Potential involvement of galectin-3 and SNAP23 in Aeromonas hydrophila cytotoxic enterotoxin-induced host cell apoptosis.
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.
Galindo
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Department of Microbiology and Immunology, The University of Texas Medical Branch, Medical Research Building, 301 University Boulevard, Galveston, Texas 77555-1070, USA.
Gutierrez
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C
Chopra
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D016980:Aeromonas hydrophila / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D053148:Caspase 3; D020169:Caspases / Q000032:analysis; D002460:Cell Line; D004768:Enterotoxins / Q000302:isolation & purification / Q000378:metabolism*; D037502:Galectin 3 / Q000378:metabolism*; D051528:Guanylate Kinases / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008264:Macrophages / Q000166:cytology / Q000378:metabolism; D051379:Mice; D040081:Protein Array Analysis / Q000379:methods; D011485:Protein Binding; D050766:Qb-SNARE Proteins / Q000378:metabolism*; D050767:Qc-SNARE Proteins / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism
D001426:Bacterial Proteins; D004768:Enterotoxins; D037502:Galectin 3; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D034741:RNA, Small Interfering; C100493:SNAP23 protein, human; C082174:cytolytic enterotoxin protein, Aeromonas; D007770:L-Lactate Dehydrogenase; D051528:Guanylate Kinases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases