Chlamydia Inhibit Host Cell Apoptosis by Degradation of Proapoptotic BH3-only Proteins

Chlamydia are obligate intracellular bacteria that replicate in a vacuole inside a host cell. Chlamydial infection has been shown to protect the host cell against apoptotic stimuli. This is likely important for the ability of Chlamydia to reproduce in human cells. Here we show that resistance to apoptosis is conveyed by the destruction of the proapoptotic BH3-only proteins Bim/Bod, Puma, and Bad during infection. Apoptotic stimuli were blocked upstream of the mitochondrial activation of Bax/Bak. During infection with both species, Chlamydia trachomatis and Chlamydia pneumoniae, Bim protein gradually disappeared without noticeable changes in Bim mRNA. The disappearance was blocked by inhibitors of the proteasome. Infected cells retained sensitivity to Bim expressed by transfection, indicating functional relevance of the Bim disappearance. Fusion to Bim targeted the green fluorescent protein for destruction during infection. Analysis of truncation mutants showed that a short region of Bim containing the BH3 domain was sufficient for destruction during chlamydial infection. Like Bim, Puma and Bad proteins disappeared during infection. These results reveal a novel way by which microbes can interfere with the host cell's apoptotic machinery, and provide a molecular explanation of the cellular resistance to apoptosis during infection with Chlamydia

BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptotic Bcl-2 proteins

Release of apoptogenic proteins such as cytochrome c from mitochondria is regulated by pro- and anti-apoptotic Bcl-2 family proteins, with pro-apoptotic BH3-only proteins activating Bax and Bak. Current models assume that apoptosis induction occurs via the binding and inactivation of anti-apoptotic Bcl-2 proteins by BH3-only proteins or by direct binding to Bax. Here, we analyze apoptosis induction by the BH3-only protein BimS. Regulated expression of BimS in epithelial cells was followed by its rapid mitochondrial translocation and mitochondrial membrane insertion in the absence of detectable binding to anti-apoptotic Bcl-2 proteins. This caused mitochondrial recruitment and activation of Bax and apoptosis. Mutational analysis of BimS showed that mitochondrial targeting, but not binding to Bcl-2 or Mcl-1, was required for apoptosis induction. In yeast, BimS enhanced the killing activity of Bax in the absence of anti-apoptotic Bcl-2 proteins. Thus, cell death induction by a BH3-only protein can occur through a process that is independent of anti-apoptotic Bcl-2 proteins but requires mitochondrial targeting

Apoptosis Is Essential for Neutrophil Functional Shutdown and Determines Tissue Damage in Experimental Pneumococcal Meningitis

During acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. Neutrophils show pro-inflammatory activity and may contribute to tissue damage. In pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. This damage may be due to excessive neutrophil activity. We here show that transgenic expression of Bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. The persistence of neutrophil brain infiltrates was accompanied by high levels of IL-1β and G-CSF as well as reduced levels of anti-inflammatory TGF-β. Significantly, Bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. In vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. The inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. In wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. These results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils

Induction of Noxa-Mediated Apoptosis by Modified Vaccinia Virus Ankara Depends on Viral Recognition by Cytosolic Helicases, Leading to IRF-3/IFN-β-Dependent Induction of Pro-Apoptotic Noxa

Viral infection is a stimulus for apoptosis, and in order to sustain viral replication many viruses are known to carry genes encoding apoptosis inhibitors. F1L, encoded by the orthopoxvirus modified vaccinia virus Ankara (MVA) has a Bcl-2-like structure. An MVA mutant lacking F1L (MVAΔF1L) induces apoptosis, indicating that MVA infection activates and F1L functions to inhibit the apoptotic pathway. In this study we investigated the events leading to apoptosis upon infection by MVAΔF1L. Apoptosis largely proceeded through the pro-apoptotic Bcl-2 family protein Bak with some contribution from Bax. Of the family of pro-apoptotic BH3-only proteins, only the loss of Noxa provided substantial protection, while the loss of Bim had a minor effect. In mice, MVA preferentially infected macrophages and DCs in vivo. In both cell types wt MVA induced apoptosis albeit more weakly than MVAΔF1L. The loss of Noxa had a significant protective effect in macrophages, DC and primary lymphocytes, and the combined loss of Bim and Noxa provided strong protection. Noxa protein was induced during infection, and the induction of Noxa protein and apoptosis induction required transcription factor IRF3 and type I interferon signalling. We further observed that helicases RIG-I and MDA5 and their signalling adapter MAVS contribute to Noxa induction and apoptosis in response to MVA infection. RNA isolated from MVA-infected cells induced Noxa expression and apoptosis when transfected in the absence of viral infection. We thus here describe a pathway leading from the detection of viral RNA during MVA infection by the cytosolic helicase-pathway, to the up-regulation of Noxa and apoptosis via IRF3 and type I IFN signalling

