Mitochondrial permeability transition and cytochrome c release induced by selenite

Cytochrome c release and mitochondrial permeability transition (MPT) play important roles in apoptosis. In this study, we found that selenium, an essential trace element, induced mitochondrial membrane potential (Delta psi(m)) loss, swelling, and cytochrome c release in isolated mitochondria. All of the above observations were blocked by cyclosporin A (CsA), which is a specific inhibitor to permeability transition pore (PTP), indicating selenite-induced mitochondrial changes were mediated through the opening of PTP. In physiological concentration, selenite could induce mitochondria at low-conductance PTP 'open' probability, which is correlated to regulate the physiological function, whereas in toxic concentration, induce mitochondria at high-conductance PTP 'open' probability and rapidly undergo a process of osmotic swelling following diffusion toward matrix as for inducer (Ca(2+)/P(i)). Selenite also induced other mitochondrial marker enzymes including monoamine oxidase (MAO) and mitochondria aspartate aminotransferase (mAST). Oligomycin inhibited the selenite-induced cytochrome c release and Delta psi(m) loss, showing that F(0)F(1)-ATPase was important in selenite or Ca(2+)/P(i)-induced MPT.     

Ca(2+)-induced high amplitude swelling and cytochrome c release from wheat (Triticum aestivum L.) mitochondria under anoxic stress

Under stress conditions, mitochondria sense metabolic changes, e.g. in pH, cytoplasmic Ca(2+), energy status, and reactive oxygen species (ROS), and respond by induction of the permeability transition pore (PTP) and by releasing cytochrome c, thus initiating the programmed cell death (PCD) cascade in animal cells. In plant cells, the presence of all the components of the cascade has not yet been shown. In wheat (Triticum aestivum L.) root mitochondria, the onset of anoxia caused rapid dissipation of the inner membrane potential, initial shrinkage of the mitochondrial matrix and the release of previously accumulated Ca(2+). Ca(2+) uptake by mitochondria was dependent on the presence of inorganic phosphate. Treatment of mitochondria with high micromolar and millimolar Ca(2+) (but not Mg(2+)) concentrations induced high amplitude swelling, indicative of PTP opening. Alterations in mitochondrial volume were confirmed by transmission electron microscopy. Mitochondrial swelling was not sensitive to cyclosporin A (CsA)-an inhibitor of mammalian PTP. The release of cytochrome c was monitored under lack of oxygen. Anoxia alone failed to induce cytochrome c release from mitochondria. Oxygen deprivation and Ca(2+) ions together caused cytochrome c release in a CsA-insensitive manner. This process correlated positively with Ca(2+) concentration and required Ca(2+) localization in the mitochondrial matrix. Functional characteristics of wheat root mitochondria, such as membrane potential, Ca(2+) transport, swelling, and cytochrome c release under lack of oxygen are discussed in relation to PCD.     

A tale of two mitochondrial channels,MAC and PTP,in apoptosis

The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial. Two mechanisms, involving opening of two different mitochondrial channels, have been proposed to be responsible for the permeabilization; the permeability transition pore (PTP) in the inner membrane and the mitochondrial apoptosis-induced channel (MAC) in the outer membrane. Opening of PTP would lead to matrix swelling, subsequent rupture of the outer membrane, and an unspecific release of intermembrane proteins into the cytosol. However, many believe PTP opening is a consequence of apoptosis and this channel is thought to principally play a role in necrosis, not apoptosis. Activation of MAC is exquisitely regulated by Bcl-2 family proteins, which are the sentinels of apoptosis. MAC provides specific pores in the outer membrane for the passage of intermembrane proteins, in particular cytochrome c, to the cytosol. The electrophysiological characteristics of MAC are very similar to Bax channels and depletion of Bax significantly diminishes MAC activity, suggesting that Bax is an essential constituent of MAC in some systems. The characteristics of various mitochondrial channels and Bax are compared. The involvement of MAC and PTP activities in apoptosis of disease and their pharmacology are discussed.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.     

