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

Daniel Vinella,Frédéric Fischer,Marie Robbe-Saule,Pierre Richaud,Hilde De Reuse,Julien Gallaud,Céline Brochier-Armanet,Egor Vorontsov,Christine Cavazza,Valérie Michel,Julia Chamot-Rooke,Christian Malosse

doi:10.1371/journal.ppat.1005312

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.

Highlights

Helicobacter pylori is a gram-negative bacterium that colonizes the stomach of about half of the human population
We showed that speciation of the gastric Helicobacter species from the other Helicobacter species occurred concomitantly with the emergence of hpn, while the duplication event at the origin of hpn-2 took place in the common ancestors of H. pylori and H. acinonychis species
As a first step for this phylogenomic analysis, we establish a core-proteome-based phylogeny on 330 proteomes of Helicobacter from various geographical origins associated with different pathologies available at the NCBI, including 305 H. pylori strains, 7 gastric and 11 enterohepatic non-pylori Helicobacter species

Summary

Introduction

Helicobacter pylori is a gram-negative bacterium that colonizes the stomach of about half of the human population. Virulence of H. pylori directly depends on its capacity to persistently colonize the stomach, a hostile and acidic niche Survival under such conditions relies on the activity of the nickel-containing urease, an enzyme that catalyzes hydrolysis of urea into ammonia and bicarbonate, two buffering compounds that allow the bacterium to maintain its cytoplasmic pH close to neutrality [4]. The only other nickeldependent enzyme in H. pylori, the [NiFe] hydrogenase [5], has been shown to be important for colonization, presumably because it provides an alternative respiratory pathway, allowing H. pylori to use molecular hydrogen as an energy source [6]. The transition metal ion Ni(II), which is an essential constituent of the active site of urease and [NiFe]hydrogenase, can be considered as an essential determinant for the virulence of H. pylori and for in vivo colonization [6,7]

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