Role of mitochondrial outer membrane permeabilization during bacterial infection.

Beyond the initial 'powerhouse' view, mitochondria have numerous functions in their mammalian cell and contribute to many physiological processes, and many of these we understand only partially. The control of apoptosis by mitochondria is firmly established. Many questions remain however how this function is embedded into physiology, and how other signaling pathways regulate mitochondrial apoptosis; the interplay of bacteria with the mitochondrial apoptosis pathway is one such example. The outer mitochondrial membrane regulates both import into mitochondria and the release of intermembrane, and in some situations also matrix components from mitochondria, and these mitochondrial components can have signaling function in the cytosol. One function is the induction of apoptotic cell death. An exciting, more recently discovered function is the regulation of inflammation. Mitochondrial molecules, both proteins and nucleic acids, have inflammatory activity when released from mitochondria, an activity whose regulation is intertwined with the activation of apoptotic caspases. Bacterial infection can have more general effects on mitochondrial apoptosis-regulation, through effects on host transcription and other pathways, such as signals controlled by pattern recognition. Some specialized bacteria have products that more specifically regulate signaling to the outer mitochondrial membrane, and to apoptosis; both pro- and anti-apoptotic mechanisms have been reported. Among the intriguing recent findings in this area are signaling contributions of porins and the sub-lethal release of intermembrane constituents. We will here review the literature and place the new developments into the established context of mitochondrial signaling during the contact of bacterial pathogens with human cells.

Mitochondria supply sub-lethal signals for cytokine secretion and DNA-damage in H. pylori infection.

The bacterium Helicobacter pylori induces gastric inflammation and predisposes to cancer. H. pylori-infected epithelial cells secrete cytokines and chemokines and undergo DNA-damage. We show that the host cell's mitochondrial apoptosis system contributes to cytokine secretion and DNA-damage in the absence of cell death. H. pylori induced secretion of cytokines/chemokines from epithelial cells, dependent on the mitochondrial apoptosis machinery. A signalling step was identified in the release of mitochondrial Smac/DIABLO, which was required for alternative NF-κB-activation and contributed to chemokine secretion. The bacterial cag-pathogenicity island and bacterial muropeptide triggered mitochondrial host cell signals through the pattern recognition receptor NOD1. H. pylori-induced DNA-damage depended on mitochondrial apoptosis signals and the caspase-activated DNAse. In biopsies from H. pylori-positive patients, we observed a correlation of Smac-levels and inflammation. Non-apoptotic cells in these samples showed evidence of caspase-3-activation, correlating with phosphorylation of the DNA-damage response kinase ATM. Thus, H. pylori activates the mitochondrial apoptosis pathway to a sub-lethal level. During infection, Smac has a cytosolic, pro-inflammatory role in the absence of apoptosis. Further, DNA-damage through sub-lethal mitochondrial signals is likely to contribute to mutagenesis and cancer development.

Chlamydia trachomatis inhibits apoptosis in infected cells by targeting the pro-apoptotic proteins Bax and Bak.

Apoptosis acts in defense against microbial infection, and many infectious agents have developed strategies to inhibit host cell apoptosis. The human pathogen Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that strongly inhibits mitochondrial apoptosis of its human host cell but there is no agreement how the bacteria achieve this. We here provide a molecular analysis of chlamydial apoptosis-inhibition in infected human cells and demonstrate that the block of apoptosis occurs during the activation of the effectors of mitochondrial apoptosis, Bak and Bax. We use small-molecule Bcl-2-family inhibitors and gene targeting to show that previous models cannot explain the anti-apoptotic effect of chlamydial infection. Although the anti-apoptotic Bcl-2-family protein Mcl-1 was strongly upregulated upon infection, Mcl-1-deficient cells and cells where Mcl-1 was pharmacologically inactivated were still protected. Ctr-infection could inhibit both Bax- and Bak-induced apoptosis. Apoptotic Bax-oligomerization and association with the outer mitochondrial membrane was reduced upon chlamydial infection. Infection further inhibited apoptosis induced conformational changes of Bak, as evidenced by changes to protease sensitivity, oligomerization and release from the mitochondrial porin VDAC2. Mitochondria isolated from Ctr-infected cells were protected against the pro-apoptotic Bcl-2-family proteins Bim and tBid but this protection was lost upon protease digestion. However, the protective effect of Ctr-infection was reduced in cells lacking the Bax/Bak-regulator VDAC2. We further found that OmpA, a porin of the outer membrane of Ctr, associated upon experimental expression with mitochondria and inhibited apoptosis, phenocopying the effect of the infection. These results identify a novel way of apoptosis inhibition, involving only the most downstream modulator of mitochondrial apoptosis and suggest that Chlamydia has a protein dedicated to the inhibition of apoptosis to secure its survival in human cells.

