A novel mode of control of nickel uptake by a multifunctional metallochaperone

Cellular metal homeostasis is a critical process for all organisms, requiring tight regulation. In the major pathogen Helicobacter pylori, the acquisition of nickel is an essential virulence determinant as this metal is a cofactor for the acid-resistance enzyme, urease. Nickel uptake relies on the NixA permease and the NiuBDE ABC transporter. Till now, bacterial metal transporters were reported to be controlled at their transcriptional level. Here we uncovered post-translational regulation of the essential Niu transporter in H. pylori. Indeed, we demonstrate that SlyD, a protein combining peptidyl-prolyl isomerase (PPIase), chaperone, and metal-binding properties, is required for the activity of the Niu transporter. Using two-hybrid assays, we found that SlyD directly interacts with the NiuD permease subunit and identified a motif critical for this contact. Mutants of the different SlyD functional domains were constructed and used to perform in vitro PPIase activity assays and four different in vivo tests measuring nickel intracellular accumulation or transport in H. pylori. In vitro, SlyD PPIase activity is down-regulated by nickel, independently of its C-terminal region reported to bind metals. In vivo, a role of SlyD PPIase function was only revealed upon exposure to high nickel concentrations. Most importantly, the IF chaperone domain of SlyD was shown to be mandatory for Niu activation under all in vivo conditions. These data suggest that SlyD is required for the active functional conformation of the Niu permease and regulates its activity through a novel mechanism implying direct protein interaction, thereby acting as a gatekeeper of nickel uptake. Finally, in agreement with a central role of SlyD, this protein is essential for the colonization of the mouse model by H. pylori. Author summary Metal ions are essential for the viability of all living organisms. Indeed, more than one-third of all proteins need metal cofactors for function. Intracellular metal concentrations require tight control as non-physiological amounts are very toxic. In particular, nickel plays a unique role in Helicobacter pylori, a bacterial pathogen that colonizes the stomach of about half of the human population worldwide and is associated with the development of gastric cancer. Nickel is essential for H. pylori as it is the cofactor of urease, an enzyme indispensable for resistance to the gastric acidity of the stomach and thus for in vivo colonization. To import nickel despite its scarcity in the human body, H. pylori requires efficient uptake mechanisms. Till now, control of nickel uptake was only reported to rely on transcriptional regulators. In the present study, we uncovered a novel mechanism of regulation of nickel acquisition. SlyD, a multifunctional enzyme was found to control, by direct protein interaction, the activity of an essential nickel uptake system in H. pylori. We revealed that the SlyD chaperone activity is mandatory for the active conformation and thus functionality of the nickel permease.

Automated summary

Metal ion homeostasis is critical for all organisms, with nickel being an essential virulence determinant in Helicobacter pylori. This study uncovered a novel post-translational regulation mechanism for the essential Niu transporter in H. pylori, controlled by the multifunctional SlyD enzyme through direct protein interaction. SlyD's chaperone activity is crucial for the active conformation of the nickel permease, indicating its role as a gatekeeper for nickel uptake.