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Potential involvement of galectin-3 and SNAP23 in Aeromonas hydrophila cytotoxic enterotoxin-induced host cell apoptosis
- Source :
- Microbial Pathogenesis. 40:56-68
- Publication Year :
- 2006
- Publisher :
- Elsevier BV, 2006.
-
Abstract
- We investigated the potential of the cytotoxic enterotoxin (Act) of Aeromonas hydrophila to bind to 1869 human and 4319 yeast proteins, using protein microarray technology. Act was capable of binding nine different human proteins, including the SNARE complex scaffolding protein synaptosomal-associated protein 23 (SNAP23), galectin-3, and guanylate kinase 1 (GUK-1). Act was also able to bind to four of the yeast proteins examined, which included the vesicle tethering protein Vsp52. We verified interaction of Act with murine and human SNAP23, galectin-3, and GUK-1 by sandwich Western blot analysis. In order to determine the physiological relevance of Act binding to these three proteins, we performed small interfering RNA (siRNA) gene knockdown experiments in RAW 264.7 cells, a murine macrophage cell line in which Act-induced signaling and cell death is well characterized. Based on real-time reverse transcriptase-polymerase chain reaction, siRNA transfection of RAW 264.7 cells with specific oligonucleotides reduced the expression of genes encoding SNAP23, galectin-3, and GUK-1 by 62, 63, and 99%, respectively. Knockdown of galectin-3 and SNAP23, but not GUK-1, significantly reduced Act-induced apoptosis of host cells, as determined by TUNEL (TdT-mediated dUTP nick end labeling) assay, lactate dehydrogenase release, Giemsa staining, and reduction in activation of caspase 3, compared to toxin-treated macrophages that were transfected with a random sequence control siRNA. We also performed these assays using a human intestinal epithelial cell line (HT-29) and observed a similar trend of galectin-3 and SNAP23 association with Act-induced apoptosis. This is the first report of putative protein binding partners for this toxin and potential mediators/regulators of Act-induced apoptosis.
- Subjects :
- Programmed cell death
Small interfering RNA
Galectin 3
Blotting, Western
Protein Array Analysis
Apoptosis
Plasma protein binding
Biology
Microbiology
Vesicle tethering
Cell Line
Enterotoxins
Mice
Bacterial Proteins
Western blot
medicine
Animals
Humans
Qc-SNARE Proteins
RNA, Small Interfering
L-Lactate Dehydrogenase
medicine.diagnostic_test
Caspase 3
Macrophages
Transfection
Qb-SNARE Proteins
Immunohistochemistry
Molecular biology
Aeromonas hydrophila
Blot
Infectious Diseases
Cell culture
Caspases
Guanylate Kinases
HT29 Cells
Protein Binding
Subjects
Details
- ISSN :
- 08824010
- Volume :
- 40
- Database :
- OpenAIRE
- Journal :
- Microbial Pathogenesis
- Accession number :
- edsair.doi.dedup.....8468cae9963604ba3d814d6c1b7aaab8