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• W2011854711 abstract "Article16 October 2000free access Targeting of the pro-apoptotic VDAC-like porin (PorB) of Neisseria gonorrhoeae to mitochondria of infected cells Anne Müller Anne Müller Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Dirk Günther Dirk Günther Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Volker Brinkmann Volker Brinkmann Central Support Unit, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Robert Hurwitz Robert Hurwitz Central Support Unit, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Thomas F. Meyer Corresponding Author Thomas F. Meyer Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Thomas Rudel Thomas Rudel Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Anne Müller Anne Müller Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Dirk Günther Dirk Günther Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Volker Brinkmann Volker Brinkmann Central Support Unit, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Robert Hurwitz Robert Hurwitz Central Support Unit, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Thomas F. Meyer Corresponding Author Thomas F. Meyer Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Thomas Rudel Thomas Rudel Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21–22, 10117 Berlin, Germany Search for more papers by this author Author Information Anne Müller1, Dirk Günther1, Volker Brinkmann2, Robert Hurwitz2, Thomas F. Meyer 1 and Thomas Rudel1 1Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstrasse 21–22, 10117 Berlin, Germany 2Central Support Unit, Schumannstrasse 21–22, 10117 Berlin, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:5332-5343https://doi.org/10.1093/emboj/19.20.5332 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Infection of cell cultures with Neisseria gonorrhoeae results in apoptosis that is mediated by the PorB porin. During the infection process porin translocates from the outer bacterial membrane into host cell membranes where its channel activity is regulated by nucleotide binding and voltage-dependent gating, features that are shared by the mitochondrial voltage-dependent anion channel (VDAC). Here we show that porin is selectively and efficiently transported to mitochondria of infected cells. Prevention of porin translocation also blocked the induction of apoptosis. Mitochondria of cells treated with porin both in vitro and in vivo were depleted of cytochrome c and underwent permeability transition. Overexpression of Bcl-2 blocked porin-induced apoptosis. The release of cytochrome c occurred independently of active caspases but was completely prevented by Bcl-2. Our data suggest that the Neisseria porin can, like its eukaryotic homologue, function at the mitochondrial checkpoint to mediate apoptosis. Introduction Apoptosis is a special form of programmed cell death that plays a pivotal role during developmental morphogenesis and cell homeostasis. The basic mechanism of apoptosis regulation is evolutionarily conserved from nematodes to man; in fact, some major lessons were learned by studies of apoptosis in the nematode Caenorhabditis elegans (Metzstein et al., 1998). The major executioners of apoptosis are the caspases (cysteinyl aspartate-directed proteases), which comprise a family of 15 members exerting different functions in inflammation and apoptosis (Nicholson and Thornberry, 1997; Salvesen and Dixit, 1997). The pathways by which caspases are activated have been pinned down to two major branches: one is initiated by cell surface receptors, the other by mitochondria. Ligation of receptors of the tumour necrosis factor (TNF) receptor family (Wallach et al., 1997) results in the recruitment and activation of initiator caspases, e.g. caspase-8 (Boldin et al., 1996; Muzio et al., 1996). Active caspase-8 is able to process and activate other caspases, which initiate apoptosis by cleavage of cellular substrates (Salvesen and Dixit, 1997). Substrates include the DNA-repair enzyme poly(ADP-ribose) polymerase (PARP) (Tewari et al., 1995) and the cytoskeleton-associated protein fodrin (Cryns et al., 1996; Janicke et al., 1998), both of which are cleaved by caspase-3. For many of the pathways induced by diverse stimuli such as cytotoxic or environmental stress or toxic cell metabolites, the mitochondria appear to be the main integrators of apoptotic signalling. They respond to these stimuli with the release of caspases-2 and -9 (Susin et al., 1999a) or caspase-activating proteins like cytochrome c (Liu et al., 1996) and apoptosis initiator factor (AIF) (Susin et al., 1999b). Cytochrome c is required as cofactor in a complex with the adapter molecule Apaf-1 and dATP for caspase-9 activation (Zou et al., 1997). Like caspase-8, active caspase-9 cleaves