scholarly journals Cholesterol Depletion Reduces Entry of Campylobacter jejuni Cytolethal Distending Toxin and Attenuates Intoxication of Host Cells

2011 ◽  
Vol 79 (9) ◽  
pp. 3563-3575 ◽  
Author(s):  
Chia-Der Lin ◽  
Cheng-Kuo Lai ◽  
Yu-Hsin Lin ◽  
Jer-Tsong Hsieh ◽  
Yu-Ting Sing ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Lipid Rafts ◽  
Campylobacter Jejuni ◽  
Host Cells ◽  
Membrane Cholesterol ◽  
Cholesterol Depletion ◽  
Cytolethal Distending Toxin ◽  
Content Type ◽  
Cycle Arrest

ABSTRACTCampylobacter jejuniis a common cause of pediatric diarrhea worldwide. Cytolethal distending toxin, produced byCampylobacter jejuni, is a putative virulence factor that induces cell cycle arrest and apoptosis in eukaryotic cells. Cellular cholesterol, a major component of lipid rafts, has a pivotal role in regulating signaling transduction and protein trafficking as well as pathogen internalization. In this study, we demonstrated that cell intoxication byCampylobacter jejunicytolethal distending toxin is through the association of cytolethal distending toxin subunits and membrane cholesterol-rich microdomains. Cytolethal distending toxin subunits cofractionated with detergent-resistant membranes, while the distribution reduced upon the depletion of cholesterol, suggesting that cytolethal distending toxin subunits are associated with lipid rafts. The disruption of cholesterol using methyl-β-cyclodextrin not only reduced the binding activity of cytolethal distending toxin subunits on the cell membrane but also impaired their delivery and attenuated toxin-induced cell cycle arrest. Accordingly, cell intoxication by cytolethal distending toxin was restored by cholesterol replenishment. These findings suggest that membrane cholesterol plays a critical role in theCampylobacter jejunicytolethal distending toxin-induced pathogenesis of host cells.

scholarly journals The Active Subunit of the Cytolethal Distending Toxin, CdtB, Derived From Both Haemophilus ducreyi and Campylobacter jejuni Exhibits Potent Phosphatidylinositol-3,4,5-Triphosphate Phosphatase Activity

2021 ◽  
Vol 11 ◽  
Author(s):  
Grace Huang ◽  
Kathleen Boesze-Battaglia ◽  
Lisa P. Walker ◽  
Ali Zekavat ◽  
Zachary P. Schaefer ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Phosphatase Activity ◽  
Campylobacter Jejuni ◽  
Human Lymphocytes ◽  
Haemophilus Ducreyi ◽  
Cell Protein ◽  
Cytolethal Distending Toxin ◽  
Induce Cell Cycle Arrest ◽  
Cycle Arrest

Human lymphocytes exposed to Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) undergo cell cycle arrest and apoptosis. In previous studies, we demonstrated that the active Cdt subunit, CdtB, is a potent phosphatidylinositol (PI) 3,4,5-triphosphate phosphatase. Moreover, AaCdt-treated cells exhibit evidence of PI-3-kinase (PI-3K) signaling blockade characterized by reduced levels of PIP3, pAkt, and pGSK3β. We have also demonstrated that PI-3K blockade is a requisite of AaCdt-induced toxicity in lymphocytes. In this study, we extended our observations to include assessment of Cdts from Haemophilus ducreyi (HdCdt) and Campylobacter jejuni (CjCdt). We now report that the CdtB subunit from HdCdt and CjCdt, similar to that of AaCdt, exhibit potent PIP3 phosphatase activity and that Jurkat cells treated with these Cdts exhibit PI-3K signaling blockade: reduced levels of pAkt and pGSK3β. Since non-phosphorylated GSK3β is the active form of this kinase, we compared Cdts for dependence on GSK3β activity. Two GSK3β inhibitors were employed, LY2090314 and CHIR99021; both inhibitors blocked the ability of Cdts to induce cell cycle arrest. We have previously demonstrated that AaCdt induces increases in the CDK inhibitor, p21CIP1/WAF1, and, further, that this was a requisite for toxin-induced cell death via apoptosis. We now demonstrate that HdCdt and CjCdt also share this requirement. It is also noteworthy that p21CIP1/WAF1 was not involved in the ability of the three Cdts to induce cell cycle arrest. Finally, we demonstrate that, like AaCdt, HdCdt is dependent upon the host cell protein, cellugyrin, for its toxicity (and presumably internalization of CdtB); CjCdt was not dependent upon this protein. The implications of these findings as they relate to Cdt’s molecular mode of action are discussed.

scholarly journals CdtA, CdtB, and CdtC Form a Tripartite Complex That Is Required for Cytolethal Distending Toxin Activity

2001 ◽  
Vol 69 (7) ◽  
pp. 4358-4365 ◽  
Author(s):  
Marı́a Lara-Tejero ◽  
Jorge E. Galán
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Campylobacter Jejuni ◽  
Bacterial Pathogens ◽  
Target Cells ◽  
Cytolethal Distending Toxin ◽  
Toxic Activity ◽  
Cellular Toxicity ◽  
B Subunit ◽  
Cycle Arrest

