Calore, Federica (2008) Caratterizzazione dei meccanismi molecolari coinvolti nell'apoptosi indotta da VacA, tossina prodotta da Helicobacter pylori. [Tesi di dottorato]
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The attested association of Helicobacter pylori to gastro-duodenal pathologies such as chronic gastritis, peptic ulcer and gastric carcinoma has always been of great interest. The bacterium produces several virulence factors: the vacuolating cytotoxin (known as VacA) is definitely one of the most interesting ones, for the multiplicity of the effects on mammalian cells. For this reason VacA has been defined a multifunctional toxin [D’Elios et al., 2007; Cover et Blanke, 2005].
VacA is secreted by the bacterium as a mature protein of 95 kDa, and it can furher be cleaved in the N-terminal domain (called p37), and a C-terminal domain (called p58) which can remain non covalently linked. Secreted toxin is a flower-shaped oligomer, formed by 6 or 7 monomers, which can insert into lipid bilayers forming voltage-dependent anion channels [Tombola et al., 1999]. Several properties of VacA are strictly dependent on the formation of these channels: cellular vacuolation [Willhite et al., 2003], a rapid drop of the trans-epithelial resistance in epithelial monolayers [Papini et al., 1998], and finally apoptosis in epithelial cells [Willhite et Blanke, 2004]. In the last few years several efforts have been devoted to the characterization of the latter. It was reported that VacA intoxication is responsible for the decrease in mitochondrial membrane potential [Kimura et al., 1999] and cytochrome c release from the organelle [Galmiche et al., 2000]. Mitochondria have been reported to be the intracellular destination of VacA, as assessed when the toxin or its p37 domain were intracellularly expressed through transient transfection [Galmiche et al., 2000] and also when the toxin was extracellularly applied [Willhite et Blanke, 2004]. Mutant forms of VacA, lacking channel activity (VacA P9A, G14A) are incapable of inducing either the decrease of the mitochondrial trans-membrane potential, or cytochrome c release [McClain et al., 2003; Willhite et Blanke, 2003 and 2004].
In 2006, Yamasaki et al. showed that VacA-induced cell death and cytochrome c release depend on the activation of pro-apoptotic proteins Bax and Bak.
However, the molecular mechanisms that regulate VacA-induced apoptosis and by which mechanism VacA is targeted to mitochondria remained two unanswered questions.
In order to address these questions we first performed cell viability experiments and verified the involvement of pro-apoptotic proteins BAX and BAK in our cell model (Mouse Embryonic Fibroblasts, MEFs). Considering also that endoplasmic reticulum Ca2+ level is crucial to control the fate of cells in response to certain apoptotic stimuli and following sensitization of mitochondria [Breckenridge et al., 2003], we verified whether VacA-induced cell death could primarily involve the reticulum or mitochondria.
As the apoptogenic role of VacA depends on its channel activity, we first performed cell death analyses inducing the intracellular expression of the full length toxin (p95), or its p37 domain where resides the membrane-channel forming ability. We also performed the same experiments with the toxin extracellularly applied: MEFs were then incubated with H. pylori supernatant containing the wt toxin, and with the supernatant containing a mutant form of the toxin, VacA P9A, which was unable to form any channel.
It is known that after being endocytosed VacA localizes to late endosomes [Molinari et al., 1997], where vacuoles arise. It is not known the mechanism which allows VacA to further target mitochondria. We analyzed, though immunofluorescence and subcellular fractionation assays, the pathway followed by the toxin inside the cell, from its endocytosis to cell death.
Here we report that BAX and BAK proteins are required for VacA-induced cell death; this process involves only mitochondria, and not the endoplasmic reticulum so it is Ca2+ independent. The role of BAX is crucial: it is recruited by VacA on early endosomes and activated by the toxin. Its following translocation to mitochondria, and VacA targeting to the organelle, seem to be the consequence of the cell trafficking alteration induced by the toxin. This process is characterized by the migration of endosomal membranes to the mitochondrial ones, and maybe by their fusion.
All the effects so far described depend on the channel activity of VacA: it is not responsible of the release of apoptogenic factors from mitochondria, but also of all the events that precede this process.
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