and activates effector caspases that coordinate the execution phase of the death programme (Slee et al., 1999). Thus, although the initial phase of receptor-mediated and mitochondria-mediated apoptotic pathways appears to be rather different, a similar set of caspases is eventually activated and executes the death programme. Although the major molecular effects of apoptotic activation of mitochondria have been elucidated, the trigger initiating the release of cytochrome c and other factors is still not well understood. Many investigators find a collapse of the mitochondrial inner membrane potential (Δψm) during apoptosis, indicating the opening of a large conductance channel generally known as the permeability transition (PT) pore complex (Zoratti and Szabo, 1995; Green and Reed, 1998). The complex involved in PT regulation consists of several factors including the mitochondrial porin, also known as voltage-dependent anion channel (VDAC) (Zoratti and Szabo, 1995; Beutner et al., 1998), the adenine nucleotide translocator (ANT) (Brustovetsky and Klingenberg, 1996), the peripheral benzodiazepine receptor (Pastorino et al., 1994) and probably kinases like hexokinase and/or creatine kinase (Beutner et al., 1998). Interestingly, PT pore opening is induced by several pro-apoptotic second messengers like Ca2+, pro-oxidants, nitric oxide, ceramide and caspases (Zamzami et al., 1995; Bernardi and Petronilli, 1996; Marzo et al., 1998a), suggesting a direct involvement of PT pore opening in apoptosis induction. Moreover, it is regulated by the anti-apoptotic members of the Bcl-2 family, Bcl-2 and Bcl-XL, which stabilize mitochondrial membranes (Decaudin et al., 1997; Kroemer, 1997a) and by the pro-apoptotic member, Bax, which induces PT (Xiang et al., 1996). Recently, we demonstrated the induction of apoptosis during the infection of epithelial cells and phagocytes by Neisseria gonorrhoeae (Ngo), a human-specific bacterial pathogen (Müller et al., 1999). Gonococci attach to their target cells via pili, hair-like protein appendages (Swanson et al., 1987) or by Opa proteins, which in addition to adhesion induce the receptor-mediated uptake by the host cell (Makino et al., 1991; Gray-Owen et al., 1997; Hauck et al., 1998). Although adhesion or invasion is a prerequisite, neither pili nor Opa proteins, but the PorB porin, induces apoptosis of target cells, when added in its purified form (Müller et al., 1999). Porins form integral diffusion channels in the outer membrane of Gram-negative bacteria. Some porins contain binding sites for certain substrates found in the environment of these bacteria (Benz, 1995). The binding of nucleotides by porin is unusual in this respect (Rudel et al., 1996) since nucleotides are not a natural substrate for Neisseria. However, during an infection porin translocates into the membrane of target cells (Weel and van Putten, 1991), where its channel activity is tightly regulated by cytosolic nucleotides that increase the voltage-dependent gating of the porin channel (Rudel et al., 1996). The properties of PorB such as voltage dependence and nucleotide binding closely resemble mitochondrial VDAC functions (Benz, 1994) and are not found in other porins of Gram-negative bacteria. Since PorB exhibits similar properties to mitochondrial VDACs and since both are involved in apoptosis induction, we investigated the influence of PorB on mitochondrial function during apoptosis induction. Here we show that infection or PorB treatment causes loss of membrane potential and the release of cytochrome c from mitochondria of intact cells. Surprisingly, PorB also induces the release of cytochrome c from purified mitochondria. The release of cytochrome c as well as PT and the induction of apoptosis is blocked by Bcl-2 in intact cells. Furthermore, in porin-treated cells as well as in infected cells PorB is specifically and efficiently targeted to the mitochondria. Results Porin induces structural and biochemical changes of mitochondria that are typical of apoptotic cells When epithelial or immune cells are treated with porin purified from N.gonorrhoeae they undergo apoptosis (Müller et al., 1999). The underlying mechanism involves release of Ca2+ into the cytosol and the activation of proteases of the caspase and calpain families. Since mitochondria play a central integrative role in the regulation of apoptosis (reviewed by Kroemer et al., 1998) we investigated the effects that porin treatment has on the structural and biochemical integrity of mitochondria (Figure 1). Jurkat cells were treated with 7 μg/ml porin for 15 h, the mitochondria were isolated and the cytochrome c content was determined by western blot analysis. Mitochondria of untreated and buffer-treated cells (Figure 1A) contained the same amount of cytochrome c