<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> glycosylphosphatidylinositols are not involved in <Emphasis Type="Italic">T. gondii</Emphasis>-induced host cell survival

Toxoplasma gondii is an intracellular parasite able to both promote and inhibit apoptosis. T. gondii renders infected cells resistant to programmed cell death induced by multiple apoptotic triggers. On the other hand, increased apoptosis of immune cells after in vivo infection with T. gondii may suppress the immune response to the parasite. Glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of T. gondii tachyzoites and GPIs are involved in the pathogenicity of protozoan parasites. In this report, we determine if GPIs are responsible for inhibition or induction of host cell apoptosis. We show here that T. gondii GPIs fail to block apoptosis that was triggered in human-derived cells via extrinsic or intrinsic apoptotic pathways. Furthermore, characteristics of apoptosis, e.g. caspase-3/7 activity, phosphatidylserine exposition at the cell surface or DNA strand breaks, were not observed in the presence of T. gondii GPIs. These results indicate that T. gondii GPIs are not involved in survival or in apoptosis of host cells. This absence of effect on apoptosis could be a feature common to GPIs of other parasites.     

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.     

Recombinant Galectins of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> Parasite Induces Apoptosis in the Peripheral Blood Lymphocytes of Goat

The effect of Haemonchus contortus galectin peptides rHco-gal-m/f to induce apoptosis in the peripheral blood lymphocytes (PBLCs) of goats was investigated. Analysis of apoptosis was carried out with agarose gel electrophoresis, flow cytometry and transmission electron microscopy. The results indicated that there were visible apoptosis bodies and typical DNA ladders by genomic DNA fragmentation. The quantitative analysis of apoptosis by flow cytometry indicated that rHco-gal-m/f peptides induced apoptosis was time and dose dependent. Ultrastructural studies of the PBLCs revealed that a large number of apoptotic cells were present in galectin-treated cells, which had the typical morphologic changes of apoptosis such as reduction of the cytoplasmic volume, loss of cell surface microvilli, chromatin condensation and fragmentation of the apoptotic cells into small apoptotic bodies.     

Overexpression of <Emphasis Type="Italic">Drosophila</Emphasis> Rad51 protein (DmRad51) disrupts cell cycle progression and leads to apoptosis

Among proteins involved in homologous recombination, Rad51 is an essential enzyme in DNA repair and recombination. However, little is known about its role in cell cycle regulation and apoptosis. To examine the function of Drosophila Rad51 (DmRad51) in cell cycle regulation and apoptosis, DmRad51 protein was overexpressed using a heat shock-inducible promoter or the UAS–GAL4 binary expression system. We observed that ubiquitous expression of DmRad51 protein in flies carrying hsp26–Rad51 or UAS–Rad51 transgenes was lethal. Induction of DmRad51—more specifically in eye or wing imaginal discs—caused tissue-specific cell death in the domains of DmRad51 expression. Cell death was due to apoptosis, as shown by staining with the TdT-mediated dUTP nick-end labeling assay. Immunocytochemistry revealed that cells expressing DmRad51 colocalized with apoptotic cells. In addition, the phenotypes caused by the overexpression of DmRad51 were similar to those caused by ectopic expression of Reaper, a proapoptotic protein, and were partially suppressed by the coexpression of p35, an antiapoptotic protein. Using an antiphosphohistone H3 antibody, we also observed that the overexpression of DmRad51 protein disrupted normal cell cycle progression in eye imaginal discs. Taken together, these results show that ectopically expressed DmRad51 disrupts cell cycle regulation and induces apoptosis.     

Cadmium induces a heterogeneous and caspase-dependent apoptotic response in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The toxic metal cadmium is linked to a series of degenerative disorders in humans, in which Cd-induced programmed cell death (apoptosis) may play a role. The yeast, Saccharomyces cerevisiae, provides a valuable model for elucidating apoptosis mechanisms, and this study extends that capability to Cd-induced apoptosis. We demonstrate that S. cerevisiae undergoes a glucose-dependent, programmed cell death in response to low cadmium concentrations, which is initiated within the first hour of Cd exposure. The response was associated with induction of the yeast caspase, Yca1p, and was abolished in a yca1Δ mutant. Cadmium-dependent apoptosis was also suppressed in a gsh1Δ mutant, indicating a requirement for glutathione. Other apoptotic markers, including sub-G₁ DNA fragmentation and hyper-polarization of mitochondrial membranes, were also evident among Cd-exposed cells. These responses were not distributed uniformly throughout the cell population, but were restricted to a subset of cells. This apoptotic subpopulation also exhibited markedly elevated levels of intracellular reactive oxygen species (ROS). The heightened ROS levels alone were not sufficient to induce apoptosis. These findings highlight several new perspectives to the mechanism of Cd-dependent apoptosis and its phenotypic heterogeneity, while opening up future analyses to the power of the yeast model system.     

