TOM-independent complex formation of Bax and Bak in mammalian mitochondria during TNFalpha-induced apoptosis.

The Bcl-2 family proteins Bax and Bak are activated in response to many apoptotic stimuli. As a consequence of activation, Bax and Bak oligomerize and permeabilize the outer mitochondrial membrane to permit the release of apoptosis-inducing factors. It still remains unclear whether these proteins require components of the mitochondrial protein import machinery for their function at the mitochondria. Here, we addressed this question by using inducible RNA interference for the study of protein import in mammalian mitochondria. After induction of apoptosis, we could not detect any impact of the absence of Tom22, Tom70, Tom40, Sam50 or metaxins on the translocation of Bax and formation of Bax and Bak complexes in mitochondria. In in vitro import studies, loss of these import and assembly proteins had no or only slight effect on the formation of complexes by radiolabeled Bax and Bak. We conclude that the import and assembly machineries of mammalian mitochondria have no impact on the translocation and complex assembly of Bax and Bak upon apoptosis induction.

Hydrogen peroxide produced by Aplysia ink toxin kills tumor cells independent of apoptosis via peroxiredoxin I sensitive pathways.

Marine snails of the genus Aplysia possess numerous bioactive substances. We have purified a 60 kDa protein, APIT (Aplysia punctata ink toxin), from the defensive ink of A. punctata that triggers cell death with profound tumor specificity. Tumor cell death induced by APIT is independent of apoptosis but is characterized by the rapid loss of metabolic activity, membrane permeabilization, and shrinkage of nuclei. Proteome analysis of APIT-treated tumor cells indicated a modification of peroxiredoxin I, a cytoplasmic peroxidase involved in the detoxification of peroxides. Interestingly, knockdown of peroxiredoxin I expression by RNA interference sensitized cells for APIT-induced cell death. APIT induced the death of tumor cells via the enzymatic production of H2O2 and catalase completely blocked APITs' activity. Our data suggest that H2O2 induced stress and the modulation of peroxiredoxins might be a promising approach for tumor therapy.

Requirement of caspase-mediated cleavage of c-Abl during stress-induced apoptosis.

c-Abl protein tyrosine kinase plays an important role in cell cycle control and apoptosis. Furthermore, induction of apoptosis correlates with the activation of c-Abl. Here, we demonstrate the cleavage of c-Abl by caspases during apoptosis. Caspases separate c-Abl into functional domains including a Src-kinase, a fragment containing nuclear import sequences, a fragment with an actin-binding domain and nuclear export sequence. Caspase cleavage increases the kinase activity of c-Abl as demonstrated by in vitro kinase assays as well as by auto- and substrate phosphorylation. Cells in which c-Abl expression was knocked down by RNA interference resisted cisplatin- but not TNFalpha-induced apoptosis. A similar selective resistance against cisplatin-induced apoptosis was observed when cleavage resistant c-Abl was overexpressed in treated cells. Our data suggest the selective requirement of c-Abl cleavage by caspases for stress-induced, but not for TNFalpha-induced apoptosis.

Epithelial cells infected with Chlamydophila pneumoniae (Chlamydia pneumoniae) are resistant to apoptosis.

The obligate intracellular pathogen Chlamydophila pneumoniae (Chlamydia pneumoniae) initiates infections in humans via the mucosal epithelia of the respiratory tract. Here, we report that epithelial cells infected with C. pneumoniae are resistant to apoptosis induced by treatment with drugs or by death receptor ligation. The induction of protection from apoptosis depended on the infection conditions since only cells containing large inclusions were protected. The underlying mechanism of infection-induced apoptosis resistance probably involves mitochondria, the major integrators of apoptotic signaling. In the infected cells, mitochondria did not respond to apoptotic stimuli by the release of apoptogenic factors required for the activation of caspases. Consequently, active caspase-3 was absent in infected cells. Our data suggest a direct modulation of apoptotic pathways in epithelial cells by C. pneumoniae.

Signal transduction pathways induced by virulence factors of Neisseria gonorrhoeae.

