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. 2004 May;72(5):2521-7.
doi: 10.1128/IAI.72.5.2521-2527.2004.

In vitro and in vivo characterization of Helicobacter hepaticus cytolethal distending toxin mutants

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In vitro and in vivo characterization of Helicobacter hepaticus cytolethal distending toxin mutants

Vincent B Young et al. Infect Immun. 2004 May.

Abstract

Helicobacter hepaticus expresses a member of the cytolethal distending toxin (CDT) family of bacterial cytotoxins. To investigate the role of CDT in the pathogenesis of H. hepaticus, transposon mutagenesis was used to generate a series of isogenic mutants in and around the cdtABC gene cluster. An H. hepaticus transposon mutant with a disrupted cdtABC coding region no longer produced CDT activity. Conversely, a transposon insertion outside of the cluster did not affect the CDT activity. An examination of these mutants demonstrated that CDT represents the previously described granulating cytotoxin in H. hepaticus. Challenge of C57BL/6 interleukin 10(-/-) mice with isogenic H. hepaticus mutants revealed that CDT expression is not required for colonization of the murine gut. However, a CDT-negative H. hepaticus mutant had a significantly diminished capacity to induce lesions in this murine model of inflammatory bowel disease.

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Figures

FIG. 1.
FIG. 1.
Transposon mutagenesis of the CDT gene cluster from H. hepaticus. Plasmid pVBY9 harboring the cdtABC gene cluster from H. hepaticus was mutagenized in vitro with a commercial Tn5-based transposon system. Mutagenized plasmids were introduced into E. coli and scored for the ability to transfer CDT activity. The insertion site of the transposon was determined by restriction mapping and DNA sequence analysis.
FIG. 2.
FIG. 2.
CPE in HeLa cells 72 h after treatment with bacterial extracts. Compared to untreated control cells (A), cells treated with extracts from wild-type H. hepaticus strain 3B1 exhibit cytoplasmic and nuclear enlargement along with nuclear abnormalities (B). Extracts from the isogenic H. hepaticus mutant 3B1::Tn16, which has a transposon inserted downstream of the cdtABC gene cluster, retain cytopathic activity (C), while an extract from the H. hepaticus mutant 3B1::Tn20, which has a transposon-disrupted CDT cluster, did not possess cytotoxic activity (D).
FIG. 3.
FIG. 3.
CPE in CCL-9.1 cells 72 h after treatment with bacterial sonicates. Compared to untreated control cells (A), cells treated with sonicates from wild-type H. hepaticus strain 3B1 exhibit cytoplasmic and nuclear enlargement along with nuclear abnormalities (B). Cell cycle analysis demonstrated G2/M arrest (inset). A sonicate of an E. coli clone carrying pVBY9 (with the entire cdtABC gene cluster from H. hepaticus) had the ability to produce CPE and cell cycle arrest (C), whereas a sonicate from an isogenic CDT mutant lost both of these abilities (D).
FIG. 4.
FIG. 4.
Histopathologic lesions in IL-10−/− mice infected with isogenic strains of H. hepaticus. IL-10−/− mice were sham infected (A) or infected with wild-type H. hepaticus strain 3B1 (B), the CDT-positive mutant 3B1::Tn16 (C), or the CDT-negative mutant 3B1::Tn20 (D). Mice infected with CDT-producing strains exhibited marked inflammation and hyperplasia. Mice infected with the CDT-negative mutant developed diminished disease. Compared to uninfected mice and H. hepaticus-infected mice with minimal colitis (E), mice that developed severe colitis also developed a mesenteric perivasculitis (F).
FIG. 5.
FIG. 5.
Categorical inflammation scores 6 weeks after challenge for uninfected (control) IL-10−/− mice, mice infected with wild-type H. hepaticus strain 3B1, mice infected with the isogenic mutant strain 3B1::Tn16 (CDT+), and mice infected with the isogenic mutant strain 3B1::Tn20 (CDT). Comparisons between the groups were performed using the nonparametric Wilcoxon rank sum test.

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