while mitochondria of porin-treated cells had lost all their cytochrome c (Figure 1A). To ensure equal loading of proteins the western blots were also developed with an antibody directed against cytochrome c oxidase. The same result was obtained with mitochondria isolated from porin-treated monocytic and epithelial cell lines (not shown). Redistribution of cytochrome c was also analysed microscopically by double staining Jurkat cells with an antibody directed against native cytochrome c and MitoTracker, a potential-sensitive dye specific for mitochondria (Figure 2). In control or buffer-treated cells both dyes colocalized completely, indicating an intact mitochondrial membrane potential and a cytochrome c distribution typical of healthy cells (Figure 2, vector control and vector + buffer). In contrast, upon porin treatment a large population of cells (80–90%) no longer stained with MitoTracker and in addition had lost the granular staining of cytochrome c (Figure 2, vector + porin). These cells also showed clear signs of apoptosis, i.e. apoptotic body formation, condensation of the cytoplasm and cell shrinkage. Similarly, infection of HeLa cells with N.gonorrhoeae strain N242 also resulted in PT, the release of cytochrome c from mitochondria and the typical morphological alterations of apoptotic cells (Figure 3). This occurred in ∼40–50% of the population. Figure 1.Effects of porin on the release of cytochrome c and PT of mitochondria in vivo. (A) Mitochondria were isolated from Jurkat T cells treated with either 7 μg/ml purified porin or an equal volume of porin purification buffer for 15 h. Mitochondrial lysates were subjected to SDS–PAGE followed by western blotting using monoclonal antibodies against human cytochrome c oxidase subunit II (Cyto C Ox) and denatured human cytochrome c (Cyto C), respectively. (B) Jurkat T cells were treated with porin or an equal volume of porin purification buffer for 15 h in the presence and absence of 1 mM Ca2+ as indicated, stained with rhodamine 123 for 30 min at 37°C and analysed by flow cytometry. The histogram shows the analysis of 10 000 cells per sample. Download figure Download PowerPoint Figure 2.Bcl-2 overexpression and caspase inhibition block porin-induced apoptosis by different mechanisms. Jurkat T cells either expressing Bcl-2 or carrying an empty vector were treated with 7 μg/ml porin or purification buffer for 15 h or left untreated. They were then subjected to immunocytochemistry using an anti-cytochrome c-specific antibody and an Alexa-488-coupled secondary antibody. Cells were stained with MitoTracker before fixation. A phase contrast image, single colours and overlays are shown for every section. Cells in the lowest panel were treated with 50 μM zVAD-fmk (Bachem) for 1 h before addition of porin. Download figure Download PowerPoint Figure 3.Cytochrome c release and PT also occurs in HeLa cells infected with N.gonorrhoeae. HeLa cells were infected with gonococcal strain N242 for 15 h at an m.o.i. of 1. Cells were then subjected to immunocytochemistry using an anti-cytochrome c-specific antibody and an Alexa-488-coupled secondary antibody. MitoTracker staining was performed before fixation. A phase contrast image, single colours and overlays are shown for infected cells and uninfected control cells. Note the swollen mitochondria in infected, non-apoptotic cells. Loss of or diffuse cytochrome c staining in apoptotic cells is indicative of cytochrome c release. Download figure Download PowerPoint We confirmed the loss of the mitochondrial membrane potential after porin treatment by staining live cells with the potential-sensitive dye rhodamine 123 for flow-cytometric analysis (Figure 1B). This dye is reportedly only incorporated into mitochondria with intact membrane potential (Van der Heiden et al., 1997). Cells that were treated with porin for 15 h showed a shift towards lower intensity (Figure 1B), the cells have therefore undergone PT. Since porin provokes a rapid influx of extracellular Ca2+ in treated cells (Müller et al., 1999) and Ca2+ alone is sufficient to induce PT in other systems in vitro (Marzo et al., 1998a), we tested whether Ca2+ is required for PT induced by porin. PT occurred with the same kinetics in cells treated with porin in Ca2+-free medium compared with the control with Ca2+ (Figure 1B), thus excluding a direct effect of Ca2+ on porin-induced PT. Porin induces cytochrome c release and PT in purified mitochondria Isolated mitochondria have been used previously to demonstrate apoptogenic effects of purified proteins such as Bax on cytochrome c content and membrane potential (Jürgensmeier et al., 1998). In the case of porin-induced apoptosis it is especially relevant to investigate the effects of the porin on isolated mitochondria because of its