The obligate human pathogen Neisseria gonorrhoeae infects a variety of human tissues. In recent years, several host cell receptors for the major bacterial adhesins have been identified. While the knowledge of the molecular mechanism of colonisation has helped to understand special aspects of the infection, like the explicit tropism of gonococci for human tissues, the long-term consequences of engaging these receptors are still unknown. A variety of signalling pathways initiated by the activated receptors and by bacterial proteins transferred to the infected cell have been defined which include lipid second messenger, protein kinases, proteases and GTPases. These pathways control important steps of the infection, such as tight adhesion and invasion, the induction of cytokine release, and apoptosis. The detailed knowledge of bacteria-induced signalling pathways could allow the design of new therapeutic approaches which might be advantageous over the classical antibiotics therapy.

Modification of host cell apoptosis by viral and bacterial pathogens.

Bacterial and viral pathogens have evolved sophisticated strategies to manipulate the host cell's life span to their own advantage. This is achieved by modifications of the intrinsic apoptosis program at several checkpoints: kinases, caspases and mitochondria. The goal of this review is to give an overview over the molecular mechanisms involved.

Low iron availability modulates the course of Chlamydia pneumoniae infection.

Chlamydiae are obligate intracellular bacteria residing exclusively in host cell vesicles termed inclusions. We have investigated the effects of deferoxamine mesylate (DAM)-induced iron deficiency on the growth of Chlamydia pneumoniae and Chlamydia trachomatis serovar L2. In epithelial cells subjected to iron starvation and infected with either C. pneumoniae or C. trachomatis L2, small inclusions were formed, and the infectivity of chlamydial progeny was impaired. Moreover, for C. trachomatis L2, we observed a delay in homotypic fusion of inclusions. The inhibitory effects of DAM were reversed by adding exogenous iron-saturated transferrin, which restored the production of infectious chlamydiae. Electron microscopy examination of iron-deprived specimens revealed that the small inclusions contained reduced numbers of C. pneumoniae that were mostly reticulate bodies. We have previously reported specific accumulation of transferrin receptors (TfRs) around C. pneumoniae inclusions within cells grown under normal conditions. Using confocal and electron microscopy, we show here a remarkable increase in the amount of TfRs surrounding the inclusions in iron-starved cultures. It has been shown that iron is an essential factor in the growth and survival of C. trachomatis. Here, we postulate that, for C. pneumoniae also, iron is an indispensable element and that Chlamydia may use iron transport pathways of the host by attracting TfR to the phagosome.

Predominant identification of RNA-binding proteins in Fas-induced apoptosis by proteome analysis.

Proteome analysis of Jurkat T cells was performed in order to identify proteins that are modified during apoptosis. Subtractive analysis of two-dimensional gel patterns of apoptotic and nonapoptotic cells revealed differences in 45 protein spots. 37 protein spots of 21 different proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. The hnRNPs A0, A2/B1, A3, K, and R; the splicing factors p54(nrb), SRp30c, ASF-2, and KH-type splicing regulatory protein (FUSE-binding protein 2); and alpha NAC, NS1-associated protein 1, and poly(A)-binding protein 4 were hitherto unknown to be involved in apoptosis. The putative cleavage sites of the majority of the proteins could be calculated by the molecular masses and isoelectric points in the two-dimensional electrophoresis gel, the peptide mass fingerprints, and after translation by treatment with recombinant caspase-3. Remarkably, 15 of the 21 identified proteins contained the RNP or KH motif, the best characterized RNA-binding motifs.

Targeting of the pro-apoptotic VDAC-like porin (PorB) of Neisseria gonorrhoeae to mitochondria of infected cells.

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.

Neisseria gonorrhoeae porin modifies the oxidative burst of human professional phagocytes.

A hallmark of infection with the gram-negative bacterium Neisseria gonorrhoeae is the local infiltration and subsequent activation of polymorphonuclear neutrophils. Several gonococcal outer membrane proteins are involved in the interaction with and the activation of these phagocytes, including gonococcal porin, the most abundant protein in the outer membrane. Previous work suggests that this porin plays a role in various cellular processes, including inhibiting neutrophils activation and phagosome maturation in professional phagocytes. Here we investigated the ability of porin to modify the oxidative metabolism of human peripheral blood neutrophils and monocytes in response to particulate stimuli (including live gonococci) and soluble agents. The activation of the oxidative metabolism was determined by chemiluminescence amplified with either luminol or lucigenin. We found that treatment of the phagocytes with porin inhibits the release of reactive oxygen species measured as luminol-enhanced chemiluminescence in response to zymosan, latex particles, and gonococci. The engulfment of these particles was not, however, affected by porin treatment. Similar effects of porin on the chemiluminescence response were observed in cytochalasin B-treated neutrophils exposed to the soluble chemotactic peptide N-formylmethionyl-leucyl-phenylalanine. This indicates that porin selectively inhibits granule fusion with those cellular membranes that are in direct contact with porin, namely, the phagosomal and plasma membranes. This porin-induced downregulation of oxidative metabolism may be a potent mechanism by which gonococci modulate oxygen-dependent reactions by activated phagocytes at inflammation sites.

