Mcl-1 is a key regulator of apoptosis resistance in Chlamydia trachomatis-infected cells

Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.     

Apoptosis of host cells regulation by Chlamydia

In a study on the impact of chlamydial infection on host cell apoptosis, C. trachomatis were shown to protect host cell against staurosporin-induced apoptosis only at the middle stage of infection development (at 20 hours post infection), C. pneumoniae--at different stages of its growth cycle (from 2 to 7 day post infection). We found, that C. trachomatis elementary bodies fail to inhibit staurosporin-induced apoptotic stimuli. The clear antiapoptotic effect of cell lysate filtrate, infected with C. trachomatis, was demonstrated by cytometric analysis and luminescent microscopy. Our findings make it possible to use biochemical approach to identification of chlamydial antiapoptotic factors in future. Investigations directed at chlamydial antiapoptotic activities may aim to create the therapies of chronic chlamydial infection.     

The neuropeptide FMRFamide can protect cells against apoptosis in the snail digestive gland

FMRFamide-related peptides are widespread neurotransmitters or neurohormones regulating somatic or visceral motor activity. Some recent data indicate that these neuropeptides may be involved in the control of cell proliferation and apoptosis. In this work we investigated the possible effect of FMRFamide on cell viability in an invertebrate-type proliferating tissue. As a model, we used the midintestinal gland of the snail, Helix lucorum Linnaeus. Immunohistochemistry demonstrated the direct innervation of the gland cells by FMRFamide-containing nerve fibers. Midintestinal glands of snails were injected with 50 μM FMRFamide and the control with sterile deionised water or bovine serum albumin (BSA). Injections were administrated 4 times. Transmission electron microscopy, annexin V-labeling, thiazolyl blue (MTT) viability tests and ploidy analyses were carried out to define the viable/dead cell ratio in the tissue samples. FMRFamide increased the MTT-reduction of tissues, reduced the amount of apoptotic nuclei and annexin V-labeled cells. Deionised water or BSA injection induced cell death. Cell cyle analysis revealed that FMRFamide significantly elevated the amount of cells in G₀/G₁ phase, but did not induce mitosis. We conclude, that the FMRFamide can be a life-signal for cells, protect them from apoptosis without altering mitosis. The project was supported by OTKA grant No. T 042762.     

Sphingosine 1-phosphate induces Mcl-1 upregulation and protects multiple myeloma cells against apoptosis

Sphingosine 1-phosphate (S1P) is a bioactive lysophospholipid which is known to induce diverse cellular responses through at least five G-protein-coupled receptors on various cell types. However, neither the distribution of S1P receptors nor the effects of S1P on multiple myeloma (MM) cells are fully understood. Here, we show that MM cells express the S1P receptors, S1P1, S1P2, and S1P3. Furthermore, S1P protects MM cells against Dex-induced apoptosis. Importantly, S1P upregulates Mcl-1 expression in a time- and concentration-dependent manner in human MM cell lines. Treatment of MM cells with pertussis toxin (PTX), a pan-S1P receptor inhibitor, results in blockage of S1P-induced upregulation of Mcl-1. These data demonstrate that S1P upregulates the expression of Mcl-1 and protects MM cells from Dex-induced apoptosis, providing the preclinical framework for novel therapeutics targeting at both Mcl-1 and/or S1P to improve the patient outcome in MM.     

Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis

We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E₂ analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients.     

Expression and Targeting of Secreted Proteins from Chlamydia trachomatis

Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a vacuole termed the inclusion. Many of the interactions of chlamydiae with the host cell are dependent upon bacterial protein synthesis and presumably exposure of these proteins to the cytosol. Because of the dearth of genetic tools for chlamydiae, previous studies examining secreted proteins required the use of heterologous bacterial systems. Recent advances in genetic manipulation of chlamydia now allow for transformation of the bacteria with plasmids. We describe here a shuttle vector system, pBOMB4, that permits expression of recombinant proteins under constitutive or conditional promoter control. We show that the inclusion membrane protein IncD is secreted in a type III-dependent manner from Yersinia pseudotuberculosis and also secreted from C. trachomatis in infected cells where it localizes appropriately to the inclusion membrane. IncD truncated of the first 30 amino acids containing the secretion signal is no longer secreted and is retained by the bacteria. Cytosolic exposure of secreted proteins can be confirmed by using CyaA, GSK, or microinjection assays. A protein predicted to be retained within the bacteria, NrdB is indeed localized to the chlamydia. In addition, we have shown that the chlamydial effector protein, CPAF, which is secreted into the host cell cytosol by a Sec-dependent pathway, also accesses the cytosol when expressed from this system. These assays should prove useful to assess the secretion of other chlamydial proteins that are potentially exposed to the cytosol of the host cell.     