similarities to mitochondrial VDAC, which is known to participate in PT and cytochrome c release (Beutner et al., 1998; Shimizu et al., 1999). Purified mitochondria from 1 × 107 Jurkat T cells were treated with 2 μg of porin and cytochrome c release was monitored by western blotting (Figure 4A). Mitochondria treated with porin were completely depleted of cytochrome c, whereas addition of porin purification buffer alone did not trigger this release. Interestingly, amounts of porin (0.5 μg) that were insufficient to induce a cytochrome c release reproducibly had an opposite effect: the mitochondria were protected from the spontaneous loss of cytochrome c that is otherwise observed after extended incubation at 37°C (not shown). We also looked for PT as a putative response of isolated mitochondria to porin treatment. For this purpose, the mitochondria were stained with rhodamine 123 and fluorescence intensity was monitored by flow cytometry (Figure 4B). Indeed, a clear shift to lower intensity is observed in mitochondria treated with porin as compared with the control, which is indicative of loss of membrane potential. Also, purified mitochondria treated with porin increased in volume compared with an untreated control, which was monitored in a swelling assay over time (Figure 4C). These data therefore provide clear evidence that neisserial porin alone is sufficient for eliciting an in vitro mitochondrial response similar to that typically observed in the course of apoptosis in vivo. Figure 4.Effects of porin on the release of cytochrome c, PT and swelling of mitochondria in vitro. (A) Mitochondria were isolated from 5 × 107 Jurkat cells. Porin (2 μg) or an equal volume of porin purification buffer was applied for 30 min at 37°C in vitro. The pellet (P) and the supernatant (SN) of every sample were then analysed by western blotting using monoclonal antibodies against human cytochrome c oxidase subunit II (Cyto C Ox) and denatured human cytochrome c (Cyto C), respectively. (B) Mitochondria obtained as described in (A) were stained with rhodamine 123 for 30 min at 37°C and analysed by flow cytometry. (C) Mitochondria were purified from mouse liver and subjected to a swelling assay as described in Materials and methods. The increase in mitochondrial volume was monitored by determining the optical density at 600 nm. The increase in volume after addition of the stimulus is visible as a decline in the OD600 over 10 min. Note that the untreated mitochondria swell spontaneously, a process that is not increased by low amounts of porin. However, higher concentrations of porin lead to an increase in the volume of mitochondria similar to treatment of the mitochondria with Ca2+, a well known inducer of mitochondrial swelling. Download figure Download PowerPoint Overexpression of Bcl-2 or Bcl-XL blocks porin-induced apoptosis Bcl-2 and Bcl-XL are anti-apoptotic members of the Bcl family. They are localized to organelle membranes as a result of their C-terminal membrane anchor (Yang and Korsmeyer, 1996) and Bcl-XL in addition is able to form ion channels in synthetic lipid membranes (Antonsson et al., 1997; Minn et al., 1997). Overexpression of these proteins has repeatedly been shown to affect all the apoptotic phenotypes seen at the mitochondrial level, yet it remains unclear if this is due to a direct function or if it is a consequence of inhibition of an earlier step in the death pathway (Zamzami et al., 1996; Kluck et al., 1997). Jurkat T cells stably expressing Bcl-2 and the control cell line were treated with porin for 15 h. Control transfected Jurkat cells showed clear morphological signs of apoptosis including cell shrinkage and extensive apoptotic body formation (Figure 2, phase contrast). These features were completely abolished by overexpression of Bcl-2. In order to investigate whether the observed protection from apoptosis induction was due to an inhibition of cytochrome c release by Bcl-2, the cells were subjected to immunocytochemistry using an anti-cytochrome c antibody and additional staining of the mitochondria by MitoTracker (Figure 2). While the control cell line completely released cytochrome c upon porin treatment and had undergone PT, Jurkat–Bcl cells retained cytochrome c and membrane potential just like the untreated control (Figure 2). These results were confirmed with two additional cell lines, CEM-Bcl-XL and SKW6-Bcl-2, which both stably expressed anti-apoptotic Bcl proteins (data not shown). Caspases are known inducers of cytochrome c release and PT (Marzo et al., 1998b) and are known to be activated during porin-induced apoptosis (Müller et al., 1999). We therefore assessed their role in porin-mediated mitochondrial damage. Interestingly, the inhibition of caspases by the broad range caspase inhibitor zVAD-fmk blocked