Cytolethal distending toxin is essential for Helicobacter hepaticus colonization in outbred Swiss Webster mice

Abstract

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.

FIG. 1.

Characterization of the transcriptional start and termination sites of the H. hepaticus cdt operon. (A) The gene organization of the cdt operon: the primers for primer extension (PEP) and for RT-PCR as well as the insertional site of TnCm4 within the cdtB are denoted. (B) Primer extension demonstrating the transcriptional start site (A₁₄₅ upstream of the start codon of the cdtA) is indicated by an arrow. Lanes C, T, A, G: dideoxy-terminated sequence using plasmid pVBY9 DNA as template. This recombinant plasmid containing the cdt operon and the portion of the upstream and downstream regions (45). Lane 1, RNA plus RNase prior to cDNA synthesis; lane 2, RNA minus RNase. (C) RT-PCR-based detection of the transcriptional termination of the cdt opron. Lanes: 1, cDNA with cdtCF/TM1; 2, cDNA with cdtCF/TM2; 3, pVBY9 with cdtCF/TM1; 4, pVBY9 with cdtCF/TM2. A 1-kb ladder (Invitrogen) is located on the left (M).

FIG. 2.

Q-PCR-based detection of H. hepaticus in four segments of the mouse intestinal tract. Symbols: F, female mice; M, male mice; WT, WT H. hepaticus dosed; cdtB inact., • HhcdtBm7-dosed cdtB inactivated); *, P values for the compared groups. The levels of H. hepaticus in the respective samples are expressed as its genomic copies per μg of mouse DNA. Ileal and jejunal tissues from two male mice of all the groups at 16 wpi were lost during processing. Therefore, the 16-wpi data on the colonization levels and cytokine profiles in the ileum and jejunum of the male mice were based on three mice of each group. The samples negative for H. hepaticus by culture are indicated in boxes.

FIG. 3.

IgG1, IgG2a, IgA antibody responses to the H. hepaticus antigens. The sera for measuring IgG1 and IgG2a were collected from the mice orally dosed with either WT H. hepaticus or HhcdtBm7 (cdtB inactivated) for 16 wpi. The IgA antibody response to H. hepaticus was measured in fecal extracts from mice orally dosed with either WT H. hepaticus or HhcdtBm7 (cdtB inactivated) by 16 wpi. *, P values for the compared groups. OD, optical density.

FIG. 4.

mRNA levels of IL-10 and IFN-γ in the respective intestinal segments. The mice were orally dosed with either Brucella broth, WT H. hepaticus, or HhcdtBm7 (cdtB inactivated) and necropsied at 8 wpi. Symbols: *, P values for the compared groups; C, the controls; WT, WT H. hepaticus-dosed; cdtB inact., HhcdtBm7 (the cdtB inactivated); F, female mice; M, male mice. The levels of the IL-10 and IFN-γ transcripts were expressed as their copy numbers per 10⁶ of the GAPDH transcripts in the same samples.

FIG. 5.

mRNA levels of IL-10 and IFN-γ in the colon at 16 wpi. Symbols: *, P values for the compared groups; C, sham dosed; WT, WT H. hepaticus-dosed; cdtB inact., HhcdtBm7 dosed (the cdtB inactivated); F, female mice; M, male mice. The levels of the IL-10 and IFN-γ transcripts were expressed as their copy numbers per 10⁶ of the GAPDH transcripts in the same samples.