Mitochondria and cell death

Mitochondria play a central role in both apoptosis and necrosis through the opening of the mitochondrial permeability transition pore (MPTP). This is thought to be formed through a Ca(2+)-triggered conformational change of the adenine nucleotide translocase (ANT) bound to matrix cyclophilin-D and we have now demonstrated this directly by reconstitution of the pure components. Opening of the MPTP causes swelling and uncoupling of mitochondria which, unrestrained, leads to necrosis. In ischaemia/reperfusion injury of the heart we have shown MPTP opening directly. Recovery of hearts correlates with subsequent closure, and agents that prevent opening or enhance closure protect from injury. Transient MPTP opening may also be involved in apoptosis by initially causing swelling and rupture of the outer membrane to release cytochrome c (cyt c), which then activates the caspase cascade and sets apoptosis in motion. Subsequent MPTP closure allows ATP levels to be maintained, ensuring that cell death remains apoptotic rather than necrotic. Apoptosis in the hippocampus that occurs after a hypoglycaemic or ischaemic insult is triggered by this means. Other apoptotic stimuli such as cytokines or removal of growth factors also involve mitochondrial cyt c release, but here there is controversy over whether the MPTP is involved. In many cases cyt c release is seen without any mitochondrial depolarization, suggesting that the MPTP does not open. Recent data of our own and others have revealed a specific outer-membrane cyt c-release pathway involving porin that does not release other intermembrane proteins such as adenylate kinase. This is opened by pro-apoptotic members of the Bcl-2 family such as BAX and prevented by anti-apoptotic members such as Bcl-X(L). Our own data suggest that this pathway may interact directly with the ANT in the inner membrane at contact sites.     

Cardiolipin deficiency releases cytochrome c from the inner mitochondrial membrane and accelerates stimuli-elicited apoptosis

Cardiolipin (CL) is a mitochondria-specific phospholipid synthesized by CL synthase (CLS). We describe here a human gene for CLS and its analysis via RNAi knockdown on apoptotic progression. Although mitochondrial membrane potential is unchanged in cells containing only 25% of the normal amount of CL, free cytochrome c (cyt. c) is detected in the intermembrane space and the mitochondria exhibit signs of reorganized cristae. However, the release of cyt. c from the mitochondria still requires apoptotic stimulation. Increased sensitivity to apoptotic signals and accelerated rates of apoptosis are observed in CL-deficient cells, followed by elevated levels of secondary necrosis. Apoptosis is thought to progress via binding of truncated Bid (tBid) to mitochondrial CL, followed by CL oxidation which results in cyt. c release. The exaggerated and accelerated apoptosis observed in CL-deficient cells is matched by an accelerated reduction in membrane potential and increased cyt. c release, but not by decreased tBid binding. This study suggests that the CL/cyt. c relationship is important in apoptotic progression and that regulating CL oxidation or/and deacylation could represent a possible therapeutic target.     

Cyclosporin A-insensitive permeability transition in brain mitochondria: inhibition by 2-aminoethoxydiphenyl borate

The mitochondrial permeability transition pore (PTP) may operate as a physiological Ca2+ release mechanism and also contribute to mitochondrial deenergization and release of proapoptotic proteins after pathological stress, e.g. ischemia/reperfusion. Brain mitochondria exhibit unique PTP characteristics, including relative resistance to inhibition by cyclosporin A. In this study, we report that 2-aminoethoxydiphenyl borate blocks Ca2+-induced Ca2+ release in isolated, non-synaptosomal rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+. Ca2+ release was not mediated by the mitochondrial Na+/Ca2+ exchanger or by reversal of the uniporter responsible for energy-dependent Ca2+ uptake. Loss of mitochondrial Ca2+ was accompanied by release of cytochrome c and pyridine nucleotides, indicating an increase in permeability of both the inner and outer mitochondrial membranes. Under these conditions, Ca2+-induced opening of the PTP was not blocked by cyclosporin A, antioxidants, or inhibitors of phospholipase A2 or nitric-oxide synthase but was abolished by pretreatment with bongkrekic acid. These findings indicate that in the presence of adenine nucleotides and Mg2+,Ca2+-induced PTP in non-synaptosomal brain mitochondria exhibits a unique pattern of sensitivity to inhibitors and is particularly responsive to 2-aminoethoxydiphenyl borate.     