Chlamydia trachomatis inclusions induce asymmetric cleavage furrow formation and ingression failure in host cells

Chlamydia trachomatis infection has been suggested to induce host genome duplication and is linked to increased risks of cervical cancer. We describe here the mechanism by which Chlamydia causes a cleavage furrow defect that consistently results in the formation of multinucleated host cells, a phenomenon linked to tumorigenesis. Host signaling proteins essential for cleavage furrow initiation, ingression, and stabilization are displaced from one of the prospective furrowing cortices after Chlamydia infection. This protein displacement leads to the formation of a unique asymmetrical, unilateral cleavage furrow in infected human cells. The asymmetrical distribution of signaling proteins is caused by the physical presence of the Chlamydia inclusion at the cell equator. By using ingested latex beads, we demonstrate that the presence of a large vacuole at the cell equator is sufficient to cause furrow ingression failure and can lead to multinucleation. Interestingly, internalized latex beads of similar size do not localize to the cell equator as efficiently as Chlamydia inclusions; moreover, inhibition of bacterial protein synthesis with antibiotic reduces the frequency at which Chlamydia localizes to the cell equator. Together, these results suggest that Chlamydia effectors are involved in strategic positioning of the inclusion during cell division.     

NF-kappa B activation is not required for Chlamydia trachomatis inhibition of host epithelial cell apoptosis

Chlamydia trachomatis, an obligate intracellular bacterial species, is known to inhibit host cell apoptosis. However, the chlamydial antiapoptotic mechanism is still not clear. Because NF-kappaB activation is antiapoptotic, we tested the potential role of NF-kappaB activation in chlamydial antiapoptotic activity in the current study. First, no obvious NF-kappaB activation was detected in the chlamydia-infected cells when these cells were resistant to apoptosis induced via either the intrinsic or extrinsic apoptosis pathways. Second, inhibition of NF-kappaB activation with pharmacologic reagents failed to block the chlamydial antiapoptotic activity. Finally, NF-kappaB p65 gene deletion did not prevent chlamydia from inhibiting host cell apoptosis. These observations together have demonstrated that NF-kappaB activation is not required for the chlamydial antiapoptotic activity.     

Naf1alpha is phosphorylated in mitotic phase and required to protect cells against apoptosis

Naf1α is an HIV Nef-associated factor expressed ubiquitously in human cells. Previously, we reported that Naf1α is phosphorylated with EGF through MEK/ERK2 pathway. In this study, we found an additional phosphorylation of Naf1α when cells are in mitotic phase (M phase) or arrested in M phase with anti-mitosis reagents, and disappeared when the cells exit from mitotic phase to G1 phase. Furthermore, we demonstrated that Naf1α plays an important role in preventing cells from apoptosis: over-expression of Naf1α in Saos-2 cells suppressed trichostatin A (TSA)-induced apoptosis either of random culture or of cell population synchronized in M phase. In addition, knock-down of Naf1α expression with small interfering RNA sensitized Saos-2 cells to TSA-induced apoptosis. Physiological significance of these findings is discussed in relation to protection of cells from the apoptosis induction.     

Inhibitor of Apoptosis Proteins Physically Interact with and Block Apoptosis Induced by Drosophila Proteins HID and GRIM

Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183–10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins.     

Nitrites can be reduced in retinal vessels during hypoxia and protect the retina against ischemia and apoptosis

The possibility and the mechanism of the reduction of nitrites in retinal vessels under acute hypoxia in vivo have been investigated. An experimental model of rat retinal ischemia was elaborated using laser coagulation of retinal vessels. It was demonstrated that vessel thrombosis does not occur if the nitrite concentration in the vessels is increased. It was proposed that, under acute hypoxia, nitrites are reduced to NO, which results in drastic vasodilatation. Considering that the effect takes less than a minute, this reduction cannot be due to hypoxic acidosis but is more likely associated with NO reduction by heme proteins. It was found that increased concentration of nitrites protects the retina from the development of ischemia progress and that preliminary administration of nitrites prevents apoptosis in the retina and a decrease in its photoelectric activity.     