Identification of a siderophore utilization locus in nontypeable <Emphasis Type="Italic">Haemophilus influenzae</Emphasis>

Background  

Haemophilus influenzae has an absolute aerobic growth requirement for either heme, or iron in the presence of protoporphyrin IX. Both iron and heme in the mammalian host are strictly limited in their availability to invading microorganisms. Many bacterial species overcome iron limitation in their environment by the synthesis and secretion of small iron binding molecules termed siderophores, which bind iron and deliver it into the bacterial cell via specific siderophore receptor proteins on the bacterial cell surface. There are currently no reports of siderophore production or utilization by H. influenzae.     

Rapid cold-hardening protects <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> from cold-induced apoptosis

The rapid cold-hardening (RCH) response increases the cold tolerance of insects by protecting against non-freezing, cold-shock injury. Apoptosis, or programmed cell death, plays important roles in development and the elimination of sub-lethally damaged cells. Our objectives were to determine whether apoptosis plays a role in cold-shock injury and, if so, whether the RCH response protects against cold-induced apoptosis in Drosophila melanogaster. The present study confirmed that RCH increased the cold tolerance of the adults at the organismal level. No flies in the cold-shocked group survived direct exposure to ‒7°C for 2 h, whereas significantly more flies in the RCH group survived exposure to ‒7°C for 2 h after a 2-h exposure to 5°C. We used a TUNEL assay to detect and quantify apoptotic cell death in five groups of flies including control, cold-shocked, RCH, heat-shocked (37.5°C, 30 min), and frozen (‒20°C, 24 h) and found that apoptosis was induced by cold shock, heat shock, and freezing. The RCH treatment significantly improved cell viability by 38% compared to the cold-shocked group. Cold shock-induced DNA fragmentation shown by electrophoresis provided further evidence for apoptosis. SDS-PAGE analysis revealed an RCH-specific protein band with molecular mass of ∼150 kDa. Western-blotting revealed three proteins that play key roles in the apoptotic pathway: caspase-9-like (apoptotic initiator), caspase-3-like (apoptotic executioner) and Bcl-2 (anti-apoptotic protein). Consequently, the results of this study support the hypothesis that the RCH response protects against cold-shock-induced apoptosis.     

Spatiotemporal activation of caspase‐dependent and ‐independent pathways in staurosporine‐induced apoptosis of p53wt and p53mt human cervical carcinoma cells

Background information. Caspase‐dependent and ‐independent death mechanisms are involved in apoptosis in a variety of human carcinoma cells treated with antineoplastic compounds. Our laboratory has reported that p53 is a key contributor of mitochondrial apoptosis in cervical carcinoma cells after staurosporine exposure. However, higher mitochondrial membrane potential dissipation and greater DNA fragmentation were observed in p53^wt (wild‐type p53) HeLa cells compared with p53^mt (mutated p53) C‐33A cells. Here, we have studied events linked to the mitochondrial apoptotic pathway. Results. Staurosporine can induce death of HeLa cells via a cytochrome c/caspase‐9/caspase‐3 mitochondrial‐dependent apoptotic pathway and via a delayed caspase‐independent pathway. In contrast with p53^wt cells, p53^mt C‐33A cells exhibit firstly caspase‐8 activation leading to caspase‐3 activation and Bid cleavage followed by cytochrome c release. Attenuation of PARP‐1 [poly(ADP‐ribose) polymerase‐1] cleavage as well as oligonucleosomal DNA fragmentation in the presence of z‐VAD‐fmk points toward a major involvement of a caspase‐dependent pathway in staurosporine‐induced apoptosis in p53^wt HeLa cells, which is not the case in p53^mt C‐33A cells. Meanwhile, the use of 3‐aminobenzamide, a PARP‐1 inhibitor known to prevent AIF (apoptosis‐inducing factor) release, significantly decreases staurosporine‐induced death in these p53^mt carcinoma cells, suggesting a preferential implication of caspase‐independent apoptosis. On the other hand, we show that p53, whose activity is modulated by pifithrin‐α, isolated as a suppressor of p53‐mediated transactivation, or by PRIMA‐1 (p53 reactivation and induction of massive apoptosis), that reactivates mutant p53, causes cytochrome c release as well as mitochondrio—nuclear AIF translocation in staurosporine‐induced apoptosis of cervical carcinoma cells. Conclusions. The present paper highlights that staurosporine engages the intrinsic mitochondrial apoptotic pathway via caspase‐8 or caspase‐9 signalling cascades and via caspase‐independent cell death, as well as through p53 activity.     