ABSTRACT Campylobacter jejuni encodes a cytolethal distending toxin (CDT) that causes cells to arrest in the G2/M transition phase of the cell cycle. Highly related toxins are also produced by other important bacterial pathogens. CDT activity requires the function of three genes: cdtA, cdtB, andcdtC. Recent studies have established that CdtB is the active subunit of CDT, exerting its effect as a nuclease that damages the DNA and triggers cell cycle arrest. Microinjection of CdtB into target cells led to G2/M arrest and cytoplasmic distention, in a manner indistinguishable from that caused by CDT treatment. Despite this progress, nothing is known about the composition of the CDT holotoxin or the function of CdtA and CdtC. We show here that, when applied individually, purified CdtA, CdtB, or CdtC does not exhibit toxic activity. In contrast, CdtA, CdtB, and CdtC when combined, interact with one another to form an active tripartite holotoxin that exhibits full cellular toxicity. CdtA has a domain that shares similarity with the B chain of ricin-related toxins. We therefore proposed that CDT is a tripartite toxin composed of CdtB as the enzymatically active subunit and of CdtA and CdtC as the heterodimeric B subunit required for the delivery of CdtB.

scholarly journals Campylobacter jejuni Cytolethal Distending Toxin Causes a G2-Phase Cell Cycle Block

1998 ◽  
Vol 66 (5) ◽  
pp. 1934-1940 ◽  
Author(s):  
Chris A. Whitehouse ◽  
Paul B. Balbo ◽  
Everett C. Pesci ◽  
Daniel L. Cottle ◽  
Peter M. Mirabito ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Campylobacter Jejuni ◽  
Hela Cells ◽  
Chromatin Condensation ◽  
Phase Cell ◽  
Specific Cell ◽  
Cytolethal Distending Toxin ◽  
Cycle Arrest ◽  
M Phase

ABSTRACT Cytolethal distending toxin (CDT) from the diarrheagenic bacteriumCampylobacter jejuni was shown to cause a rapid and specific cell cycle arrest in HeLa and Caco-2 cells. Within 24 h of treatment, CDT caused HeLa cells to arrest with a 4N DNA content, indicative of cells in G2 or early M phase. Immunofluorescence studies indicated that the arrested cells had not entered M phase, since no evidence of tubulin reorganization or chromatin condensation was visible. CDT treatment was also shown to cause HeLa cells to accumulate the inactive, tyrosine-phosphorylated form of CDC2. These results indicated that CDT treatment results in a failure to activate CDC2, which leads to cell cycle arrest in G2. This mechanism of action is novel for a bacterial toxin and provides a model for the generation of diarrheal disease byC. jejuni and other diarrheagenic bacteria that produce CDT.

scholarly journals Role of lipid rafts in E-cadherin– and HGF-R/Met–mediated entry of Listeria monocytogenes into host cells

2004 ◽  
Vol 166 (5) ◽  
pp. 743-753 ◽  
Author(s):  
Stéphanie Seveau ◽  
Hélène Bierne ◽  
Stéphanie Giroux ◽  
Marie-Christine Prévost ◽  
Pascale Cossart
Keyword(s):  
Listeria Monocytogenes ◽  
Lipid Rafts ◽  
Host Cells ◽  
Bacterial Invasion ◽  
Membrane Cholesterol ◽  
Cholesterol Depletion ◽  
Lipid Domains ◽  
Initial Interaction ◽  
E Cadherin

Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-β-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.

Inhibition of DNA repair for sensitizing resistant glioma cells to temozolomide

2003 ◽  
Vol 99 (6) ◽  
pp. 1047-1052 ◽  
Author(s):  
Takao Kanzawa ◽  
Joshua Bedwell ◽  
Yasuko Kondo ◽  
Seiji Kondo ◽  
Isabelle M. Germano
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Viability ◽  
Cell Cycle Arrest ◽  
Glioma Cells ◽  
Glioblastoma Cell ◽  
Content Type ◽  
Cycle Arrest ◽  
M Phase ◽  
Fine Print

Object. Temozolomide (TMZ) is a DNA alkylating agent currently used as adjuvant treatment for anaplastic astrocytomas. Its use in managing glioblastoma multiforme has been halted because of the lack of therapeutic effects due to cell resistance. Note that O6-alkylguanine—DNA alkyltranferase (AGT) is a DNA repair enzyme that limits the efficacy of TMZ. In this study the authors investigated the ability of O6-benzylguanine (BG), an AGT inhibitor, to sensitize a glioblastoma cell line resistant to TMZ. Methods. The effects of TMZ alone (100 µg) and after exposure to BG (50 µg) were assessed in two glioblastoma cell lines, U373-MG and T98G, respectively, sensitive and resistant to TMZ. Cell viability was assessed using trypan blue; cell cycle analysis by fluorescence-activated cell sorter; and apoptosis and autophagy by terminal deoxynucleotidyl transferase—mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and acridine orange staining, respectively. Furthermore, the involvement of an autophagy marker, microtubule-associated light chain 3 (LC3), was assessed. Temozolomide suppressed the growth of and caused cell cycle arrest in the G2—M phase of U373-MG cells but not T98G cells. Exposure to BG prior to TMZ resulted in a significant decrease in cell viability as well as cell cycle arrest in the G2—M phase in T98G cells (p < 0.05). Although apoptosis was not detected on TUNEL staining, programmed cell death Type II (autophagy) was detected after exposure to BG and TMZ in T98G cells. Conclusions. These results indicate that inhibition of AGT by BG can render previously resistant glioma cells sensitive to TMZ treatment. The mechanism of cell demise following BG-TMZ treatment seems to be autophagy and not apoptosis. Combination therapy involving TMZ and an AGT inhibitor may be an effective strategy to treat resistant gliomas.

