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

IAP-IAP Complexes Required for Apoptosis Resistance of C. trachomatis–Infected Cells

Host cells infected with obligate intracellular bacteria Chlamydia trachomatis are profoundly resistant to diverse apoptotic stimuli. The molecular mechanisms underlying the block in apoptotic signaling of infected cells is not well understood. Here we investigated the molecular mechanism by which apoptosis induced via the tumor necrosis factor (TNF) receptor is prevented in infected epithelial cells. Infection with C. trachomatis leads to the up-regulation of cellular inhibitor of apoptosis (cIAP)-2, and interfering with cIAP-2 up-regulation sensitized infected cells for TNF-induced apoptosis. Interestingly, besides cIAP-2, cIAP-1 and X-linked IAP, although not differentially regulated by infection, are required to maintain apoptosis resistance in infected cells. We detected that IAPs are constitutively organized in heteromeric complexes and small interfering RNA–mediated silencing of one of these IAPs affects the stability of another IAP. In particular, the stability of cIAP-2 is modulated by the presence of X-linked IAP and their interaction is stabilized in infected cells. Our observations suggest that IAPs are functional and stable as heteromers, a thus far undiscovered mechanism of IAP regulation and its role in modulation of apoptosis

Antisense repression of the mitochondrial nadh-binding subunit of complex I in transgenic potato plants affects male fertility

Mitochondrial respiratory chain complex I is a large multi-subunit enzyme composed of both organellar and nuclear encoded proteins. To investigate the role of the nuclear encoded components, expression of the gene for the 55 kDa NADH-binding subunit of complex I was disturbed by antisense repression in transgenic potato plants. The antisense construct driven by the CaMV 35S promoter decreases the steady-state mRNA levels of transcripts for the 55 kDa subunit to 33% of the wild type levels. Quantities of the 55 kDa protein in mitochondrial protein extracts are lowered to about 50% in these plants. Transgenic plants show normal vegetative growth and tuber formation, but pollen maturation is found to be disturbed. The reduced male fertility of the transgenic 55 kDa antisense plants may be caused by an insufficient mitochondrial respiratory chain, impaired by the decreased expression of the NADH-binding component of mitochondrial complex I

Reduced Activation of Caspase-3 in Infected Cells

<div><p>(A) HeLa cells were infected (+) for 16 h or not (−) and treated for an additional 8 h with TNFα and CHX. Lysates of these cells were separated and probed with an antibody specifically recognizing the active p19/17 fragment of caspase-3 by immunoblot analysis.</p><p>(B) Blots from the same lysates used in (A) were probed for caspase-8 (casp-8 p18) and caspase-3 (casp-3 p19/17). The processing of BID as a specific substrate for caspase-8 is also shown.</p><p>(C) HeLa cells were infected and treated with TNF/CHX as mentioned before. Caspase-8 p60 (Casp-8) and −3 p32 (Casp-3) was detected by immunoblot analysis of the lysates. β-Actin staining was included as equal loading control.</p><p>(D) Active caspase-8 and caspase-3 were precipitated from the same lysates as in (A) using Biotin-VAD-fmk, and precipitated proteins were detected with antibodies for caspase-8 and caspase-3 as indicated.</p><p>(E) Active caspase-8 was monitored in living HeLa cells infected with Ctr and treated with TNFα/CHX (TNF) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020114#ppat-0020114-sd001" target="_blank">Protocol S1</a> (Detection of active caspases). Shown is the mean ± SD of three experiments.</p><p>(F) Caspase-3 p19 and p17 fragments were detected by immunoblotting in lysates of cells infected for the indicated times with Ctr and treated for 5 or 10 h with TNFα/CHX.</p><p>(G) Cells infected for the indicated time points with Ctr were treated with TNFα/CHX for 10 h, and the full-size caspase-8 (p60) and the cleavage products (p41/43; p18) were analyzed by immunoblotting.</p></div

Infection-Induced Up-regulation of cIAP-2

<div><p>(A) mRNA levels of different IAPs were monitored by qRT-PCR in HeLa cells infected (+) or not (−) with Ctr. The amount of cIAP-2 mRNA increases strongly in infected cells.</p><p>(B) The level of IAP expression was determined by immunoblot analysis in noninfected (−) and infected (+) cells. Note that cIAP-2 and <i>survivin</i> are strongly up-regulated in infected cells.</p><p>(C) Time course of cIAP-2 and <i>survivin</i> up-regulation was tested in infected cells by immunoblot analysis. Cells were infected and lysed at different time points postinfection as indicated.</p></div

IAPs Cross-Regulate Each Other

<p>siRNAs directed against (A) luciferase or XIAP, (B) cIAP-1, (C) cIAP-2, or (D) <i>survivin</i> were transfected in HeLa cells, and the protein levels of all IAPs were determined in infected (+) or uninfected (−) samples by immunoblot analysis as indicated.</p

IAPs Are Organized in Heteromeric Complexes

<div><p>(A) Endogenous cIAP-2 and XIAP were immunoprecipitated (IP) from control and apoptotic cells as indicated. The level of endogenous cIAP-2 and XIAP was determined in precipitates by immunoblotting.</p><p>(B) Endogenous IAPs were precipitated from control and TNF/CHX-treated cells and the coprecipitation of active caspase-3 was monitored by immunoblotting.</p><p>(C) Endogenous cIAP-2 was immunoprecipitated from control and infected cells treated or not with TNF/CHX as indicated. The level of endogenous cIAP-2 and XIAP was determined in precipitates (IP) and in lysates.</p><p>(D) The IAP complexes were isolated by gel filtration as mentioned in Materials and Methods. The proteins from different fractions were TCA-precipitated and resolved in SDS-PAGE, and the presence of IAPs was checked by immunoblot analysis as indicated. Note that high amounts of cIAP-1, XIAP, and cIAP-2 were present together in a protein complex of around 400 kDa. Shown are the fraction numbers (fractions) and the approximate size of proteins and complexes in the respective fractions in kDa.</p><p>(E) The cytosol from HeLa cells induced to apoptosis with TNF/CHX was isolated by subcellular fractionation, and gel filtration was performed as mentioned in Materials and Methods. The proteins were separated by SDS-PAGE, and the immunoblot analyses were performed as before.</p><p>(F) Gel filtration experiments were performed in XIAP-silenced cells and the presence of cIAP-1 and cIAP-2 in various fractions was tested by immunoblot analysis. Shown in (C), (D), and (E) are the data from one representative experiment.</p></div