.

Original languageEnglish (US)
Pages (from-to)2628-2643
Number of pages16
JournalInfection and Immunity
Volume73
Issue number5
DOIs
StatePublished - May 2005

Fingerprint

Aeromonas hydrophila
HT29 Cells
Enterotoxins
Microarray Analysis
Interleukin-8
Proteomics
Epithelial Cells
Phosphorylation
Cyclic AMP Response Element-Binding Protein
Calcium
Proto-Oncogene Proteins c-jun
STAT3 Transcription Factor
Proto-Oncogene Proteins c-akt
Mitogen-Activated Protein Kinase 3
JNK Mitogen-Activated Protein Kinases
Mitogen-Activated Protein Kinase 1
Transcription Factor AP-1
Mitogen-Activated Protein Kinases
Reverse Transcriptase Polymerase Chain Reaction
Oncogenes

ASJC Scopus subject areas

  • Immunology

Cite this

Microarray and proteomics analyses of human intestinal epithelial cells treated with the Aeromonas hydrophila cytotoxic enterotoxin. / Galindo, C. L.; Fadl, A. A.; Sha, Jian; Pillai, L.; Gutierrez, C.; Chopra, Ashok.

In: Infection and Immunity, Vol. 73, No. 5, 05.2005, p. 2628-2643.

Research output: Contribution to journalArticle

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