A two dimensional electrophoresis database of a human Jurkat T-cell line.

About 2000 protein spots of human Jurkat T-cells were detected by high resolution two-dimensional gel electrophoresis (2-DE) and were characterized in terms of their isoelectric point and molecular mass. A 2-DE database was constructed and is available at http://www.mpiib-berlin.mpg.de/2D-PAGE/. At present the database contains 67 identified protein spots. These proteins were identified after tryptic digestion by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). Proteins with a sequence coverage of at least 30% were introduced in the database. This sequence coverage could not always be obtained by using only the matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) for the mass analysis. Therefore, an additional mass spectrum was recorded by using 2,5-dihydroxybenzoic acid (DHB). Usually, additional mass peaks were detected and together with the mass spectrum of CHCA this resulted in the desired sequence coverage.

Analysis of missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation after in-gel tryptic digestion.

Peptide mass fingerprinting is a powerful tool for the identification of proteins. Trypsin is the most widely used enzyme for this purpose. Therefore, 104 protein digests from human Jurkat T cells and Mycobacterium were analyzed considering missed cleavage sites, tryptophan oxidation and N-terminal pyroglutamylation. About 90% of the matched peptides with missed cleavage sites could be classified into three groups: (i) lysine and arginine with a neighbouring proline on the carboxy-terminal side, (ii) neighboring lysines/arginines, and (iii) lysines and arginines with an aspartic acid or glutamic acid residue on either the amino- or carboxy-terminal side. The first group is already accounted for by search programs. The number of missed cleavage sites can be increased without reducing the precision of the database search by taking the other two groups into consideration. Peptides with tryptophan were observed in non, singly (+16 Da) and doubly (+32 Da) oxidized forms. The higher oxidized form was only observed with lower intensity in the presence of the lower oxidized form. Peptides with N-terminal glutamine were found always as pyroglutamate (-17 Da), and in the majority of cases in pairs with unmodified glutamine. These data can be used for the refinement of protein searches by peptide mass fingerprinting.

Caspase inhibitors in prevention of apoptosis.

Apoptosis, a morphological distinct form of programmed cell death, is a crucial process during development, the maintenance of cell homeostasis and the regulation of the immune system. A variety of diseases have been found to correlate with physiological apoptosis including cancer, autoimmune disease, viral infection and degenerative disorders. Although very different signals initiate apoptosis, the phenotype of apoptosis is surprisingly similar even in different cell types suggesting that the final stages of apoptotic death are highly conserved. The execution of the death program is coordinated by a recently identified class of cysteine proteases termed caspases. The finding that caspases are mainly involved in regulation of this conserved part of the death program has boosted the search for caspase inhibitors which might offer a therapeutic approach to treat apoptotic disorders. Synthetic peptide inhibitors have been developed which exhibit some selectivity for the different caspases. In the last years several natural inhibitors have been discovered which either prevent caspase activation or caspase activity. This review will present the recent advances and discuss the potential of caspase inhibitors as future therapeutics.

Mutagenesis of the Neisseria gonorrhoeae porin reduces invasion in epithelial cells and enhances phagocyte responsiveness.

Porin (PorB), the major outer membrane protein of Neisseria gonorrhoeae, has been implicated in pathogenesis previously. However, the fact that porin deletion mutants are not viable has complicated investigations. Here, we describe a method of manipulating the porin gene site-specifically. N. gonorrhoeae MS11, which harbours the porB1B (P.1B) porin allele, was used to generate mutants carrying deletions in the surface loops 1 and 5. An 11-amino-acid deletion in loop 1 impaired Opa50-dependent invasion into human Chang epithelial cells, whereas loop 5 deletion exhibited no apparent phenotype. In a second approach, the complete gonococcal porB1B was replaced by the porBNia gene of Neisseria lactamica. Such mutants were unable to induce efficient uptake by epithelial cells but induced an enhanced respiratory response in HL60 phagocytic cells. The increased respiratory burst was accompanied by an enhanced phagocytic uptake of the mutant compared with the wild-type strain. Our data extend previous evidence for multiple central functions of PorB in the infection process.