Prevention of cytokine withdrawal-induced apoptosis by Mcl-1 requires interaction between Mcl-1 and Bim

Growth factor withdrawal from hemopoietic cells results in activation of the mitochondrial pathway of apoptosis. Members of the Bcl-2 family regulate this pathway, with anti-apoptotic members counteracting the effects of pro-apoptotic members. We investigated the effect on Mcl-1 function of mutation at a conserved threonine 163 residue (T163) in its proline, glutamate, serine, and threonine rich (PEST) region. Under normal growth conditions, Mcl-1 half-life increased with alteration of T163 to glutamic acid, but decreased with mutation to alanine. However, both T163 mutants exhibited greater pro-survival effects compared with the wild type, which can be explained by an increased stability of the T163A mutant in cytokine-starved conditions. Both the mutant forms exhibited prolonged binding to pro-apoptotic Bim in cytokine-deprived cells. The extent to which Mcl-1 mutants were able to exert their anti-apoptotic effects correlated with their ability to associate with Bim. We further observed that primary bone marrow derived macrophages survived following cytokine withdrawal as long as Bim and Mcl-1 remained associated. In our study, we were unable to detect a role for GSK-3-mediated regulation of Mcl-1 expression. Based on these results we propose that upon cytokine withdrawal, survival of hemopoietic cells depends on association between Mcl-1 and Bim. Furthermore, alteration of T163 of Mcl-1 may change the protein such that its association with Bim is affected, resulting in prolonged association and increased survival.     

The effect of Chlamydia trachomatis infection on the host cell cytoskeleton and membrane compartments

Human epithelial cells and the McCoy cell line were infected with Chlamydia trachomatis, serotype E. The organization of the cytoplasm was then studied with probes which stained cytoskeletal components and membrane compartments. The major actin-containing stress fibre bundles were not associated with inclusions due to the peri-basal and peri-apical location of these bundles within the host cell. The cytokeratin network was distorted by the presence of inclusions so that a common basket of these intermediate filaments surrounded both nucleus and peri-nuclear inclusions. The microtubule network was similarly distorted, but the nucleus and inclusion were surrounded by separate rather than joint baskets of tubules. After reversible depolymerization by nocadazole the microtubules in amniotic epithelial cells began to reassemble at the peri-nuclear microtubule-organizing centre, so that independent microtubule networks were rapidly regenerated around the nucleus and inclusion. Mitochondria of amniotic epithelial cells were vitally stained with the fluorescent probe DiOC6 (3,3'-dihexyloxacarbocyanine iodide) after 48 h of infection and found to be widely distributed throughout the host cytoplasm. When the morphology of the Golgi complex was examined with C6-NBD-ceramide (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)] aminocaproyl sphingosine) the main cisternae were retained in a juxta-nuclear position, although scattered stained structures were also present close to the cytoplasmic surface of the inclusion. These results demonstrate that the peri-nuclear position of inclusions is determined by the configuration of the cytoskeleton, and that normal host-cell architecture is maintained during infection, albeit in a distorted form.     

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia -containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia -containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis .     

Chlamydia trachomatis co-opts GBF1 and CERT to acquire host sphingomyelin for distinct roles during intracellular development

The obligate intracellular pathogen Chlamydia trachomatis replicates within a membrane-bound inclusion that acquires host sphingomyelin (SM), a process that is essential for replication as well as inclusion biogenesis. Previous studies demonstrate that SM is acquired by a Brefeldin A (BFA)-sensitive vesicular trafficking pathway, although paradoxically, this pathway is dispensable for bacterial replication. This finding suggests that other lipid transport mechanisms are involved in the acquisition of host SM. In this work, we interrogated the role of specific components of BFA-sensitive and BFA-insensitive lipid trafficking pathways to define their contribution in SM acquisition during infection. We found that C. trachomatis hijacks components of both vesicular and non-vesicular lipid trafficking pathways for SM acquisition but that the SM obtained from these separate pathways is being utilized by the pathogen in different ways. We show that C. trachomatis selectively co-opts only one of the three known BFA targets, GBF1, a regulator of Arf1-dependent vesicular trafficking within the early secretory pathway for vesicle-mediated SM acquisition. The Arf1/GBF1-dependent pathway of SM acquisition is essential for inclusion membrane growth and stability but is not required for bacterial replication. In contrast, we show that C. trachomatis co-opts CERT, a lipid transfer protein that is a key component in non-vesicular ER to trans-Golgi trafficking of ceramide (the precursor for SM), for C. trachomatis replication. We demonstrate that C. trachomatis recruits CERT, its ER binding partner, VAP-A, and SM synthases, SMS1 and SMS2, to the inclusion and propose that these proteins establish an on-site SM biosynthetic factory at or near the inclusion. We hypothesize that SM acquired by CERT-dependent transport of ceramide and subsequent conversion to SM is necessary for C. trachomatis replication whereas SM acquired by the GBF1-dependent pathway is essential for inclusion growth and stability. Our results reveal a novel mechanism by which an intracellular pathogen redirects SM biosynthesis to its replicative niche.     

