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Abstract
Helicobacter pylori nickel metabolism and trafficking are necessarily complex; the bacterium inhabits an environment where nickel concentration is in constant flux while simultaneously requiring nickel for survival. H. pylori utilizes nickel transport mechanisms, a nickel-specific regulator, two essential nickel-containing enzymes and their associated accessory proteins, and two nickel storage proteins Hpn and Hpnl. Hpn and Hpnl modulate activity of one of the two nickel-containing enzymes, urease, through their interaction with accessory proteins HypAB. It was hypothesized, but unstudied, that the storage proteins contribute to other aspects of nickel metabolism. I show, through a crosslinking and affinity pulldown approach, that Hpn and Hpnl interact with up to 215 putative partners. Among these interacting partners are proteins known to be involved in nickel management, including UreAB, UreG, and HypB. Interestingly, many of the most strongly enriched interacting proteins have no known connection to nickel homeostasis. Interactions were explored by several approaches, including phenotypic characterization of mutant strains (hpn, hpnl, or hpnhpnl) and interaction verification by tryptophan fluorescence. Hydrogenase activity of the hpnhpnl strain was severely impacted by nickel deprivation in contrast to this effect on the wild type (WT). Interactions of two enzymes with both nickel storage proteins were confirmed: leucyl aminopeptidase (PepA) and aliphatic amidase (AmiE) interaction with the storage proteins was validated by observing a shift in tryptophan fluorescence upon incubation with either storage protein. The nickel storage proteins had opposite effects on PepA activity; deletion of hpn resulted in a three-fold decrease in activity while deletion of hpnl resulted in a seven-fold increase relative to WT. Hpn and Hpnl synergistically suppressed AmiE activity in a nickel-dependent manner AmiE activity in the mutant strains was higher than in their WT counterpart, and this activity was reduced by supplementation with nickel. Both storage proteins were required for full suppression the addition of pure Hpn or Hpnl to the extract lacking that protein resulted in suppression back to WT levels. Additionally, I show that recombinant amidase can bind the divalent metals zinc, cadmium, manganese, cobalt, and nickel. I show herein that Hpn and Hpnl may play more diverse roles than previously thought.