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Published on: October 1, 2012
Morphological and intracellular alterations induced by Serratia marcescens cytotoxin
Gleize Villela Carbonell1, Rosabel Falcón, Aureo T Yamada
1Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), São Paulo, SP, Brazil. gleize@yahoo.com
Abstract:
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.
Insights
Serratia marcescens cytotoxin rapidly induces apoptosis in Chinese hamster ovary cells by binding to the cell surface. The toxin disrupts mitochondrial function and cytoskeletal organization, leading to cell death without internalization.
Area of Science:
- Microbiology
- Cell Biology
- Toxicology
Background:
- Serratia marcescens is a bacterium known to produce toxins.
- Understanding the mechanism of cytotoxin action is crucial for assessing its pathogenicity.
Purpose of the Study:
- To investigate the in vitro toxicity of Serratia marcescens cytotoxin on Chinese hamster ovary (CHO) cells.
- To determine the time course of cellular alterations, including apoptosis, mitochondrial dysfunction, and cytoskeletal changes.
- To examine the internalization process of the cytotoxin by CHO cells.
Main Methods:
- In vitro assays using cultured CHO cells.
- Microscopy to observe cellular morphology and cytoskeletal changes.
- TUNEL and propidium iodide staining for DNA fragmentation.
- DNA electrophoresis to detect apoptosis.
- Lactate dehydrogenase leakage assay for cell membrane integrity.
- Mitochondrial activity assays.
- Fluorescence microscopy to track toxin internalization.
Main Results:
- CHO cells exhibited rapid morphological changes (rounding, nuclear shrinkage, blebbing) within 10-15 minutes.
- Apoptosis was confirmed by DNA fragmentation (TUNEL, electrophoresis) after 30 minutes.
- Cytoskeletal actin organization was lost within 10 minutes.
- Mitochondrial activity decreased, and lactate dehydrogenase leakage increased over time.
- The cytotoxin was detected only on the cell surface, indicating no internalization.
Conclusions:
- Serratia marcescens cytotoxin induces rapid apoptosis in CHO cells.
- Toxin binding to the cell surface is sufficient to trigger intracellular metabolic changes and cell death.
- The cytotoxin does not appear to be internalized by CHO cells.
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