Roles of His-Rich Hpn and Hpn-Like Proteins in Helicobacter pylori Nickel Physiology

Abstract

Individual gene-targeted hpn and hpn-like mutants and a mutant with mutations in both hpn genes were more sensitive to nickel, cobalt, and cadmium toxicity than was the parent strain, with the hpn-like strain showing the most metal sensitivity of the two individual His-rich protein mutants. The mutant strains contained up to eightfold more urease activity than the parent under nickel-deficient conditions, and the parent strain was able to achieve mutant strain activity levels by nickel supplementation. The mutants contained 3-to 4-fold more and the double mutant about 10-fold more Ni associated with their total urease pools, even though all of the strains expressed similar levels of total urease protein. Hydrogenase activities in the mutants were like those in the parent strain; thus, hydrogenase is fully activated under nickel-deficient conditions. The histidine-rich proteins appear to compete with the Ni-dependent urease maturation machinery under lownickel conditions. Upon lowering the pH of the growth medium from 7.3 to 5, the wild-type urease activity increased threefold, but the activity in the three mutant strains was relatively unaffected. This pH effect was attributed to a nickel storage role for the His-rich proteins. Under low-nickel conditions, the addition of a nickel chelator did not significantly affect the urease activity of the wild type but decreased the activity of all of the mutants, supporting a role for the His-rich proteins as Ni reservoirs. These nickel reservoirs significantly impact the active urease activities achieved. The His-rich proteins play dual roles, as Ni storage and as metal detoxification proteins, depending on the exogenous nickel levels.

The Helicobacter pylori GroES Cochaperonin HspA Functions as a Specialized Nickel Chaperone and Sequestration Protein through Its Unique C-Terminal Extension

“…Thus, the presence of a nickel-binding tail on the highly abundant and constantly expressed GroES proteins could enable H. pylori to balance efficient nickel uptake and to deal with the constant intracellular presence of nickel ions. We have found that nickel binding by HspA has a role similar to the nickel sequestration function of the Hpn protein (14,15,27,35). However, the hpn deletion strain showed a greater sensitivity to nickel overload, in agreement with the fact that Hpn binds 2 to 3 more nickel ions per monomer than HspA, having-binding affinities of the same range.…”

“…However, despite this interconnectivity, it seems that different nickel-binding proteins of H. pylori specifically sustain nickel incorporation into either urease or hydrogenase. Hpn and Hpn-like proteins have been shown to act as nickel reservoirs that under certain conditions supply this metal to urease but not to hydrogenase (3,23,35). In the present study, we demonstrated that the nickel-binding domain of HspA is required for hydrogenase activation and not for urease activity.…”

“…Furthermore, it has recently been proposed that Hpn and Hpn-like can compete for nickel ions with the urease enzyme and thus regulate its enzymatic activity. In vivo and in vitro experiments indicate that Hpn and Hpn-like sequester nickel ions at neutral pH but donate them for urease activation under acidic pH conditions (14,35,44). Hydrogenase activity was unchanged in the ⌬hpn and ⌬hpn-like mutants (35).…”

An Intact Urease Assembly Pathway Is Required To Compete with NikR for Nickel Ions in Helicobacter pylori

“…De novo synthesis of NikR was not required for the mutant phenotypes, because inhibition of translation by chloramphenicol or erythromycin had no effect (data not shown). Single or double mutant strains with deletions of genes involved in Ni 2ϩ transport (nixA [28] and exbBD tonB [36]), Ni 2ϩ storage (hpn and hpn-like [6,19,29,37]), and Ni 2ϩ efflux (cznABC [39]) also showed no changes in NikR-dependent regulation (data not shown). This contrasts with results from E. coli, where the Ni 2ϩ efflux protein RcnA impedes NikR activation under nickel-limiting conditions (23).…”

Metal-binding characteristics of the protein which shows the highest histidine content in the Arabidopsis genome

“…For example, Hpn, which is a histidine-rich metal-binding peptide in Helicobacter pylori, has 28 histidine residues out of 60 amino acids of the total residues (histidine content; 46.7%) (Gilbert et al 1995). Because Hpn binds Ni 2ϩ , Hpn has been proposed to play a role in Ni storage and homeostasis in H. pylori to produce Ni-dependent enzymes, such as urease, which is necessary to their gastric infection Seshadri et al 2007). Histatin 5, which is a human salivary metallopeptide showing antimicrobial activity against Candida albicans, is 24 amino acids in length and contains 7 histidine residues (histidine content; 29.1%) (Oppenheim et al 1988).…”