Partial Inactivation of the Chromatin Remodelers SMARCA2 and SMARCA4 in Virus-Infected Cells by Caspase-Mediated Cleavage.

The BAF-chromatin remodeling complex, with its mutually exclusive ATPases SMARCA2 and SMARCA4, is essential for the transcriptional activation of numerous genes, including a subset of interferon-stimulated genes (ISGs). Here, we show that C-terminally truncated forms of both SMARCA2 and SMARCA4 accumulate in cells infected with different RNA or DNA viruses. The levels of truncated SMARCA2 or SMARCA4 strongly correlate with the degree of cell damage and death observed after virus infection. The use of a pan-caspase inhibitor and genetically modified cell lines unable to undergo apoptosis revealed that the truncated forms result from the activity of caspases downstream of the activated intrinsic apoptotic pathway. C-terminally cleaved SMARCA2 and SMARCA4 lack potential nuclear localization signals as well as the bromo- and SnAC domain, with the latter two domains believed to be essential for chromatin association and remodeling. Consistent with this belief, C-terminally truncated SMARCA2 was partially relocated to the cytoplasm. However, the remaining nuclear protein was sufficient to induce ISG expression and inhibit the replication of vesicular stomatitis virus and influenza A virus. This suggests that virus-induced apoptosis does not occur at the expense of an intact interferon-mediated antiviral response pathway.IMPORTANCE Efficient induction of interferon-stimulated genes (ISGs) prior to infection is known to effectively convert a cell into an antiviral state, blocking viral replication. Additionally, cells can undergo caspase-mediated apoptosis to control viral infection. Here, we identify SMARCA2 and SMARCA4 to be essential for the efficient induction of ISGs but also to be targeted by cellular caspases downstream of the intrinsic apoptotic pathway. We find that C-terminally cleaved SMARCA2 and SMARCA4 accumulate at late stages of infection, when cell damage already had occurred. Cleavage of the C terminus removes domains important for nuclear localization and chromatin binding of SMARCA2 and SMARCA4. Consequently, the cleaved forms are unable to efficiently accumulate in the cell nucleus. Intriguingly, the remaining nuclear C-terminally truncated SMARCA2 still induced ISG expression, although to lower levels. These data suggest that in virus-infected cells caspase-mediated cell death does not completely inactivate the SMARCA2- and SMARCA4-dependent interferon signaling pathway.

Infection of epithelial cells with Chlamydia trachomatis inhibits TNF-induced apoptosis at the level of receptor internalization while leaving non-apoptotic TNF-signalling intact.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of medical importance. C. trachomatis develops inside a membranous vacuole in the cytosol of epithelial cells but manipulates the host cell in numerous ways. One prominent effect of chlamydial infection is the inhibition of apoptosis in the host cell, but molecular aspects of this inhibition are unclear. Tumour necrosis factor (TNF) is a cytokine with important roles in immunity, which is produced by immune cells in chlamydial infection and which can have pro-apoptotic and non-apoptotic signalling activity. We here analysed the signalling through TNF in cells infected with C. trachomatis. The pro-apoptotic signal of TNF involves the activation of caspase-8 and is controlled by inhibitor of apoptosis proteins. We found that in C. trachomatis-infected cells, TNF-induced apoptosis was blocked upstream of caspase-8 activation even when inhibitor of apoptosis proteins were inhibited or the inhibitor of caspase-8 activation, cFLIP, was targeted by RNAi. However, when caspase-8 was directly activated by experimental over-expression of its upstream adapter Fas-associated protein with death domain, C. trachomatis was unable to inhibit apoptosis. Non-apoptotic TNF-signalling, particularly the activation of NF-κB, initiates at the plasma membrane, while the activation of caspase-8 and pro-apoptotic signalling occur subsequently to internalization of TNF receptor and the formation of a cytosolic signalling complex. In C. trachomatis-infected cells, NF-κB activation through TNF was unaffected, while the internalization of the TNF-TNF-receptor complex was blocked, explaining the lack of caspase-8 activation. These results identify a dichotomy of TNF signalling in C. trachomatis-infected cells: Apoptosis is blocked at the internalization of the TNF receptor, but non-apoptotic signalling through this receptor remains intact, permitting a response to this cytokine at sites of infection.