all described morphological alterations but could not prevent the release of cytochrome c and PT in treated cells (Figure 2, zVAD + porin). In order to quantify the anti-apoptotic effect of the Bcl proteins (Figure 5A, left panels) we measured phosphatidylserine (PS) exposure. The majority (>90%) of vector-transfected Jurkat cells treated with porin for 15 h bound annexin V, which is indicative of PS exposure on their surface (upper left panel), whereas no increase in the annexin V-positive population was observed with Jurkat–Bcl (lower left panel). These results were confirmed by quantifying DNA degradation via propidium iodide incorporation (not shown). Figure 5.Bcl-2 overexpression inhibits porin-induced PT, caspase activation and apoptosis. (A) Jurkat cells either expressing Bcl-2 or carrying an empty vector were treated with 7 μg/ml porin (as indicated in the histograms) for 15 h and split: one half was subjected to an annexin V binding protocol in order to determine PS exposure, the other was stained with rhodamine 123 to assess mitochondrial membrane potential. Both samples were analysed by flow cytometry. (B) Aliquots of the samples in (A) were lysed and subjected to western blotting for assessment of caspase activation. Cleavage of full-length fodrin (240 kDa) into signature fragments of 120 and 150 kDa was monitored by using a monoclonal antibody against fodrin that recognizes all three forms. Download figure Download PowerPoint When the same cells were monitored for mitochondrial membrane potential by staining with rhodamine 123, no porin-induced shift towards lower fluorescence intensity was observed in cells expressing Bcl-2, indicating that PT did not occur in these cells (Figure 5A, right panels) and thereby confirming the conclusions drawn from microscopic analysis. Downstream caspases were not active in Jurkat–Bcl-2, as judged by the absence of substrate cleavage of PARP (data not shown) and fodrin (Figure 5B). Interestingly, whereas the generation of the 120 kDa caspase signature fragment of fodrin (Nath et al., 1996) was completely blocked, the 150 kDa calpain fragment did appear, indicating that calpains are active in the presence of overexpressed Bcl-2, but their activity is not sufficient to drive the cells into apoptosis. Porin is targeted to the mitochondria Due to the similarities between eukaryotic VDAC and neisserial porin it seemed a promising hypothesis that porin is targeted to the mitochondria where it then exerts its cytotoxic effects directly by inducing PT and cytochrome c release. When isolated mitochondria were treated with porin in vitro, the vast majority of the porin amount applied was detected in the mitochondrial pellet, whereas hardly any porin was found in the supernatant (not shown). Thus, porin associated with mitochondria in vitro. We then addressed the question of whether porin is localized to mitochondria of porin-treated cells. Cells were either treated with porin purification buffer or with porin for 15 h and washed extensively before preparation of whole cell lysates and mitochondria. We also treated cells for the same period of time with purified Staphylococcus aureus α-toxin which, like porin, consists of amphipathic β-barrels and induces apoptosis by forming pores in the cytoplasmic membrane of the cells (Jonas et al., 1994; Song et al., 1996). Purified gonococcal PilC adhesin, which binds to several human cell lines, served as additional control. Porin, as well as PilC and α-toxin, were present in whole cell lysates, indicating their insertion into or tight attachment to membranes (Figure 6A and B). However, large amounts of porin purified with the mitochondrial fraction whereas PilC and α-toxin were always absent from this compartment. Pro-cathepsin D (Figure 6A) and transferrin receptor (not shown) did not copurify with the mitochondrial compartment, suggesting the absence of endosomal and cytoplasmic membranes. To test whether porin is also transported to mitochondria during infection with N.gonorrhoeae, HeLa cells were infected with either the adherent and invasive Opa-expressing strain N242 (Figure 6C) or the adherent, piliated strain N138 (Figure 6D), both of which induce apoptosis in HeLa cells (Müller et al., 1999). Highly purified mitochondria from these cells contain large amounts of porin protein. Indeed, porin was the only neisserial antigen detectable in mitochondria from infected cells. Neither Opa outer membrane proteins (Figure 6C) nor PilC (Figure 6D) were found in the same mitochondrial preparations, suggesting a selective transport of porin to the mitochondria of infected cells. Porin was not present in mitochondria of HeLa cells infected with non-adherent gonococcal strains, e.g. N898 (Figure 6D) and commensal Neisseria spp. (not shown), which are