Changes in mitochondrial membrane potential during staurosporine-induced apoptosis in Jurkat cells

Cytochrome c release from mitochondria is central to apoptosis, but the events leading up to it are disputed. The mitochondrial membrane potential has been reported to decrease, increase or remain unchanged during cytochrome c release. We measured mitochondrial membrane potential in Jurkat cells undergoing apoptosis by the uptake of the radiolabelled lipophilic cation TPMP, enabling small changes in potential to be determined. The ATP/ADP ratio, mitochondrial and cell volumes, plasma membrane potential and the mitochondrial membrane potential in permeabilised cells were also measured. Before cytochrome c release the mitochondrial membrane potential increased, followed by a decrease in potential associated with mitochondrial swelling and the release of cytochrome c and DDP-1, an intermembrane space house keeping protein. Mitochondrial swelling and cytochrome c release were both blocked by bongkrekic acid, an inhibitor of the permeability transition. We conclude that during apoptosis mitochondria undergo an initial priming phase associated with hyperpolarisation which leads to an effector phase, during which mitochondria swell and release cytochrome c.     

Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore.

The overexpression of Bax kills cells by a mechanism that depends on induction of the mitochondrial permeability transition (MPT) (Pastorino, J. G., Chen, S.-T., Tafani, M., Snyder, J. W., and Farber, J. L. (1998) J. Biol. Chem. 273, 7770-7775). In the present study, purified, recombinant Bax opened the mitochondrial permeability transition pore (PTP). Depending on its concentration, Bax had two distinct effects. At a concentration of 125 nM, Bax caused the release of the intermembranous proteins cytochrome c and adenylate kinase and the release from the matrix of sequestered calcein, effects prevented by the inhibitor of the PTP cyclosporin A (CSA). At this concentration of Bax, there was no detectable mitochondrial swelling or depolarization. These effects of low Bax concentrations are interpreted as the consequence of transient, non-synchronous activation of the PTP followed by a prompt recovery of mitochondrial integrity. By contrast, Bax concentrations between 250 nM and 1 microM caused a sustained opening of the PTP with consequent persistent mitochondrial swelling and deenergization (the MPT). CSA prevented the MPT induced by Bax. Increasing concentrations of calcium caused a greater proportion of the mitochondria to undergo the MPT in the presence of Bax. Importantly, two known mediators of apoptosis, ceramide and GD3 ganglioside, potentiated the induction by Bax of the MPT. The data imply that Bax mediates the opening of the mitochondrial PTP with the resultant release of cytochrome c from the intermembranous space.     

Fluoride curcumin derivatives: new mitochondrial uncoupling agents

The mitochondrial effects of two fluoride curcumin derivatives were studied. They induced the collapse of mitochondrial membrane potential (DeltaPsi), increased mitochondrial respiration, and decreased O(2)*- production and promoted Ca(2+) release. These effects were reversed by the recoupling agent 6-Ketocholestanol, but not by cyclosporin A, an inhibitor of the permeability transition pore (PTP), suggesting that these compounds act as uncoupling agents. This idea was reinforced by the analysis of the physico-chemical properties of the compounds indicating, that they are mainly in the anionic form in the mitochondrial membrane. Moreover, they are able to induce PTP opening by promoting the oxidation of thiol groups and the release of cytochrome c, making these two molecules potential candidates for induction of apoptosis.     

Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.     

The "pro-apoptotic genies" get out of mitochondria: oxidative lipidomics and redox activity of cytochrome c/cardiolipin complexes

One of the prominent consequences of the symbiogenic origin of eukaryotic cells is the unique presence of one particular class of phospholipids, cardiolipin (CL), in mitochondria. As the product originated from the evolution of symbiotic bacteria, CL is predominantly confined to the inner mitochondrial membrane in normally functioning cells. Recent findings identified CL and its oxidation products as important participants and signaling molecules in the apoptotic cell death program. Early in apoptosis, massive membrane translocations of CL take place resulting in its appearance in the outer mitochondrial membrane. Consequently, significant amounts of CL become available for the interactions with cyt c, one of the major proteins of the intermembrane space. Binding of CL with cytochrome c (cyt c) yields the cyt c/CL complex that acts as a potent CL-specific peroxidase and generates CL hydroperoxides. In this review, we discuss the catalytic mechanisms of CL oxidation by the peroxidase activity of cyt c as well as the role of oxidized CL (CLox) in the release of pro-apoptotic factors from mitochondria into the cytosol. Potential implications of cyt c/CL peroxidase intracellular complexes in disease conditions (cancer, neurodegeneration) are also considered. The discovery of the new role of cyt c/CL complexes in early mitochondrial apoptosis offers interesting opportunities for new targets in drug discovery programs. Finally, exit of cyt c from damaged and/or dying (apoptotic) cells into extracellular compartments and its accumulation in biofluids is discussed in lieu of the formation of its peroxidase complexes with negatively charged lipids and their significance in the development of systemic oxidative stress in circulation.     

