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1.
Saskia Lehr Juliane Vier Georg Häcker Susanne Kirschnek 《Microbes and infection / Institut Pasteur》2018,20(5):284-292
The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial agent of sexually transmitted disease world-wide. Chlamydia trachomatis primarily infects epithelial cells of the genital tract but the infection may be associated with ascending infection. Infection-associated inflammation can cause tissue damage resulting in female infertility and ectopic pregnancy. The precise mechanism of inflammatory tissue damage is unclear but earlier studies implicate the chlamydial cryptic plasmid as well as responding neutrophils. We here rebuilt the interaction of Chlamydia trachomatis-infected epithelial cells and neutrophils in-vitro. During infection of human (HeLa) or mouse (oviduct) epithelial cells with Chlamydia trachomatis, a soluble factor was produced that attracted neutrophils and prolonged neutrophil survival, independently of Toll-like receptor signaling but dependent on the chlamydial plasmid. A number of cytokines, but most strongly GM-CSF, were secreted at higher amounts from cells infected with plasmid-bearing, compared to plasmid-deficient, bacteria. Blocking GM-CSF removed the secreted pro-survival activity towards neutrophils. A second, neutrophil TNF-stimulatory activity was detected in supernatants, requiring MyD88 or TRIF independently of the plasmid. The results identify two pro-inflammatory activities generated during chlamydial infection of epithelial cells and suggest that the epithelial cell, partly through the chlamydial plasmid, can initiate a myeloid immune response and inflammation. 相似文献
2.
Kawana K Quayle AJ Ficarra M Ibana JA Shen L Kawana Y Yang H Marrero L Yavagal S Greene SJ Zhang YX Pyles RB Blumberg RS Schust DJ 《The Journal of biological chemistry》2007,282(10):7368-7375
Chlamydia trachomatis is an obligate intracellular pathogen that can persist in the urogenital tract. Mechanisms by which C. trachomatis evades clearance by host innate immune responses are poorly described. CD1d is MHC-like, is expressed by epithelial cells, and can signal innate immune responses by NK and NKT cells. Here we demonstrate that C. trachomatis infection down-regulates surface-expressed CD1d in human penile urethral epithelial cells through proteasomal degradation. A chlamydial proteasome-like activity factor (CPAF) interacts with the CD1d heavy chain, and CPAF-associated CD1d heavy chain is then ubiquitinated and directed along two distinct proteolytic pathways. The degradation of immature glycosylated CD1d was blocked by the proteasome inhibitor lactacystin but not by MG132, indicating that degradation was not via the conventional proteasome. In contrast, the degradation of non-glycosylated CD1d was blocked by lactacystin and MG132, consistent with conventional cellular cytosolic degradation of N-linked glycoproteins. Immunofluorescent microscopy confirmed the interruption of CD1d trafficking to the cell surface, and the dislocation of CD1d heavy chains into both the cellular cytosol and the chlamydial inclusion along with cytosolic CPAF. C. trachomatis targeted CD1d toward two distinct proteolytic pathways. Decreased CD1d surface expression may help C. trachomatis evade detection by innate immune cells and may promote C. trachomatis persistence. 相似文献
3.
During infection of epithelial cells, the obligate intracellular pathogen Chlamydia trachomatis secretes the serine protease Chlamydia protease-like activity factor (CPAF) into the host cytosol to regulate a range of host cellular processes through targeted proteolysis. Here we report the development of an in vitro assay for the enzyme and the discovery of a cell-permeable CPAF zymogen-based peptide inhibitor with nanomolar inhibitory affinity. Treating C. trachomatis-infected HeLa cells with this inhibitor prevented CPAF cleavage of the intermediate filament vimentin and led to the loss of vimentin cage surrounding the intracellular vacuole. Because Chlamydia is a genetically intractable organism, this inhibitor may serve as a tool for understanding the role of CPAF in pathogenesis. 相似文献
4.
