Abstract
The prokaryotic ATP-dependent Lon protease participates in the turnover of misfolded proteins and mediates the activity of regulatory proteins in diverse cellular processes. The lon gene of Helicobacter pylori strains is constitutively expressed during growth. The biological role of Lon protease in H. pylori (HpLon) is unclear so far. In this study, we found that polyphosphate (polyP) enhanced the protease and peptidase activity of HpLon. PolyP also competed with double strand DNA (dsDNA) for HpLon binding. In order to elucidate the function and physiological role of HpLon in H. pylori, a trapping approach was used to identify putative Lon binding partners in the bacterium. Many of HpLon-interacting partners were found to be associated with the activation of metronidazole (Mtz), which is widely used in first-line therapy against H. pylori infections. In addition, we constructed a lon-deficient mutant more susceptible to Mtz than the wild-type form. A proteomic analysis identified RdxA, which encodes oxygen-insensitive NADPH nitroreductase, a major Mtz-activating enzyme in H. pylori. RdxA is a physiological interacting partner of HpLon and is not cleaved by the protease. We found that the AAA+ module (ATPases Associated with diverse Activities) in HpLon causes a decrease in both NADPH oxidase and Mtz reductase activity in RdxA. This result explains why the protease-defective lon mutant (lonS741A mutant) exhibits the same level of Mtz susceptibility as wild-type H. pylori. Our results are consistent with a model in which HpLon recruits the ADP moiety of NADPH through the AAA+ module to interact with RdxA and thereby further decreases its NADPH-oxidizing and Mtz-reducing activities. This is the first study to figure out how HpLon regulates RdxA activity and participate the process of Mtz activation in H. pylori.