Phospholipase A(2) Functions in Pseudomonas aeruginosa- Induced Apoptosis

Pseudomonas aeruginosa, a gram-negative, facultative pathogen, causes severe and often even lethal infections in immunocompromised patients, as well as cystic fibrosis patients. We show here that a variety of P. aeruginosa strains activate phospholipase A(2) (PLA(2)), cultured epithelial cells, and fibroblasts, resulting in increased intracellular and extracellular arachidonic acid release. The use of different PLA(2) inhibitors revealed that P. aeruginosa-induced arachidonic acid release is mediated by activation of cytosolic PLA(2) (cPLA2), whereas iPLA(2) or sPLA(2) do not seem to be involved in the response to P. aeruginosa. Likewise, the cPLA(2)-specific inhibitors MAFP and AACOCF3 prevented apoptosis of cultured epithelial cells upon P. aeruginosa infection, whereas inhibitors specific for iPLA(2) or sPLA(2) were without effect. The physiological significance of these findings is indicated by an inhibition of apoptosis in tracheal epithelial cells upon in vivo infection with P. aeruginosa. The data indicate that arachidonic acid generation by activation of cPLA(2) during P. aeruginosa infection plays an important role in the induction of host cell death

Interferon-γ-dependent control of Anaplasma phagocytophilum by murine neutrophil granulocytes

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that is transmitted by ticks of the Ixodes ricinus complex. It replicates in neutrophils and elicits febrile disease in humans and animals. Because of its striking tropism for neutrophils, A. phagocytophilum has been used as a model organism to study the immune response against obligate intracellular pathogens. In mice, the control of A. phagocytophilum in the early phase of infection is dependent on natural killer cell-derived interferon-γ (IFN-γ). In contrast, the final elimination strictly requires CD4+ T-cells. It is a matter of debate, whether neutrophils serve only as host cells or as killer cells as well

Interferon-γ-dependent control of Anaplasma phagocytophilum by murine neutrophil granulocytes

Abstract Background Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that is transmitted by ticks of the Ixodes ricinus complex. It replicates in neutrophils and elicits febrile disease in humans and animals. Because of its striking tropism for neutrophils, A. phagocytophilum has been used as a model organism to study the immune response against obligate intracellular pathogens. In mice, the control of A. phagocytophilum in the early phase of infection is dependent on natural killer cell-derived interferon-γ (IFN-γ). In contrast, the final elimination strictly requires CD4+ T-cells. It is a matter of debate, whether neutrophils serve only as host cells or as killer cells as well. Results To study this, we used in vitro generated murine neutrophils with defects in major antimicrobial molecules such as NADPH-oxidase (gp91phox−/−), myeloperoxidase (MPO−/−) and inducible nitric oxide synthase (iNOS−/−). However, bacterial growth in gene-deficient neutrophils was comparable to that in wild-type cells. Whereas gp91phox and MPO expression remained unchanged, the infection led to an induction of iNOS. In neutrophils stimulated with IFN-γ, bacterial growth was significantly impaired, and iNOS was induced. However, the antibacterial effect of IFN-γ was still seen in iNOS−/− neutrophils. Conclusion Thus, murine in vitro generated neutrophils stimulated with IFN-γ seem to act as killer cells by an iNOS-independent mechanism