Pirbhai M Dong F Zhong Y Pan KZ Zhong G 《The Journal of biological chemistry》2006,281(42):31495-31501
Chlamydia trachomatis has evolved a profound anti-apoptotic activity that may aid in chlamydial evasion of host defense. The C. trachomatis anti-apoptotic activity has been correlated with blockade of mitochondrial cytochrome c release, inhibition of Bax and Bak activation, and degradation of BH3-only proteins. This study presents evidence that a chlamydia-secreted protease factor designated CPAF is both necessary and sufficient for degrading the BH3-only proteins. When the C. trachomatis-infected cell cytosolic extracts were fractionated by column chromatography, both the CPAF protein and activity elution peaks overlapped with the BH3-only protein degradation activity peak. Depletion of CPAF with a CPAF-specific antibody removed the BH3-only protein degradation activity from the infected cell cytosolic extracts, whereas depletion with control antibodies failed to do so. Notably, recombinant CPAF expressed in bacteria was able to degrade the BH3-only proteins, whereas CPAF mutants similarly prepared from bacteria failed to do so. Finally, bacterium-expressed CPAF also degraded the human BH3-only protein Pumaalpha purified from bacteria. These results demonstrate that CPAF contributes to the chlamydial anti-apoptotic activity by degrading the pro-apoptotic BH3-only Bcl-2 subfamily members. 相似文献
5.
Murphey C Murthy AK Meier PA Neal Guentzel M Zhong G Arulanandam BP 《Cellular immunology》2006,242(2):110-117
We have previously determined the protective efficacy of intranasal vaccination with chlamydial protease-like activity factor (CPAF) against genital chlamydial infection. Since T-helper 1 (Th1) responses are important for anti-chlamydial immunity, we examined the contribution of CD4(+) T cells in CPAF mediated immunity against intravaginal (i.vag.) Chlamydia muridarum infection in C57BL/6 mice. CPAF+IL-12 vaccination induced antigen-specific CD4(+) T cells that secreted elevated levels of IFN-gamma, and generated strong humoral responses. The protective effects of CPAF vaccination against genital chlamydial challenge were abrogated by anti-CD4 neutralizing antibody treatment. Moreover, anti-chlamydial immunity could be adoptively transferred to na?ve recipients using CPAF-specific CD4(+) T cells. Therefore, CPAF mediated anti-chlamydial immunity is highly dependent upon antigen-specific CD4(+) T cells. 相似文献
6.
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. 相似文献
7.
Giraud A Chabaud O Lejeune PJ Barbaria J Mallet B 《Biochemical and biophysical research communications》2006,346(3):746-750
Plasminogen (Pl), a circulating protease synthesized in the liver, is also present in several tissues. In the thyroid gland a Pl-like protease was found in the apical lumen where it is involved, through its proteolytic activity, in luminal degradation of thyroglobulin (Tg). Here, we showed for the first time that the Pl-like protease apically secreted by epithelial thyroid cells is sulfated, both on tyrosine residue(s) and on oligosaccharide side chains. The Pl molecule is composed of a large N-terminal moiety made of five distinct Kringle domains (K1-K5) separated by small peptidic fragments, and of a C-terminal domain with serine protease activity. Using a software tool able to predict tyrosine sulfation sites in protein sequences we localized the potential tyrosine sulfation sites of Pl. Then, we became aware that, whatever the species considered, at least three of the four potential tyrosine sulfation sites of Pl were located on Kringle sites, and more precisely, for K1, on the highly conserved binding domain of K1. We determined with the same software tool which potential sulfation sites were the most likely to be really sulfated. We hypothesize that the sulfation of these sites modulates the binding properties of Pl. 相似文献
8.
Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-beta1 (transforming growth factor beta1). Here we demonstrate a novel role of Id-1 in promoting TGF-beta1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-beta1, which was associated with increased cell-substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-beta1-induced cell motility was mediated through activation of MEK-ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-beta1-treated cells through down-regulation of E-cadherin, redistribution of beta-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell-cell adhesion in response to TGF-beta1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-beta1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis. 相似文献
9.
Hiiragi T Sasaki H Nagafuchi A Sabe H Shen SC Matsuki M Yamanishi K Tsukita S 《The Journal of biological chemistry》1999,274(48):34148-34154
Transglutaminase type 1 was identified as a tyrosine-phosphorylated protein from the isolated junctional fraction of the mouse liver. This enzyme was reported to be involved in the covalent cross-linking of proteins in keratinocytes, but its expression and activity in other cell types have not been examined. Northern blotting revealed that transglutaminase type 1 was expressed in large amounts in epithelial tissues (lung, liver, and kidney), which was also confirmed by immunoblotting with antibodies raised against mouse recombinant protein. Immunoblotting of the isolated junctional fraction revealed that transglutaminase type 1 was concentrated in the fraction not only as a 97-kDa form but also as forms of various molecular masses cross-linked to other proteins. In agreement with this finding, endogenous transglutaminase type 1 was immunofluorescently colocalized with E-cadherin in cultured simple epithelial cells. In the liver and kidney, immunoelectron microscopy revealed that transglutaminase type 1 was concentrated, albeit not exclusively, at cadherin-based adherens junctions. Furthermore, by in vitro and in vivo labeling, transglutaminase cross-linking activity was also shown to be concentrated at intercellular junctions of simple epithelial cells. These findings suggested that the formation of covalently cross-linked multimolecular complexes by transglutaminase type 1 is an important mechanism for maintenance of the structural integrity of simple epithelial cells, especially at cadherin-based adherens junctions. 相似文献
10.
