Morphological and intracellular alterations induced by Serratia marcescens cytotoxin.
In the present work, in vitro assays were used to investigate the toxicity of Serratia marcescens cytotoxin in cultured Chinese hamster ovary (CHO) cells. The time necessary to detect cellular alterations such as the onset of apoptosis, the perturbation of mitochondrial function, and cytoskeletal changes was assessed. The internalization of the cytotoxin by CHO cells was also examined. Within 10-15 min of exposure to cytotoxin, CHO cells became round, the nucleus shrank, the chromatin became more compact, and cytoplasmic blebs appeared on the cell surface. TUNEL (TdT-mediated dUTP nick end labeling) and propidium iodide staining identified some nuclei with fragmented DNA, and electrophoresis of CHO cell DNA obtained after 30-min exposure to S. marcescens toxin showed a pattern of DNA fragments typically associated with apoptosis. The cells also lost their characteristic actin organization within 10 min of exposure to cytotoxin. Lactate dehydrogenase leakage was detected after 20-min exposure to the cytotoxin and increased with time thereafter. Concomitantly, there was a time-dependent reduction in mitochondrial activity. Fluorescein-labeled S. marcescens cytotoxin was detected only on the surface of CHO cells, even after 30-min exposure to the toxin. These results show that there was no internalization of the toxin by CHO cells, and that, once bound to the cell surface, the toxin was able to induce changes in intracellular metabolism and to trigger cell death by apoptosis.
Carbonell
Gleize Villela
GV
Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), São Paulo, SP, Brazil. gleize@yahoo.com
Falcón
Rosabel
R
Yamada
Aureo T
AT
da Fonseca
Benedito Antonio Lopes
BA
Yano
Tomomasa
T
D000818:Animals; D017209:Apoptosis; D001427:Bacterial Toxins / Q000378:metabolism / Q000633:toxicity*; D016466:CHO Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000648:ultrastructure; D048429:Cell Size; D002843:Chromatin / Q000648:ultrastructure; D006224:Cricetinae; D003593:Cytoplasm / Q000648:ultrastructure; D003599:Cytoskeleton / Q000648:ultrastructure; D003603:Cytotoxins / Q000378:metabolism / Q000633:toxicity*; D053938:DNA Fragmentation; D005562:Formazans / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D011419:Propidium / Q000378:metabolism; D011485:Protein Binding; D012706:Serratia marcescens / Q000378:metabolism*; D013778:Tetrazolium Salts / Q000378:metabolism
D001427:Bacterial Toxins; D002843:Chromatin; D003603:Cytotoxins; D005562:Formazans; D013778:Tetrazolium Salts; C079782:MTT formazan; D011419:Propidium; D007770:L-Lactate Dehydrogenase