Evolution of Helicobacter: Acquisition by Gastric Species of Two Histidine-Rich Proteins Essential for Colonization

Abstract

Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2) play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn and Hpn-2 by gastric Helicobacter species constituted a decisive evolutionary event to allow Helicobacter to colonize the hostile gastric environment, in which no other bacteria persistently thrives. This acquisition was key for the emergence of one of the most successful bacterial pathogens, H. pylori.

Characterization in Helicobacter pylori of a Nickel Transporter Essential for Colonization That Was Acquired during Evolution by Gastric Helicobacter Species

“…pylori genes were randomly inactivated in vitro by the insertion of a transposable element conferring chloramphenicol resistance, was used to transform H . pylori strain G27 [50] and the isogenic Δhpn mutant that is more sensitive to nickel [22]. We first established the nickel concentrations at which the plating efficiencies of strains G27 and G27 Δhpn were strongly affected.…”

“…Using a previously described method [53], we constructed unmarked deletions mutants of niuD ( hpB8_663 ) in the B128-S and B128-S Δ hpn strains, (a genetic background that we previously characterized [22], S1 and S2 Tables). Tolerance of these mutants to nickel exposure was evaluated at neutral pH by following growth inhibition in liquid medium.…”

“…These include NikR, a nickel responsive pleiotropic transcriptional regulator [15–17], CznABC a metal efflux pump [18] and also three Histidine-rich proteins Hpn, Hpn-2 and HspA that are found only in the Helicobacter genus [19–22]. Hpn and HspA are involved in sequestration of nickel ions and the corresponding mutants are highly sensitive to nickel exposure [21,22]. In addition, the Hpn/Hpn-2 and HspA nickel-binding proteins contribute to the production of active urease and hydrogenase, respectively [22–24].…”

“…Hpn and HspA are involved in sequestration of nickel ions and the corresponding mutants are highly sensitive to nickel exposure [21,22]. In addition, the Hpn/Hpn-2 and HspA nickel-binding proteins contribute to the production of active urease and hydrogenase, respectively [22–24]. …”

In Vivo Analysis of the Viable Microbiota and Helicobacter pylori Transcriptome in Gastric Infection and Early Stages of Carcinogenesis

“…Among the H. pylori genes with highest overall expression were genes involved in pH regulation and ammonia production, including those encoding urease subunits A and B (ureA and ureB), a pH-regulated permease (ureI), and an aliphatic amidase responsible for ammonia production, acylamide amidohydrolase (amiE). Also, two genes encoding the recently characterized histidine-rich, metal-binding polypeptides Hpn (HP1427) and Hpn2 (HP1432) (28), involved in sequestration and intracellular trafficking of nickel, were among the most highly expressed genes.…”