Tight junction formation in cultured epithelial cells (MDCK) 总被引:21,自引:0,他引:21
L. Gonzalez-Mariscal B. Chávez de Ramírez M. Cereijido 《The Journal of membrane biology》1985,86(2):113-125
Summary Synthesis and assembly of tight junctions are studied in monolayers of MDCK cells plated at a density sufficient for confluence, allowed to attach for 1 hr, and transferred to fresh media without cells containing or not Ca2+, 20 hr later, while monolayers with Ca2+ have fully developed junctions that confer an electrical resistance across of 346±51 cm2, those without Ca2+ have a negligible resistance. If at this time Ca2+ is added, junctions assemble and seal with a fast kinetics, that can be followed through the development of electrical resistance, penetration of ruthenium red, and electron microscopy. Drugs that impair synthesis, maturation and transport of proteins (cycloheximide, tunicamycin, monensin) indicate that protein components are synthesized early upon plating, do not seem to require N-glycosylation, and are stored in the Golgi compartment. Upon addition of Ca2+ they are transferred to the membrane with the participation of microfilaments but not of microtubules. These components seem to insert directly in the position they occupy in the strands, and the cell circles its perimeter with one strand as early as 15 min, even if in some segments it only consists of a row of particles. New strands develop in association with previous ones, and the pattern completes in 4 to 6 hr. Ca2+ is required for the maintenance of the assembly and also for the sealing with neighboring cells. These processes cannot occur below 25°C. Serum is not required. Polarized distribution of intramembrane particles (IMP) in apical and basolateral regions follows the same time course as junction formation, in spite of the fence constituted by those strands that are already assembled. This suggests that IMP do not redistribute by lateral displacements in the plane of the membrane, but by removal and insertion in the apical and basolateral domains. 相似文献
11.
Yoko Ito Rachel Zemans Kelly Correll Ivana V. Yang Aftab Ahmad Bifeng Gao Robert J. Mason 《Biochemical and biophysical research communications》2014
Alveolar type II (ATII) cells remain differentiated and express surfactant proteins when cultured at an air–liquid (A/L) interface. When cultured under submerged conditions, ATII cells dedifferentiate and change their gene expression profile. We have previously shown that gene expression under submerged conditions is regulated by hypoxia inducible factor (HIF) signaling due to focal hypoxia resulting from ATII cell metabolism. Herein, we sought to further define gene expression changes in ATII cells cultured under submerged conditions. We performed a genome wide microarray on RNA extracted from rat ATII cells cultured under submerged conditions for 24–48 h after switching from an A/L interface. We found significant alterations in gene expression, including upregulation of the HIF target genes stanniocalcin-1 (STC1), tyrosine hydroxylase (Th), enolase (Eno) 2, and matrix metalloproteinase (MMP) 13, and we verified upregulation of these genes by RT-PCR. Because STC1, a highly evolutionarily conserved glycoprotein with anti-inflammatory, anti-apoptotic, anti-oxidant, and wound healing properties, is widely expressed in the lung, we further explored the potential functions of STC1 in the alveolar epithelium. We found that STC1 was induced by hypoxia and HIF in rat ATII cells, and this induction occurred rapidly and reversibly. We also showed that recombinant human STC1 (rhSTC1) enhanced cell motility with extended lamellipodia formation in alveolar epithelial cell (AEC) monolayers but did not inhibit the oxidative damage induced by LPS. We also confirmed that STC1 was upregulated by hypoxia and HIF in human lung epithelial cells. In this study, we have found that several HIF target genes including STC1 are upregulated in AECs by a submerged condition, that STC1 is regulated by hypoxia and HIF, that this regulation is rapidly and reversibly, and that STC1 enhances wound healing moderately in AEC monolayers. However, STC1 did not inhibit oxidative damage in rat AECs stimulated by LPS in vitro. Therefore, alterations in gene expression by ATII cells under submerged conditions including STC1 were largely induced by hypoxia and HIF, which may be relevant to our understanding of the pathogenesis of various lung diseases in which the alveolar epithelium is exposed to relative hypoxia. 相似文献
12.