scholarly journals MicroRNA miR-27 Inhibits Adenovirus Infection by Suppressing the Expression of SNAP25 and TXN2

2017 ◽  
Vol 91 (12) ◽  
Author(s):  
Mitsuhiro Machitani ◽  
Fuminori Sakurai ◽  
Keisaku Wakabayashi ◽  
Kosuke Nakatani ◽  
Masashi Tachibana ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Gene Silencing ◽  
Cell Cycle Arrest ◽  
Target Genes ◽  
Host Cells ◽  
Microarray Gene Expression ◽  
Posttranscriptional Gene Silencing ◽  
Gene Therapies ◽  
Cycle Arrest ◽  
Efficient Suppression

ABSTRACT Recent studies have reported that host microRNAs (miRNAs) regulate infections by several types of viruses via various mechanisms and that inhibition of the miRNA processing factors enhances or prevents viral infection. However, it has not been clarified whether these effects of miRNAs extend to adenovirus (Ad) infection. Here we show that miR-27a and -b efficiently inhibit infection with an Ad via the downregulation of SNAP25 and TXN2, which are members of the SNARE proteins and the thioredoxin family, respectively. Approximately 80% reductions in Ad genomic copy number were found in cells transfected with miR-27a/b mimics, whereas there were approximately 2.5- to 5-fold larger copy numbers of the Ad genome following transfection with miR-27a/b inhibitors. Microarray gene expression analysis and in silico analysis demonstrated that SNAP25 and TXN2 are target genes of miR-27a/b. A reporter assay using plasmids containing the 3′ untranslated regions of the SNAP25 and TXN2 genes showed that miR-27a/b directly suppressed SNAP25 and TXN2 expression through posttranscriptional gene silencing. Knockdown of SNAP25 led to a significant inhibition of Ad entry into cells. Knockdown of TXN2 induced cell cycle arrest at G1 phase, leading to a reduction in Ad replication. In addition, overexpression of Ad-encoded small noncoding RNAs (VA-RNAs) restored the miR-27a/b-mediated reduction in infection level with a VA-RNA-lacking Ad mutant due to the VA-RNA-mediated inhibition of miR-27a/b expression. These results indicate that miR-27a and -b suppress SNAP25 and TXN2 expression via posttranscriptional gene silencing, leading to efficient suppression of Ad infection. IMPORTANCE Adenovirus (Ad) is widely used as a platform for replication-incompetent Ad vectors (Adv) and replication-competent oncolytic Ad (OAd) in gene therapy and virotherapy. Regulation of Ad infection is highly important for efficient gene therapies using both Adv and OAd. In this study, we demonstrate that miR-27a and -b, which are widely expressed in host cells, suppress SNAP25 and TXN2 expression through posttranscriptional gene silencing. Suppression of SNAP25 and TXN2 expression leads to inhibition of Ad entry into cells and to cell cycle arrest, respectively, leading to efficient suppression of Ad infection. Our findings provide important clues to the improvement of gene therapies using both Adv and OAd.

scholarly journals Escherichia coli CdtB Mediates Cytolethal Distending Toxin Cell Cycle Arrest

2001 ◽  
Vol 69 (5) ◽  
pp. 3418-3422 ◽  
Author(s):  
Cherilyn Elwell ◽  
Kinlin Chao ◽  
Kamlesh Patel ◽  
Lawrence Dreyfus
Keyword(s):  
Escherichia Coli ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Hela Cells ◽  
Functional Relationship ◽  
Type I ◽  
Cytolethal Distending Toxin ◽  
E Coli ◽  
Cycle Arrest ◽  
Dna Nicking

ABSTRACT We previously reported that the CdtB polypeptide ofEscherichia coli cytolethal distending toxin (CDT) shares significant pattern-specific homology with mammalian type I DNases. In addition, the DNase-related residues of CdtB are required for cellular toxicity. Here we demonstrate that purified CdtB converts supercoiled plasmid DNA to relaxed and linear forms and promotes cell cycle arrest when combined with an E. coli extract containing CdtA and CdtC. CdtB alone had no effect on HeLa cells, however; introduction of the polypeptide into HeLa cells by electroporation resulted in cellular distension, chromatin fragmentation, and cell cycle arrest, all of which are consequences of CDT action. In contrast to these findings, purified CdtBH154A lacked both DNA-nicking and cell cycle arrest activities. These results suggest a functional relationship between DNase-related residues in CdtB and CDT biological activity.

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