Rotenone inhibits the mitochondrial permeability transition-induced cell death in U937 and KB cells

The permeability transition pore (PTP) is a mitochondrial inner membrane Ca(2+)-sensitive channel that plays a key role in different models of cell death. Because functional links between the PTP and the respiratory chain complex I have been reported, we have investigated the effects of rotenone on PTP regulation in U937 and KB cells. We show that rotenone was more potent than cyclosporin A at inhibiting Ca(2+)-induced PTP opening in digitonin-permeabilized cells energized with succinate. Consistent with PTP regulation by electron flux through complex I, the effect of rotenone persisted after oxidation of pyridine nucleotides by duroquinone. tert-butyl hydroperoxide induced PTP opening in intact cells (as shown by mitochondrial permeabilization to calcein and cobalt), as well as cytochrome c release and cell death. All these events were prevented by rotenone or cyclosporin A. These data demonstrate that respiratory chain complex I plays a key role in PTP regulation in vivo and confirm the importance of PTP opening in the commitment to cell death.     

Drug-induced Ca2+ release from isolated sarcoplasmic reticulum. III. Block of Ca2+-induced Ca2+ release by organic polyamines

Calcium ions that have been preloaded into isolated SR subfractions in the presence of ATP and pyrophosphate may be released upon addition of a large number of diverse pharmacologic substances in a manner that is effectively blocked by ruthenium red and other organic polyamines. Effective blocking substances include certain antibiotics (neomycin, gentamicin, streptomycin, clindamycin, kanamycin, and tobramycin), naturally occurring polyamines (spermine and spermidine), and a number of basic polypeptides and proteins (polylysine, polyarginine, certain histones, and protamine). These agents have only one feature in common: the presence of several amino groups. Ruthenium red, neomycin, spermine, and protamine all appear to act by blocking SR Ca2+ channels since unidirectional 45Ca2+ efflux from the vesicles is strongly inhibited by these agents. Functions ascribable to the SR Ca2+ pump are largely unaffected by these agents. Since inositol 1,4,5-trisphosphate is ineffective at inducing Ca2+ release under these conditions, we conclude that these polyamines may directly block SR Ca2+ channels at very low concentrations by a mechanism unrelated to effects on inositol 1,4,5-trisphosphate production.     

Gliotoxin induces Mg2+ efflux from intact brain mitochondria

Gliotoxin (GT) is a hydrophobic fungal metabolite of the epipolythiodioxopiperazine group which reacts with membrane thiols. When added to a suspension of energized brain mitochondria, it induces matrix swelling of low amplitude, collapse of membrane potential (DeltaPsi), and efflux of endogenous cations such as Ca2+ and Mg2+, typical events of mitochondrial permeability transition (MPT) induction. These effects are due to opening of the membrane transition pore. The addition of cyclosporin A (CsA) or ADP slightly reduces membrane potential collapse, matrix swelling and Ca2+ efflux; Mg2+ efflux is not affected at all. The presence of exogenous Mg2+ or spermine completely preserve mitochondria against DeltaPsi collapse, matrix swelling and Ca2+ release. Instead, Mg2+ efflux is only slightly affected by spermine. Our results demonstrate that, besides inducing MPT, gliotoxin activates a specific Mg2+ efflux system from brain mitochondria.     

Nitric oxide donors, nitrosothiols and mitochondrial respiration inhibitors induce caspase activation by different mechanisms

We investigated to what extent different types of NO donors induce caspase activation by opening of the mitochondrial permeability transition pore (PTP) or inhibition of mitochondrial respiration. We found that nitrosothiols can directly open the PTP in isolated mitochondria and cause cytochrome c release, whereas NONOate donors can not. In macrophages nitrosothiols cause caspase activation that is blocked by cyclosporin A or calcium chelation, both of which prevent PTP opening, whereas caspase activation caused by NONOates is much less sensitive to these agents. Inhibitors of mitochondrial respiration did not promote PTP opening in isolated mitochondria, and although they cause caspase activation in macrophages, this activation was slower than that caused by NO donors, and was relatively insensitive to cyclosporin and calcium chelators suggesting that PTP opening was not involved.     

Anti-apoptotic effect of cGMP in cultured astrocytes: inhibition by cGMP-dependent protein kinase of mitochondrial permeable transition pore.