Drannik AG Nag K Yao XD Henrick BM Jain S Ball TB Plummer FA Wachihi C Kimani J Rosenthal KL 《Journal of virology》2012,86(8):4599-4610
Cervicovaginal lavage fluid (CVL) is a natural source of anti-HIV-1 factors; however, molecular characterization of the anti-HIV-1 activity of CVL remains elusive. In this study, we confirmed that CVLs from HIV-1-resistant (HIV-R) compared to HIV-1-susceptible (HIV-S) commercial sex workers (CSWs) contain significantly larger amounts of serine antiprotease trappin-2 (Tr) and its processed form, elafin (E). We assessed anti-HIV-1 activity of CVLs of CSWs and recombinant E and Tr on genital epithelial cells (ECs) that possess (TZM-bl) or lack (HEC-1A) canonical HIV-1 receptors. Our results showed that immunodepletion of 30% of Tr/E from CVL accounted for up to 60% of total anti-HIV-1 activity of CVL. Knockdown of endogenous Tr/E in HEC-1A cells resulted in significantly increased shedding of infectious R5 and X4 HIV-1. Pretreatment of R5, but not X4 HIV-1, with either Tr or E led to inhibition of HIV-1 infection of TZM-bl cells. Interestingly, when either HIV-1 or cells lacking canonical HIV-1 receptors were pretreated with Tr or E, HIV-1 attachment and transcytosis were significantly reduced, and decreased attachment was not associated with altered expression of syndecan-1 or CXCR4. Determination of 50% inhibitory concentrations (IC(50)) of Tr and E anti-HIV-1 activity indicated that E is ~130 times more potent than its precursor, Tr, despite their equipotent antiprotease activities. This study provides the first experimental evidence that (i) Tr and E are among the principal anti-HIV-1 molecules of CVL; (ii) Tr and E affect cell attachment and transcytosis of HIV-1; (iii) E is more efficient than Tr regarding anti-HIV-1 activity; and (iv) the anti-HIV-1 effect of Tr and E is contextual. 相似文献
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Poly(ADP-ribose) polymerase-1 is a survival factor for radiation-exposed intestinal epithelial stem cells in vivo 下载免费PDF全文
Ishizuka S Martin K Booth C Potten CS de Murcia G Bürkle A Kirkwood TB 《Nucleic acids research》2003,31(21):6198-6205
Poly(ADP-ribose) polymerase-1 (PARP-1) is a key enzyme mediating the cellular response to DNA strand breaks. It plays a critical role in genomic stability and survival of proliferating cells in culture undergoing DNA damage. Intestinal epithelium is the most proliferative tissue in the mammalian body and its stem cells show extreme sensitivity to low-level genotoxic stress. We investigated the role of PARP-1 in the in vivo damage response of intestinal stem cells in crypts of PARP-1–/– and control mice following whole-body γ-irradiation (1 Gy). In the PARP-1–/– mice there was a significant delay during the first 6 h in the transient p53 accumulation in stem cells whereas an increased number of cells were positive for p21CIP1/WAF1. Either no or only marginal differences were noted in MDM2 expression, apoptosis, induction of or recovery from mitotic blockage, or inhibition of DNA synthesis. We further observed a dose-dependent reduction in crypt survival measured at 4 days post-irradiation in control mice, and this crypt-killing effect was significantly potentiated in PARP-1–/– mice. Our results thus establish that PARP-1 acts as a survival factor for intestinal stem cells in vivo and suggest a functional link with early p53 and p21CIP1/WAF1 responses. 相似文献
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Interactions between Chlamydia trachomatis, host cells, and the immune system are believed to involve lipopolysaccharide (LPS). We used immunogold techniques to study the distribution of chlamydial LPS in cultured cells infected with C. trachomatis LGV-L1. McCoy cells inoculated with C. trachomatis were cultured and then fixed and embedded in situ with acrylic resins. Sections were immunolabeled with a protein A-gold method using antisera to the genus-specific, periodate-sensitive epitope on chlamydial LPS. Pre-embedding immunogold labeling on permeabilized cells was also done. By post-embedding methods, labeling for LPS was equally abundant over the outer membranes of elementary (EB) and reticulate bodies (RB). By post-embedding labeling, the sub-surface side of the EB outer membrane was more heavily labeled than the surface side. By pre-embedding labeling, LPS was found to be less abundant on the surface of EBs than RBs. Labeling for LPS was found over apparent lysosomes in McCoy cells and over electron-dense blebs on or near the surface of the plasma membranes of McCoy cells. These results indicate that the concentration of LPS in chlamydial membranes is constant during development but that with development its location changes from being mostly cell-surface to sub-surface. These results show that the post-embedding immunogold technique can be a useful approach for the cell culture-based study of chlamydial LPS. 相似文献
17.
