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Perturbation of mitochondrial structure and function plays a central role in Actinobacillus actinomycetemcomitans
J Korostoff1, N Yamaguchi, M Miller
1Department of Periodontics, Leon Levy Research Center for Oral Biology, University of Pennsylvania, 4010 Locust Street, Philadelphia, PA 19104-6002, USA. jkorosto@pobox.upenn.edu
Abstract:
Certain pore-forming bacterial toxins, including the leukotoxin (Ltx) produced by Actinobacillus actinomycetemcomitans, induce apoptosis in susceptible target cells. Although binding to the target cell surface represents the first step in the initiation of this process, the downstream events leading to toxin-induced apoptotic cell death have not been identified. Perturbation of mitochondrial function has been shown to have a major role in regulating progression of apoptosis initiated by exposure to numerous stimuli. Using Ltx as a model, the aim of this study was to evaluate whether induction of apoptosis by pore-forming toxins follows a similar paradigm. After exposure to Ltx, Epstein-Barr virus transformed B cells (JY cell line) exhibited the classical morphological features of apoptosis including decreased cell size, plasma membrane blebbing, selective alterations in plasma membrane permeability and condensation of nuclear DNA. The morphologic changes were accompanied by swelling of the mitochondria, a decrease in mitochondrial transmembrane potential (Psi(m)), hyperproduction of reactive oxygen intermediates (ROIs) and release of cytochrome c from the intermembrane space. Subsequently, we detected activation of the c ysteine asp artate-specific prote ases (caspases)-3 and -9, cleavage of the nuclear DNA repair enzyme, poly(ADP-ribose)polymerase (PARP) and internucleosomal DNA fragmentation. These results indicate that perturbation of mitochondrial structure and function, in concert with activation of specific caspases, initiate the effector phase of Ltx-induced apoptosis.
Insights
Pore-forming leukotoxin (Ltx) induces apoptosis by disrupting mitochondrial function. This leads to reactive oxygen intermediates, cytochrome c release, and caspase activation, culminating in cell death.
Area of Science:
- Cell Biology
- Microbiology
- Toxicology
Background:
- Pore-forming bacterial toxins, like leukotoxin (Ltx), trigger apoptosis in target cells.
- The precise downstream mechanisms of toxin-induced apoptosis remain unclear.
- Mitochondrial dysfunction is a known regulator of apoptosis from various stimuli.
Purpose of the Study:
- To investigate if pore-forming toxins induce apoptosis via mitochondrial perturbation.
- To elucidate the downstream signaling events in Ltx-induced apoptosis.
Main Methods:
- Exposure of Epstein-Barr virus transformed B cells (JY cell line) to Ltx.
- Morphological analysis of apoptosis.
- Assessment of mitochondrial transmembrane potential (Psi(m)).
- Measurement of reactive oxygen intermediates (ROIs) and cytochrome c release.
- Detection of caspase activation (caspases-3 and -9) and PARP cleavage.
Main Results:
- Ltx induced classical apoptotic morphology, including plasma membrane blebbing and nuclear DNA condensation.
- Mitochondrial swelling, decreased Psi(m), increased ROIs, and cytochrome c release were observed.
- Activation of caspases-3 and -9, PARP cleavage, and DNA fragmentation confirmed the apoptotic pathway.
Conclusions:
- Mitochondrial structure and function perturbation are key initiators of the effector phase in Ltx-induced apoptosis.
- Caspase activation plays a critical role in mediating Ltx-induced cell death.
- This study provides a detailed mechanism for pore-forming toxin-induced apoptosis.