Potential role of the EPEC translocated intimin receptor (Tir) in host apoptotic events

Apoptosis, or programmed cell death, is a well-ordered process that allows damaged or diseased cells to be removed from an organism without severe inflammatory reactions. Multiple factors, including microbial infection, can induce programmed death and trigger reactions in both host and microbial cellular pathways. Whereas an ultimate outcome is host cell death, these apoptotic triggering mechanisms may also facilitate microbial spread and prolong infection. To gain a better understanding of the complex events of host cell response to microbial infection, we investigated the molecular role of the microorganism Enteropathogenic Escherichia coli (EPEC) in programmed cell death. We report that wild type strain of EPEC, E2348/69, induced apoptosis in cultured PtK2 and Caco-2 cells, and in contrast, infections by the intracellularly localized Listeria monocytogenes did not. Fractionation and concentration of EPEC-secreted proteins demonstrated that soluble protein factors expressed by the bacteria were capable of inducing the apoptotic events in the absence of organism attachment, suggesting adherence is not required to induce host cell death. Among the known EPEC proteins secreted via the Type III secretion (TTS) system, we identified the translocated intimin receptor (Tir) in the apoptosis-inducing protein sample. In addition, host cell ectopic expression of an EPEC GFP-Tir showed mitochondrial localization of the protein and produced apoptotic effects in transfected cells. Taken together, these results suggest a potential EPEC Tirmediated role in the apoptotic signaling cascade of infected host cells.     

Piscirickettsia salmonis induces apoptosis in macrophages and monocyte‐like cells from rainbow trout

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS) which causes significant losses in salmon production in Chile and other and in other regions in the southern hemisphere. As the killing of phagocytes is an important pathogenic mechanism for other bacteria to establish infections in vertebrates, we investigated whether P. salmonis kills trout macrophages by apoptosis. Apoptosis in infected macrophages was demonstrated by techniques based on morphological changes and host cell DNA fragmentation. Transmission electron microcopy showed classic apoptotic characteristics and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling showed fragmented DNA. Programmed cell death type I was further confirmed by increased binding of annexin V to externalized phosphatidylserine in infected macrophages. Moreover, significant increases of caspase 3 activation were detected in infected cells and treatment with caspase inhibitor caused a decrease in levels of apoptosis. This is the first evidence that P. salmonis induces cell death in trout macrophages. This could lead to bacterial survival and evasion of the host immune response and play an important role in the establishment of infection in the host. J. Cell. Biochem. 110: 468–476, 2010. © 2010 Wiley‐Liss, Inc.     

Mechanisms of programmed cell death during oogenesis in <Emphasis Type="Italic">Drosophila virilis</Emphasis>

We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers. The present study was co-financed within Op. Education by the European Social Fund and by National Resources via a grant (HRAKLEITOS 70/3/7164) to Professor L.H. Margaritis.     

Iron homeostasis in <Emphasis Type="Italic">Brucella abortus</Emphasis>: the role of bacterioferritin

Brucella abortus is the etiological agent of bovine brucellosis, an infectious disease of humans and cattle. Its pathogenesis is mainly based on its ability to survive and multiply inside macrophages. It has been demonstrated that if B. abortus ferrochelatase cannot incorporate iron into protoporphyrin IX to synthesize heme, the intracellular replication and virulence in mice is highly attenuated. Therefore, it can be hypothesized that the unavailability of iron could lead to the same attenuation in B. abortus pathogenicity. Thus, the purpose of this work was to obtain a B. abortus derivative unable to keep an internal iron pool and test its ability to replicate under iron limitation. To achieve this, we searched for iron-storage proteins in the genome of brucellae and found bacterioferritin (Bfr) as the sole ferritin encoded. Then, a B. abortus bfr mutant was built up and its capacity to store iron and replicate under iron limitation was investigated. Results indicated that B. abortus Bfr accounts for 70% of the intracellular iron content. Under iron limitation, the bfr mutant suffered from enhanced iron restriction with respect to wild type according to its growth retardation pattern, enhanced sensitivity to oxidative stress, accelerated production of siderophores, and altered expression of membrane proteins. Nonetheless, the bfr mutant was able to adapt and replicate even inside eukaryotic cells, indicating that B. abortus responds to internal iron starvation before sensing external iron availability. This suggests an active role of Bfr in controlling iron homeostasis through the availability of Bfr-bound iron.     