Prostasin is expressed at the apical surface of normal epithelial cells and suppresses in vitro invasion of cancer cells. Prostasin re-expression in the PC-3 prostate carcinoma cells down-regulated the epidermal growth factor receptor (EGFR) protein expression and EGF-induced phosphorylation of the extracellular signal-regulated kinases (Erk1/2). We report here that prostasin and its activating enzyme matriptase are capable of inducing proteolytic cleavages in the EGFR extracellular domain (ECD) when co-expressed in the FT-293 cells, generating two amino-terminally truncated fragments EGFR135 and EGFR110, at 135 and 110 kDa. Prostasin's role in EGFR cleavage is dependent on the serine active-site but not the GPI-anchor. The modifications of EGFR were confirmed to be on the primary structure by deglycosylation. EGFR135 and EGFR110 are not responsive to EGF stimulation, indicating loss of the ligand-binding domains. EGFR110 is constitutively phosphorylated and in its presence Erk1/2 phosphorylation is increased in the absence of EGF. The protease-induced EGFR cleavages are not dependent on EGFR phosphorylation. The EGFR ECD proteolytic modification by matriptase-prostasin is also observed in the BEAS-2B normal lung epithelial cells, the BPH-1 benign prostate hyperplasia and the MDA-MB-231 breast cancer cell lines; and represents a novel mechanism for epithelial cells to modulate EGF-EGFR signaling. 相似文献
18.
Dintilhac A Bihan R Guerrier D Deschamps S Bougerie H Watrin T Bonnec G Pellerin I 《The International journal of developmental biology》2005,49(7):851-858
While studies have highlighted the role of HOXA9-13 and PBX1 homeobox genes during the development of the female genital tract, the molecular mechanisms triggered by these genes are incompletely elucidated. In several developmental pathways, PBX1 binds to MEINOX family members in the cytoplasm to be imported into the nucleus where they associate with HOX proteins to form a higher complex that modulates gene expression. This concept has been challenged by a recent report showing that in some cell cultures, PBX1 nuclear localization might be regulated independently of MEINOX proteins (Kilstrup-Nielsen et al., 2003). Our work gives the first illustration of this alternative mechanism in an organogenesis process. Indeed, we show that PBX1 is mostly cytoplasmic in epithelial endometrial cells of the developing female genital tract despite the nuclear localization of MEIS1. We thus provide evidence for a control of PBX1 intracellular distribution which is independent of MEINOX proteins, but is cell cycle correlated. 相似文献
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Epstein-Barr virus latent membrane protein 1 (LMP-1) half-life in epithelial cells is down-regulated by lytic LMP-1 下载免费PDF全文
This study examined the effect of naturally occurring Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene sequence variation on the LMP-1 half-life in epithelial cells. The LMP-1 half-life was not influenced by sequence variation in amino acids 250 to 307 or amino acids 343 to 352. The LMP-1 half-life was short when the amino acid encoded at position 129 was methionine, the initiation codon product of lytic LMP-1 (lyLMP-1). The mutation of amino acid 129 to isoleucine greatly increased the LMP-1 half-life. Expression of lyLMP-1 in trans down-regulated the LMP-1 half-life in a dose-dependent manner and restored a short-half-life phenotype to the mutated LMP-1 construct lacking the cis ability to express lyLMP-1. This observed dominant negative effect of lyLMP-1 expression on the LMP-1 half-life in epithelial cells in vitro may have implications for EBV epithelial oncogenesis in vivo. 相似文献