Trifluoperazine inhibits the infectivity of Chlamydia trachomatis for McCoy cells

Abstract Trifluoperazine (TFP), an inhibitor of the Ca²⁺-binding protein calmodulin, was used to study the infectivity of Chlamydia trachomatis for McCoy cells. TFP inhibited the number of chlamydial inclusions and the chlamydia-dependent amino acid incorporation when added within 9 h after inoculation with chlamydiae. However, TFP did not affect the attachment of chlamydiae to the cells or the protease-removable fraction of cell-bound chlamydiae.
These results suggest that an early step in the intracellular development of chlamydiae, partly coinciding with the elementary body-reticulate body conversion, is sensitive to TFP and that clathrin coats are not crucial in the ingestion of chlamydiae by McCoy cells.     

The development of Chlamydia trachomatis inclusions within the host eukaryotic cell during interphase and mitosis

The dynamic nature of Chlamydia trachomatis inclusions was studied by video and 35 mm time-lapse photomicrography of live cells, and by immunolocalization of inclusions in fixed cells. A serotype E isolate was used to infect the MCCoy cell line and endometrial epithelia. Then resulting inclusions were observed over 4 d. They appeared as slowly expanding fluid-filled membrane vesicles whose growth varied considerably, and which were subject to great physical distortion by the host cell during interphase and mitosis. When this distortion became extreme the inclusion was observed to divide. However, as inclusions were mobile within the cytoplasm and thus able to come into contact with each other, there was a net tendency for the opposite process of inclusion fusion to occur when cells contained more than one inclusion. The proportion of infected cells decreased with time as a result of host cell proliferation, despite transmission of inclusions to progeny at the time of mitosis. Inclusion growth physically disrupted karyokinesis and cytokinesis so that host cell division became distorted or blocked on the second or third day of infection. Cell death eventually occurred by a very rapid lysis event.     

Clitocine potentiates TRAIL-mediated apoptosis in human colon cancer cells by promoting Mcl-1 degradation

Among anti-cancer candidate drugs, TRAIL might be the most specific agent against cancer cells due to its low toxicity to normal cells. Unfortunately, cancer cells usually develop drug resistance to TRAIL, which is a major obstacle for its clinical application. One promising strategy is co-administrating with sensitizer to overcome cancer cells resistance to TRAIL. Clitocine, a natural amino nucleoside purified from wild mushroom, is recently demonstrated that can induce apoptosis in multidrug-resistant human cancer cells by targeting Mcl-1. In the present study,we found that pretreatment with clitocine dramatically enhances TRAIL lethality in its resistant human colon cancer cells by inducing apoptosis. More importantly, combination of clitocine and TRAIL also effectively inhibits xenograft growth and induces tumor cells apoptosis in athymic mice. The disruption of the binding between Mcl-1 and Bak as well as mitochondrial translocation of Bax mediated by clitocine are identified as the key underlying mechanisms, which leading to mitochondrial membrane permeabilization. Enforced exogenous Mcl-1 can effectively attenuate clitocine/TRAIL-induced apoptosis by suppressing the activation of intrinsic apoptotic pathway. Furthermore, clitocine regulates Mcl-1 expression at the posttranslational level as no obvious change is observed on mRNA level and proteasome inhibitor MG132 almost blocks the Mcl-1 suppression by clitocine. In fact, more ubiquitinated Mcl-1 was detected under clitocine treatment. Our findings indicate that clitocine is potentially an effective adjuvant agent in TRAIL-based cancer therapy.