Reperfusion of cultured astrocytes with normal medium after exposure to H(2)O(2)-containing medium causes apoptosis. We have recently shown that ibudilast, which has been used for bronchial asthma and cerebrovascular disorders, attenuated the H(2)O(2)-induced apoptosis of astrocytes via the cGMP signaling pathway. This study examines the mechanism underlying the protective effect of cGMP. The membrane-permeable cGMP analog dibutyryl-cGMP attenuated the H(2)O(2)-induced decrease in cell viability, DNA ladder formation, nuclear condensation, reduction of the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase-3 activation in cultured astrocytes. These effects of dibutyryl-cGMP were almost completely inhibited by the cGMP-dependent protein kinase (PKG) inhibitor KT5823. In isolated rat brain mitochondria, cGMP in the presence of cytosolic extract from astrocytes inhibited the mitochondrial permeability transition pore (PTP) as determined by monitoring Ca(2+)-induced mitochondrial swelling. This ability of the cytosolic extract was inactivated by heat treatment and was mimicked by exogenous PKG. The effect of cGMP on the mitochondrial swelling was blocked by KT5823. The PTP inhibitors cyclosporin A and bongkrekic acid prevented the H(2)O(2)-induced decrease in cell viability and caspase-3 activation. These findings demonstrate that cGMP inhibits the mitochondrial PTP via the activation of PKG, and the prevention of mitochondrial dysfunction contributes to its anti-apoptotic effect.     

The effects of alkali-metal chlorides and of spermidine and spermine on the swelling pressures of DNA gels in the ultracentrifuge

The effects is solution of the alkali-metal chlorides on the gel-like phase of DNA formed in the ultracentrifuge cell have been studied. The polycations, spermidine and spermine, also were shown to affect strongly the swelling pressure of the DNA gel, with evidence for the destabilization of DNA in very dilute spermine, below 10^?6 M, and for the collapse of DNA in both spermine and spermidine solutions above 10^?6 M.     

Role of alpha-tocopherol in the regulation of mitochondrial permeability transition

We previously showed that Ca2+-induced cyclosporin A-sensitive membrane permeability transition (MPT) of mitochondria occurred with concomitant generation of reactive oxygen species (ROS) and release of cytochrome c (Free Rad. Res.38, 29-35, 2004). To elucidate the role of alpha-tocopherol in MPT, we investigated the effect of alpha-tocopherol on mitochondrial ROS generation, swelling and cytochrome c release induced by Ca2+ or hydroxyl radicals. Biochemical analysis revealed that alpha-tocopherol suppressed Ca2+-induced ROS generation and oxidation of critical thiol groups of mitochondrial adenine nucleotide translocase (ANT) but not swelling and cytochrome c release. Hydroxyl radicals also induced cyclosporin A-sensitive MPT of mitochondria. alpha-Tocopherol suppressed the hydroxyl radical-induced lipid peroxidation, swelling and cytochrome c release from mitochondria. These results indicate that alpha-tocopherol inhibits ROS generation, ANT oxidation, lipid peroxidation and the opening of MPT, thereby playing important roles in the prevention of oxidative cell death.     

Calcium-induced cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane

The mechanisms of Ca2+-induced release of Cytochrome c (Cyt c) from rat brain mitochondria were examined quantitatively using a capture ELISA. In 75 or 125 mm KCl-based media 1.4 micromol Ca2+/mg protein caused depolarization and mitochondrial swelling. However, this resulted in partial Cyt c release only in 75 mm KCl. The release was inhibited by Ru360, an inhibitor of the Ca2+ uniporter, and by cyclosporin A plus ADP, a combination of mitochondrial permeability transition inhibitors. Transmission electron microscopy (TEM) revealed that Ca2+-induced swelling caused rupture of the outer membrane only in 75 mm KCl. Koenig's polyanion, an inhibitor of mitochondrial porin (VDAC), enhanced swelling and amplified Cyt c release. Dextran T70 that is known to enhance mitochondrial contact site formation did not prevent Cyt c release. Exposure of cultured cortical neurons to 500 microM glutamate for 5 min caused Cyt c release into the cytosol 30 min after glutamate removal. MK-801 or CsA inhibited this release. Thus, the release of Cyt c from CNS mitochondria induced by Ca2+ in vitro as well as in situ involved the mPT and appeared to require the rupture of the outer membrane.