unable to induce apoptosis. Confocal immunomicroscopy of porin-treated or infected cells using purified antiserum against porin revealed the presence of porin in mitochondria that appeared extremely enlarged (Figure 7). In summary, porin is efficiently and selectively transported to the mitochondria of porin-treated or infected cells undergoing apoptosis. Figure 6.Porin is targeted to the mitochondria of porin-treated and infected cells. (A) HeLa cells were treated with 7 μg/ml porin or respective amounts of staphylococcal α-toxin for 15 h. Lysates were prepared from an aliquot of every sample. In parallel, mitochondria were prepared and both cell lysates and mitochondrial preparations were subjected to western blotting using specific antibodies against porin, α-toxin, cathepsin D and cytochrome c oxidase. Detection was performed using the ECL system (Amersham) according to the manufacturer's instructions. (B) HeLa cells were treated with 5 μg/ml purified PilC for 15 h. Cell lysates and mitochondria were prepared from treated and control cells and were subjected to western blotting using specific antibodies against PilC and cytochrome c oxidase. Detection was performed as described above. (C) HeLa cells were infected with gonococcal strain N242 for 15 h at an m.o.i. of 1. Lysates and mitochondria were prepared from infected and non-infected control cells and subjected to western blotting using specific antibodies against Opa proteins, porin, cathepsin D and cytochrome c oxidase. Detection was performed as described above. (D) HeLa cells were infected with piliated (N138) and non-piliated (N898), isogenic derivatives of gonococcal strain MS11 for 15 h at an m.o.i. of 1. Lysates and mitochondria were prepared from infected and non-infected control cells and subjected to western blotting using specific antibodies against PilC, porin and cathepsin D. Detection was performed as described above. L, lysate; M, mitochondria. Download figure Download PowerPoint Figure 7.Porin is targeted to the mitochondria of porin-treated and infected cells. HeLa cells were infected with the non-piliated, Opa-positive gonococcal strain VP1 (N242) for 3 h at an m.o.i. of 1 or treated with 7 μg/ml porin for the same time. Infected, treated and control cells were double stained with MitoTracker and a polyclonal, porin-specific antiserum followed by an Alexa-488-labelled secondary antibody. Single fluorescence pictures and overlays are shown. White arrows point to bacteria, blue arrows to mitochondria. Download figure Download PowerPoint Interference with porin translocation prevents apoptosis We noticed that induction of apoptosis by porin is highly sensitive to serum. In the presence of 10% heat-inactivated fetal calf serum (FCS) the strong pro-apoptotic effect of porin was completely blocked (Figure 8A). The inhibitory effect of serum was not due to degradation since the overall amount of porin was similar during incubation in the absence or presence of serum (data not shown). However, in the presence of serum, porin was no longer present in the cell lysate and therefore was also absent from mitochondria prepared from treated cells (Figure 8B). Thus, translocation of porin is an absolute prerequisite for its pro-apoptotic effect. Figure 8.FCS blocks apoptosis and porin insertion. (A) HeLa cells were treated with 7 μg/ml porin for 15 h in the presence or absence of 10% FCS. Cells were harvested and stained with annexin V. (B) HeLa cells were treated with porin for 15 h in the presence or absence of 10% FCS. Lysates and mitochondria were prepared and subjected to western blotting using a specific antibody against gonococcal porin variant P.IA. Download figure Download PowerPoint Discussion It has become increasingly apparent that mitochondria participate in the regulation of those forms of apoptotic cell death that do not involve activation of upstream caspases via signalling cascades triggered by death receptor ligation (Scaffidi et al., 1998; Yoshida et al., 1998). However, the significance of single events observed at the mitochondrial level for the overall regulatory process and especially the exact sequence of events is still controversially debated (for a review see Green and Reed, 1998). Here we describe a new mechanism of mitochondria-dependent apoptosis induction occurring naturally during infection of human cells by N.gonorrhoeae. As in most other complex apoptosis scenarios, cells either infected with N.gonorrhoeae or treated with neisserial porin display a release of cytochrome c in" @default.
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• W2011854711 title "Targeting of the pro-apoptotic VDAC-like porin (PorB) of Neisseria gonorrhoeae to mitochondria of infected cells" @default.
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