Helicobacter hepaticus Hydrogenase Mutants Are Deficient in Hydrogen-Supported Amino Acid Uptake and in Causing Liver Lesions in A/J Mice

Abstract

Helicobacter hepaticus, a causative agent of chronic hepatitis and hepatocellular carcinoma in mice, expresses a nickel-containing hydrogen-oxidizing hydrogenase enzyme. Growth of a hyaB gene-targeted mutant was unaffected by the presence of hydrogen, unlike the wild-type strain, which showed an enhanced growth rate when supplied with H 2 . Hydrogenase activities in H. hepaticus were constitutive and not dependent on the inclusion of H 2 during growth. Addition of nickel during growth significantly stimulated both urease (for wild-type and hyaB) and hydrogenase (for wild-type) activities. In a 5-h period, the extent of 14 C-labeled amino acid uptake by the wild type was markedly enhanced in the presence of hydrogen and was >5-fold greater than that of the hyaB mutant strain. In the presence of H 2 , the short-term whole-cell amino acid uptake V max of the parent strain was about 2.2-fold greater than for the mutant, but the half-saturation affinity for amino acid transport was the same for the parent and mutant strain. The liver-and cecum-colonizing abilities of the strains was estimated by real-time PCR quantitation of the H. hepaticus-specific cytolethal distending toxin gene and showed similar animal colonization for the hyaB mutant and the wild type. However, at 21 weeks postinoculation, the livers from mice inoculated with wild type exhibited moderate lobular lymphoplasmacytic hepatitis with hepatocytic coagulative necrosis, but the hydrogenase mutants exhibited no histological evidence of lobular inflammation or necrosis.In recent years, more than three different Helicobacter species have been recovered from rodents (24,25,31), with H. hepaticus being the most well-studied enterohepatic Helicobacter species. First isolated in 1992 from untreated A/JCr control mice, H. hepaticus is a gram-negative, microaerophilic bacterium (6) that occurs naturally in many strains of inbred mice (30). Mice infected with this bacterium develop chronic hepatic lesions and are more prone to developing hepatocellular carcinoma (25, 26), which has made H. hepaticus an excellent model for studying mechanisms of bacterium-associated liver carcinogenesis. Although H. hepaticus can be isolated from the liver of infected mice, it is more consistently recovered from the intestinal tract since the primary site of colonization is the lower bowels of mice (6, 7). Recently, Helicobacter species DNA was reported to be associated with primary hepatocellular carcinomas in human liver samples (9, 23).At the same time, a hydrogen uptake hydrogenase enzyme well studied for its roles in nonpathogenic bacteria was demonstrated to be important for colonization of animals by some human pathogens (i.e., by Salmonella enterica serovar Typhimurium and Helicobacter pylori [14,21]); this sparked our interest in studying the physiological role of this enzyme in H. hepaticus (16). The colonization deficiency of hydrogenase structural gene mutants of H. pylori was attributed to their inability to utilize hydrogen as an energy substrate. H. hepaticus hydro...

Nickel enzyme maturation in Helicobacter hepaticus: roles of accessory proteins in hydrogenase and urease activities

“…H. hepaticus also possesses both enzymes (Beckwith et al, 2001;Mehta et al, 2005;Suerbaum et al, 2003). While it is known that H. hepaticus hydrogenase mutants are deficient in hydrogen-supported amino acid uptake and cannot cause liver lesions in mice (Mehta et al, 2005), the role of urease in H. hepaticus survival in the host remains to be demonstrated.…”

“…In this well studied bacterium, two nickel-containing enzymes, the hydrogen uptake hydrogenase and urease, have been shown to be required for early colonization and persistence in the stomach (Eaton et al, 1991;Olson & Maier, 2002). H. hepaticus also possesses both enzymes (Beckwith et al, 2001;Mehta et al, 2005;Suerbaum et al, 2003). While it is known that H. hepaticus hydrogenase mutants are deficient in hydrogen-supported amino acid uptake and cannot cause liver lesions in mice (Mehta et al, 2005), the role of urease in H. hepaticus survival in the host remains to be demonstrated.…”

The NADPH quinone reductase MdaB confers oxidative stress resistance to Helicobacter hepaticus

“…The cloned plasmid was then extracted from the culture (Qiagen) and an Erythromycin cassette (Ery) [11] was inserted into the unique EcoRI restriction site within the mdaB gene. The recombinant plasmid pGEMT:mdaB:Ery was then introduced into H. hepaticus by electrotransformation [15]. Allelic exchange occurred leading to the formation of the mdaB mutant strain.…”

Differential expression of NiFe uptake-type hydrogenase genes in Salmonella enterica serovar Typhimurium

“…It was recently shown that hydrogen gas (H 2 ) is an important energy source for the survival of pathogens in vivo (Mehta et al, 2005;Olson & Maier, 2002). H 2 is produced in the host by colonic bacterial fermentations and diffuses throughout the animal's body (Maier, 2003).…”