Host cell death induced by the egress of intracellular <Emphasis Type="Italic">Plasmodium</Emphasis> parasites

Intracellular pathogens are known to inhibit host cell apoptosis efficiently to ensure their own survival. However, following replication within a cell, they typically need to egress in order to infect new cells. For a long time it was assumed that this happens by simply disrupting the host cell and in some cases, such as for Plasmodium-infected erythrocytes, this seems indeed to be true. However, recently it has been shown that in Plasmodium-infected hepatocytes, an ordered form of cell death is initiated. This cell death is parasite-dependent and can clearly be distinguished from apoptosis and necrosis. The key event, and point of no return, appears to be the rupture of the parasitophorous vacuole membrane (PVM). PVM disruption and host cell death depend on the activation of cysteine proteases. Whether these are of parasite or host cell origin seems to rely on the life cycle stage of the Plasmodium parasite and the corresponding host cell.     

Enhanced CD95-mediated apoptosis contributes to radiation hypersensitivity of <Emphasis Type="Italic">NBS</Emphasis> lymphoblasts

The molecular causes for enhanced radiosensitivity of Nijmegen Breakage Syndrome cells are unclear, especially as repair of DNA damage is hardly impeded in these cells. We clearly demonstrate that radiation hypersensitivity is accompanied by enhanced γ-radiation-induced apoptosis in NBS1 deficient lymphoblastoid cell lines. Differences in the apoptotic behavior of NBS1 ^−/− and NBS1 ^+/− cells are not due to an altered p53 stabilization or phosphorylation in NBS1 ^−/− cells. γ-radiation-induced caspase-8 activity is increased and visualization of CD95 clustering by laser scanning microscopy shows a significant higher activation of the death receptor in NBS1 ^−/− cells. Further investigation of the molecular mechanisms reveals a role for reactive oxygen species-triggered activation of CD95. These results demonstrate that NBS1 suppresses the CD95 death receptor-dependent apoptotic pathway after γ-irradiation and evidence is given that this is achieved by regulation of the PI3-K/AKT survival pathway.     

Apoptosis in pre-Bilaterians: <Emphasis Type="Italic">Hydra</Emphasis> as a model

Hydra is a member of the ancient metazoan phylum Cnidaria and is an especially well investigated model organism for questions of the evolutionary origin of metazoan processes. Apoptosis in Hydra is important for the regulation of cellular homeostasis under different conditions of nutrient supply. The molecular mechanisms leading to apoptosis in Hydra are surprisingly extensive and comparable to those in mammals. Genome wide sequence analysis has revealed the presence of large caspase and Bcl-2 families, the apoptotic protease activating factor (APAF-1), inhibitors of apoptotic proteases (IAPs) and components of a putative death receptor pathway. Regulation of apoptosis in Hydra may involve BH-3 only proteins and survival pathways, possibly including insulin signalling.     

NECROSIS OF LUNG EPITHELIAL CELLS BY FILARIAL PARASITIC PROTEIN VIA AN EARLY INDUCTION OF c-H-rasAND TNFα EXPRESSION

The direct interaction of filarial proteins with lung epithelial cells was examined to determine the possible mechanism of inducing cell death, an event that is observed in patients with tropical pulmonary eosinophilia. Exposure of lung epithelial cells to filarial parasitic proteins,Brugia malayi(BmA),Setaria digitata(Sd), and recombinant filarial protein (pGT 7)in vitrofor more than 2 days, causes the appearance of DNA fragments both in the cytoplasm and culture supernatants, while no fragmentation was observed in the untreated controls. The release of DNA fragments both in the cytoplasm and the culture supernatants simultaneously, indicates that cell death is induced by a necrotic event rather than apoptosis. Fluorescent-labelled studies also indicate the fragmentation of DNA increasing in a time-dependent manner. Normal cellular function is controlled through several oncogenes. The modulation of specific proto-oncogenes like myc, ras and TNFα during exposure to filarial parasitic proteins reveal elevated levels of expression of ras and TNFα as early as 2 hours, implicating their involvement prior to DNA fragmentation leading to pathogenesis.     