Host cell interactions and signalling with Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a highly adapted human pathogen that utilises multiple adhesins to interact with a variety of host cell receptors. Recently, substantial progress has been made in unravelling the signalling events induced by N. gonorrhoae that can lead to cytoskeletal reorganisation, invasion or phagocytic uptake, intraphagosomal accommodation, nuclear signalling, cytokine/chemokine release and apoptosis.

Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases.

The porin (PorB) of Neisseria gonorrhoeae is an intriguing bacterial factor owing to its ability to translocate from the outer bacterial membrane into host cell membranes where it modulates the infection process. Here we report on the induction of programmed cell death after prolonged infection of epithelial cells with pathogenic Neisseria species. The underlying mechanism we propose includes translocation of the porin, a transient increase in cytosolic Ca2+ and subsequent activation of the Ca2+ dependent protease calpain as well as proteases of the caspase family. Blocking the porin channel by ATP eliminates the Ca2+ signal and also abolishes its pro-apoptotic function. The neisserial porins share structural and functional homologies with the mitochondrial voltage-dependent anion channels (VDAC). The neisserial porin may be an analogue or precursor of the ancient permeability transition pore, the putative central regulator of apoptosis.

Roles of PilC and PilE proteins in pilus-mediated adherence of Neisseria gonorrhoeae and Neisseria meningitidis to human erythrocytes and endothelial and epithelial cells.

Unlike other type 4 pili, the neisserial pili consist of at least two distinct proteins, the highly variable major subunit PilE forming the pilus fiber and the tip-associated adhesin PilC. PilC protein purified either from gonococci or from Escherichia coli interacted with different human epithelial cell lines, primary epithelial and endothelial cells. The binding of PilC protein efficiently prevented the attachment of piliated Neisseria gonorrhoeae and Neisseria meningitidis to these cell types. Fluorescent beads coated with pili prepared from piliated wild-type N. gonorrhoeae also adhered to these cells, in contrast to beads coated with pili prepared from a piliated PilC-deficient mutant. In the latter case, the binding of fluorescent beads was restored after pretreatment of the pilus-loaded beads with purified PilC. Piliated wild-type N. gonorrhoeae, the piliated PilC-deficient mutant, and N. gonorrhoeae pili assembled in Pseudomonas aeruginosa agglutinated human erythrocytes, while nonpiliated gonococci did not. Consistently, purified PilC did not agglutinate or bind to human erythrocytes, suggesting that N. gonorrhoeae PilE is responsible for pilus-mediated hemagglutination.

Characterization and intracellular trafficking pattern of vacuoles containing Chlamydia pneumoniae in human epithelial cells.

Chlamydiae are obligate intracellular pathogens that reside within a membrane-bound vacuole throughout their developmental cycle. In this study, the intraphagosomal pH of Chlamydia pneumoniae (Cpn) was qualitatively assessed, and the intracellular fate of the pathogen-containing vacuole and its interaction with endocytic organelles in human epithelial cells were analysed using conventional immunofluorescence and confocal microscopy. The pH-sensitive probes acridine orange (AO), LysoTracker (LyT) and DAMP did not accumulate in the bacterial inclusion. In addition, exposure of cells to bafilomycin A1(BafA1), a potent acidification inhibitor, did not inhibit or delay chlamydial growth. The chlamydial compartment was not accessible to the fluid-phase tracer Texas Red (TR)-dextran and did not exhibit any level of staining for the late endosomal marker cation-independent mannose-6-phosphate receptor (Ci-M6PR) or for the lysosomal-associated membrane proteins (LAMP-1 and -2) and CD63. In addition, transferrin receptor (TfR)-enriched vesicles were observed close to Cpn vacuoles, potentially indicating a specific translocation of these organelles through the cytoplasm to the vicinity of the vacuole. We conclude that Cpn, like other chlamydial spp., circumvents the host endocytic pathway and inhabits a non-acidic vacuole, which is dissociated from late endosomes and lysosomes, but selectively accumulates early endosomes.