Subsequent to the recognition of H. pylori
and other gastric Helicobacter
species, the EHS were identified in the distinct environment of the intestinal tracts and/or livers of humans, other mammals, and birds (11
). EHS are emerging as important veterinary and human pathogens that are responsible for a number of acute disease syndromes, including gastroenteritis and bacteremia or sepsis. In addition, a subset of EHS also cause persistent infections associated with chronic inflammation and neoplasia, which is analogous to the association between H. pylori
infection and subsequent chronic gastritis and gastric cancer (3
). There is growing evidence that EHS can be associated with chronic liver diseases in humans, including chronic hepatitis, liver carcinoma, chronic cholecystitis, and cholangiocarcinoma (1
In order to study the molecular pathogenesis of EHS infections, genetic tools for the manipulation of these organisms are needed. We describe here a method for the generation of targeted isogenic mutations in the EHS H. hepaticus. The transposon mutagenesis system described here is also useful for the generation of random mutations throughout the genome in addition to the generation of targeted mutations in potential virulence factors.
The production of toxins is a critical feature of the pathogenesis of a number of enteric pathogens (reviewed in reference 27
). Toxins have been theorized to play a role in a number of pathogenic processes, including tissue invasion and the stimulation of fluid secretion to enhance nutrient delivery and/or to facilitate the spread to new hosts and escape from immune surveillance. Since we identified CDT in H. hepaticus
, we sought to determine if this toxin plays a role in the pathogenesis of a disease associated with murine infection by this pathogen.
was previously reported to possess GCT activity (31
). Given the ability of H. hepaticus
to colonize the murine biliary tree and cause chronic hepatitis and hepatic cancer, the organisms were tested for the ability to cause CPE on CCL-9.1 cells, which are cloned liver cells derived from a newborn C3H/An mouse.
We demonstrate here that the cytopathic activity initially described as GCT is mediated by CDT. Three lines of evidence support this conclusion. First, a transposon mutant of H. hepaticus targeting the cdtABC gene cluster lacks both CDT and GCT activity. Second, an E. coli strain that carries the cloned cdtABC gene cluster from H. hepaticus produces GCT activity as assayed on CCL-9.1 cells. Third, cell cycle analysis of CCL-9.1 cells that exhibit GCT CPE reveals that the cells arrested at G2/M.
Although CDT activity and gene sequences appear to be widely distributed among a diverse group of bacterial pathogens, there has been little experimental evidence that suggests that CDT plays a critical role in the production of disease in mammalian hosts infected with CDT-producing organisms. The elimination of CDT activity in Haemophilus ducreyi
did not alter the virulence of this organism in either the temperature-dependent rabbit model for experimental chancroid (19
) or human volunteers (34
). An isogenic C. jejuni
CDT mutant was found to be as efficient as the wild type at colonizing the gastrointestinal tracts of mice (26
). It was suggested that the C. jejuni
CDT mutant was less invasive since it was found in spleen, liver, and blood samples of only 4 of 15 infected mice 2 h after challenge, whereas wild-type C. jejuni
was found in spleen, liver, and blood samples of 8 of 15 infected mice.
The data presented here indicate that CDT plays a role in the development of IBD in immune-altered mice infected with H. hepaticus. Although an isogenic H. hepaticus CDT mutant retained the ability to colonize C57BL/6 IL-10−/− mice, animals infected with the mutant developed significantly less severe disease than littermates infected with a wild-type H. hepaticus strain. Additionally, there were notable qualitative differences in the natures of the typhlocolitis seen in mice infected with 3B1::Tn20. Although each individual mouse was infected with the mutant H. hepaticus strain, the gastrointestinal tract of one mouse was histologically normal, and another exhibited only mild, patchy mucosal inflammation. Although two mice infected with 3B1::Tn20 did develop diffuse mucosal inflammation, none of the mice infected with the CDT-negative mutant developed significant submucosal inflammation similar to that observed in a subset of the mice infected with CDT-positive strains.
In summary, we have provided a description of a transposon-mediated shuttle mutagenesis system for the construction of isogenic mutants of the murine pathogen H. hepaticus. Using this genetic system we have demonstrated that the previously described GCT activity of H. hepaticus is mediated by CDT. In addition, we have demonstrated a role for CDT in mediating the IBD seen in IL-10−/− mice infected with H. hepaticus. The mechanism by which CDT leads to intestinal inflammation is not clear, but it may be that CDT targets a particular cell type involved in innate and/or adaptive immunity to escape immune surveillance. In mice with altered immune systems, such as IL-10−/− mice, this leads to a marked dysregulation of the immune response that results in the clinical entity of IBD.