Cytolethal distending toxin sequence and activity in the enterohepatic pathogen Helicobacter hepaticus

Abstract

Little is known about the molecular pathogenesis of hepatitis and enterocolitis caused by enterohepatic Helicobacter species. Sonicates of the murine pathogen Helicobacter hepaticus were found to cause progressive cell distension, accumulation of filamentous actin, and G(2)/M cell cycle arrest in HeLa cell monolayers. The genes encoding this cytotoxic activity were cloned from H. hepaticus. Three open reading frames with closest homology to cdtA, cdtB, and cdtC from Campylobacter jejuni were identified. Sonicates of a laboratory strain of Escherichia coli carrying the cloned cdtABC gene cluster from H. hepaticus reproduced the cytotoxic activities seen with sonicates of H. hepaticus. Cytolethal distending toxin activity is a potential virulence determinant of H. hepaticus that may play a role in the pathogenesis of Helicobacter-associated hepatitis and enterocolitis.

FIG. 1

Nucleotide sequence of the cdtABC gene cluster from H. hepaticus. The three presumed ribosomal binding sites (underlined) for each open reading frame are shown along with the deduced amino acid sequences. Also indicated (bold) are the sites at which the degenerate PCR primers VAT2 and WMI1 bind. Forward primer VAT2 and the predicted site of reverse primer WMI1 are expected to yield a 505-bp amplicon. The actual binding site of the reverse primer WMI1* along with forward primer VAT2 produced an observed product of 768 bp.

FIG. 2

Comparison of the predicted amino acid sequences of CdtA, CdtB, and CdtC from H. hepaticus ATCC 51449 (Hh) and C. jejuni 81-176 (Cj). Colons indicate identical amino acids, and conserved amino acids are indicated by periods. Dashes indicate gaps introduced by the MacVector ClustalW alignment program.

FIG. 3

Cytopathic effect of H. hepaticus CDT on HeLa cells. Compared to untreated cells (A), cells treated with sonicates from H. hepaticus (B) exhibited marked cytoplasmic distension along with nuclear enlargement, multinucleation, and nuclear fragmentation. Magnification, ×250.

FIG. 4

Cytoplasmic and nuclear distension of HeLa cells treated with sonicates of H. hepaticus. Photomicrographs of untreated cells (A and B) and cells treated with sonicates of H. hepaticus (C and D) for 72 h are shown. Fixed and permeabilized cells were double labeled for epifluorescence microscopy with Texas red-labeled phalloidin (A and C) and Hoechst 33342 (B and D). Treated cells exhibited increased cell size and prominent stress fiber-like structures (C) as well, as increased nuclear size (D). Magnification, ×500.

FIG. 5

Time dependence of DNA content and cytopathic effect of HeLa cells treated with bacterial sonicates with CDT activity. Compared to control cells (A), cells treated with sonicates from H. hepaticus (D, G, and J) showed a progressive increase in the fraction of cells with a DNA content of 4 N, with a maximal effect being reached 72 h after sonicate addition (J). Compared to control cells (B and C), cells treated with sonicates from an E. coli strain harboring the cloned H. hepaticus cdt locus showed a progressive increase in size with accumulations of polymerized actin (E, H, and K) and progressive nuclear abnormalities (F, I, and L). These changes in morphology mirrored the progressive cell cycle block observed by flow cytometry. Magnification, ×400. PI, propidium iodide.

FIG. 6

Cytolethal distending toxin activity exhibited by sonicates of E. coli clones carrying plasmids containing various sections of the H. hepaticus cdtABC gene cluster. The ability of sonicates of each clone to cause a typical CDT cytopathic effect and cell cycle arrest in cultured HeLa cell monolayers is indicated. Cell cycle arrest is indicated by the percentage of cells in G₂/M relative to the percentage seen in monolayers treated with H. hepaticus sonicates. Only sonicates of a clone (pVBY9) that carries a plasmid with the entire cdtABC gene cluster were able to cause a cytopathic effect or cell cycle arrest. ND, not done.