Induction of caspase 3 activation by multiple Legionella pneumophila Dot/Icm substrates

The intracellular pathogen Legionella pneumophila is able to strike a balance between the death and survival of the host cell during infection. Despite the presence of high level of active caspase 3, the executioner caspase of apoptotic cell death, infected permissive macrophages are markedly resistant to exogenous apoptotic stimuli. Several bacterial molecules capable of promoting the cell survival pathways have been identified, but proteins involved in the activation of caspase 3 remain unknown. To study the mechanism of L. pneumophila‐mediated caspase 3 activation, we tested all known Dot/Icm substrates for their ability to activate caspase 3. Five effectors capable of causing caspase 3 activation upon transient expression were identified. Among these, by using its ability to activate caspase 3 by inducing the release of cytochrome c from the mitochondria, we demonstrated that VipD is a phospholipase A2, which hydrolyses phosphatidylethanolamine (PE) and phosphocholine (PC) on the mitochondrial membrane in a manner that appears to require host cofactor(s). The lipase activity leads to the production of free fatty acids and 2‐lysophospholipids, which destabilize the mitochondrial membrane and may contribute to the release of cytochrome c and the subsequent caspase 3 activation. Furthermore, we found that whereas it is not detectably defectively in caspase 3 activation in permissive cells, amutant lacking all of these five genes is less potent in inducing apoptosis in dendritic cells. Our results reveal that activation of host cell death pathways by L. pneumophila is a result of the effects of multiple bacterial proteins with diverse biochemical functions.     

Role of apoptosis in autoimmunity

Apoptosis is a physiological form of cell death required to ensure that the rate of cell division is balanced by the rate of cell death in multicellular organisms. Dysregulation of apoptosis is associated with the pathogenesis of a wide array of diseases: cancer, neurodegeneration, autoimmunity, heart disease and others. In this review we collect arguments supporting a hypothesis of a dysregulated apoptosis leading to development of autoimmunity like systemic lupus erythematosus (SLE). This notion is supported by occurence of known autoantigens in apoptotic blebs, in vitro findings of an increased rate of apoptotic lymphoblasts despite optimal cytokine stimulation combined with a defective in vitro clearance of apoptotic bodies by SLE phagocytes. Moreover, we and others could generate histone-specific lymphocytic cell lines from cells after activation with autologous apoptotic material. These lymphocytes could stimulate autologous B-lymphocytes to produce of anti-dsDNA antibodies, a diagnostic hallmark for SLE. Finally, antibodies against phospholipids like phosphatidylserine are often associated with systemic autoimmunopathies like SLE and others. Phosphatidylserine is exposed on apoptotic cells as early sign of programmed cell death and serves as phagocyte recognition molecule for apoptotic cells. Formation of immune complexes and deposition in tissues might lead to organ damage and disease. This scenario will be discussed in this review in detail.     

<Emphasis Type="Italic">Aeromonas</Emphasis> Spp. Human Isolates Induce Apoptosis of Murine Macrophages

Interactions of Aeromonas caviae, Aeromonas veronii biotype sobria, and Aeromonas hydrophila strains isolated from fecal specimens of humans with gastroenteritis on murine macrophages, J774 cells, were investigated. Analyses of cellular morphology and DNA fragmentation in phagocytes infected with these strains exhibited typical characteristic features of cells undergoing apoptosis. We observed the morphological changes, including condensation of nuclear chromatin, formation of apoptotic bodies and blebbing of cell membrane, and fragmentation of nuclear DNA into oligonucleosomal fragments. The lowest apoptotic index did not exceed 25%, whereas the highest reached 78% at 24 h and 96% at 48 h after infection. After incubation of J774 cells with cytotoxic enterotoxin isolated from A. veronii biotype sobria strain, we noted that the toxin was able to trigger cytotoxicity and apoptosis of macrophages. The results indicate that apoptosis could be one of the mechanisms contributing to the development of Aeromonas-associated diarrheal disease.