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Contribution of Helicobacter hepaticus cytolethal distending toxin subunits to human epithelial cell cycle arrest and apoptotic death in vitro.

ABSTRACT: Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus, the cause of chronic typhlocolitis and hepatitis leading to colonic and hepatocellular carcinomas in mice. Interaction of the tripartite polypeptide CdtA, CdtB, and CdtC subunits produced by H. hepaticus CDT (HhepCDT) causes cell cycle arrest and apoptotic death of cultured cells; however, the contribution of individual subunit to these processes has not been investigated.The temporal relationship between cell cycle and apoptotic death of human epithelial HeLa and INT407 cells intoxicated with HhepCDT holotoxin or reconstituted recombinant HhepCDT was compared by flow cytometry. The genotoxic activity of individual and combinations of recombinant HhepCDT protein subunits or increasing concentrations of individual recombinant HhepCDT protein subunits transfected into HeLa cells was assessed at 72 hours post-treatment by flow cytometry.Similar time course of HhepCDT-induced G2 /M cell cycle arrest and apoptotic death was found with both cell lines which reached a maximum at 72 hours. The presence of all three HhepCDT subunits was required for maximum cell cycle arrest and apoptosis of both cell lines. Transfection of HeLa cells with HhepCdtB, but not with HhepCdtA or HhepCdtC, resulted in a dose-dependent G2 /M arrest and apoptotic death.All three subunits of HhepCDT are required for maximum epithelial cell cycle arrest and progression to apoptotic death, and HhepCdtB subunit alone is necessary and sufficient for epithelial cell genotoxicity.

SUBMITTER: Liyanage NP

PROVIDER: S-EPMC3808484 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Contribution of Helicobacter hepaticus cytolethal distending toxin subunits to human epithelial cell cycle arrest and apoptotic death in vitro.

Liyanage Namal P M NP Dassanayake Rohana P RP Kuszynski Charles A CA Duhamel Gerald E GE

Helicobacter 20130729 6

<h4>Background</h4>Cytolethal distending toxin (CDT) is the only known virulence factor found in H. hepaticus, the cause of chronic typhlocolitis and hepatitis leading to colonic and hepatocellular carcinomas in mice. Interaction of the tripartite polypeptide CdtA, CdtB, and CdtC subunits produced by H. hepaticus CDT (HhepCDT) causes cell cycle arrest and apoptotic death of cultured cells; however, the contribution of individual subunit to these processes has not been investigated.<h4>Materials ...[more]

PMID: 23895367

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Project description:Helicobacter hepaticus, which induces chronic hepatitis and typhlocolitis in susceptible mouse strains, produces a cytolethal distending toxin (CDT) consisting of CdtA, CdtB, and CdtC. A cdtB-deficient H. hepaticus isogenic mutant (HhcdtBm7) was generated and characterized for colonization parameters in four intestinal regions (jejunum, ileum, cecum, and colon) of outbred Swiss Webster (SW) mice. Inactivation of the cdtB gene abolished the ability of HhcdtBm7 to colonize female mice at both 8 and 16 weeks postinfection (wpi), whereas HhcdtBm7 colonized all of four intestinal regions of three of five males at 8 wpi and then was eliminated by 16 wpi. Wild-type (WT) H. hepaticus was detected in the corresponding intestinal regions of both male and female mice at 8 and 16 wpi; however, colonization levels of WT H. hepaticus in the cecum and colon of male mice were approximately 1,000-fold higher than in females (P < 0.0079) at 16 wpi. Infection with WT H. hepaticus, but not HhcdtBm7, at 8 wpi was associated with significantly increased mRNA level of ileal and cecal gamma interferon (IFN-gamma) in females (P < 0.016 and 0.031 between WT H. hepaticus-infected and sham-dosed females, respectively). In contrast, the mRNA levels of IFN-gamma were significantly higher in the colon (P < 0.0079) and trended to be higher in the cecum (P < 0.15) in the HhcdtBm7-colonized male mice versus the sham-dosed controls at 8 wpi. In addition, mRNA levels of ileal IFN-gamma were significantly higher in the control females than males at 8 wpi (P < 0.016). There were significantly higher Th1-associated immunoglobulin G2a (IgG2a), Th2-associated IgG1 and mucosal IgA (P < 0.002, 0.002, 0.002, respectively) responses in the mice infected with WT H. hepaticus when compared to HhcdtBm7 at 16 wpi. Colonic interleukin-10 (IL-10) expressions at 16 wpi were significantly lower in both female and male mice colonized by WT H. hepaticus or in males transiently colonized through 8 wpi by HhcdtBm7 versus control mice (P < 0.0159). These lines of evidence indicate that (i) H. hepaticus CDT plays a crucial role in the persistent colonization of H. hepaticus in SW mice; (ii) SW female mice are more resistant to H. hepaticus colonization than male mice; (iii) there was persistent colonization of WT H. hepaticus in cecum, colon, and jejunum but only transient colonization of H. hepaticus in the ileum of female mice; (iv) H. hepaticus colonization was associated with down-regulation of colonic IL-10 production.

Project description:Cytolethal distending toxins (CDTs) are released by Gram-negative pathogens into the extracellular medium as free toxin or associated with extracellular vesicles (EVs), commonly known as outer membrane vesicles (OMVs). CDT production by the gastrointestinal pathogen Campylobacter jejuni has been implicated in colorectal tumorigenesis. Despite CDT being a major virulence factor for C. jejuni, little is known about the EV-associated form of this toxin. To address this point, C. jejuni mutants lacking each of the three CDT subunits (A, B, and C) were generated. C. jejuni cdtA, cdtB, and cdtC bacteria released EVs in similar numbers and sizes to wild-type bacteria, ranging from 5 to 530 nm (mean ± SEM = 118 ±6.9 nm). As the CdtAC subunits mediate toxin binding to host cells, we performed "surface shearing" experiments, in which EVs were treated with proteinase K and incubated with host cells. These experiments indicated that CDT subunits are internal to EVs and that surface proteins are probably not involved in EV-host cell interactions. Furthermore, glycan array studies demonstrated that EVs bind complex host cell glycans and share receptor binding specificities with C. jejuni bacteria for fucosyl GM1 ganglioside, P1 blood group antigen, sialyl, and sulfated Lewisx. Finally, we show that EVs from C. jejuni WT but not mutant bacteria induce cell cycle arrest in epithelial cells. In conclusion, we propose that EVs are an important mechanism for CDT release by C. jejuni and are likely to play a significant role in toxin delivery to host cells.ImportanceCampylobacter jejuni is the leading cause of foodborne gastroenteritis in humans worldwide and a significant cause of childhood mortality due to diarrheal disease in developing countries. A major factor by which C. jejuni causes disease is a toxin, called cytolethal distending toxin (CDT). The biology of this toxin, however, is poorly understood. In this study, we report that C. jejuni CDT is protected within membrane blebs, known as extracellular vesicles (EVs), released by the bacterium. We showed that proteins on the surfaces of EVs are not required for EV uptake by host cells. Furthermore, we identified several sugar receptors that may be required for EV binding to host cells. By studying the EV-associated form of C. jejuni CDT, we will gain a greater understanding of how C. jejuni intoxicates host cells and how EV-associated CDT may be used in various therapeutic applications, including as anti-tumor therapies.

Dataset Information

Contribution of Helicobacter hepaticus cytolethal distending toxin subunits to human epithelial cell cycle arrest and apoptotic death in vitro.

Publications

Contribution of Helicobacter hepaticus cytolethal distending toxin subunits to human epithelial cell cycle arrest and apoptotic death in vitro.

Similar Datasets

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