Helicobacter hepaticus urease is not required for intestinal colonization but promotes hepatic inflammation in male A/JCr mice

Abstract

Urease activity contributes to bacterial survival in the acidic environment of the stomach and is essential for persistent infection by known gastric helicobacters such as the human pathogen Helicobacter pylori. Several enterohepatic Helicobacter species (EHS) that primarily infect the less acidic intestine also have very active urease enzymes. The importance of urease and its contribution to pathogenesis for these EHS are poorly understood. In this study, we generated a urease-deficient, isogenic mutant (HhureNT9) of Helicobacter hepaticus 3B1 (Hh 3B1), an EHS that possesses a urease gene cluster similar to that of H. pylori. Lack of urease activity did not affect the level of cecal colonization by HhureNT9 compared to Hh 3B1 in male A/JCr mice (P=0.48) at 4 months post-inoculation (MPI). In contrast, there was no HhureNT9 detected in the livers of any infected mice, whereas all livers from the Hh 3B1-infected mice were PCR-positive for Hh 3B1. The mice infected with HhureNT9 developed significantly less severe hepatitis (P=0.017) and also produced significantly lower hepatic mRNA levels of proinflammatory cytokines IFN-gamma (P=0.0007) and TNF-alpha (P<0.0001) compared to the Hh 3B1-infected mice. The Hh 3B1-infected mice developed significantly higher total IgG, Th1-associated IgG2a and Th2-associated IgG1 responses to infection. These results indicate that H. hepaticus urease activity plays a crucial role in hepatic disease but is not required for cecal colonization by H. hepaticus.

Figure 1

Construction and characterization of urease-deficient H. hepaticus mutant HhureNT9. A, Schematic depiction of the genotype of HhureNT9. Primers used for generating and characterizing the mutant are denoted. B. Reverse-transcription PCR of the H. hepaticus ureI RNA with primers ZMG174 and ZMG175. Lanes: 1, (RNase treatment), 2 for Hh 3B1; 3 (RNase treatment), 4 for HhureN9 RNA; 5 for no template control and 6 for Hh 3B1 chromosomal DNA.

Figure 2

Q-PCR-based measurement of Hh 3B1 and HhureNT9 in cecum and liver using the primers and probe derived from the Hh distending cytolethal toxin gene B [33] . The levels of H. hepaticus are expressed as genomic copies per μg of mouse DNA.

Figure 3

Pathology in the livers of male A/JCr mice infected by Hh 3B1 or the urease-deficient Hh mutant HhureNT9. A. Liver histopathology. B. Pathological index for male A/JCr mice. No chronic hepatitis developed in the HhureNT9-infected mice at 4 MPI. In contrast, 4 out of 5 mice infected with Hh 3B1 developed severe hepatic inflammation.

Figure 4

Relative mRNA levels of proinflammatory and anti-inflammatory cytokines in the murine livers and ceca. For comparison of mRNA levels, the target mRNA was normalized to that of the house-keeping gene GAPDH. Numbers represent mean fold change of target mRNA levels in reference to the control levels (defined as 0, standard deviation represented by hatched bars).

Figure 5

Male A/JCr mouse IgG responses to Hh 3B1 or HhureNT9 infection. I, Th1-associated IgG2a and Th2-associated IgG1 to the OMPs of Hh 3B1 or HhureNT9. Sera from the individual mice: C for the control mice; NT9 for HhureNT9-infected mice; Hh for Hh 3B1-infected mice. II, Western blotting for total IgG responses to antigens from Hh 3B1 (lanes: 1, outer membrane proteins [OMP]; 2, whole-cell lysate) and HhureNT9 (lanes: 3, OMP; 4, whole-cell lysate). A, pooled sera from Hh 3B1-infected mice; B, pooled sera from HhureNT9-infected mice.