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Iron and pH homeostasis intersect at the level of Fur regulation in the gastric pathogen Helicobacter pylori.

ABSTRACT: Helicobacter pylori persistently colonizes the stomach of the majority of the world's population and is a tremendous medical burden due to its causal role in diverse gastric maladies. Since the stomach is a constantly changing environment, successful colonization of H. pylori within this niche requires regulation of bacterial gene expression to cope with the environmental fluctuations. In H. pylori, the ferric uptake regulator (Fur) has been shown to play an intricate role in adaptation of the bacterium to two conditions known to oscillate within the gastric mucosa: iron limitation and low pH. To extend our knowledge of the process of regulation and adaptation in H. pylori, we show that Fur is required for efficient colonization of the Mongolian gerbil: the mutant strain exhibits a 100-fold increase in the 50% infectious dose, as well as a 100-fold defect in competitive colonization, when coinfected with wild-type bacteria. Furthermore, we used DNA microarrays to identify genes whose expression was altered in a Fur-deficient strain. We show that the Fur regulon of H. pylori consists of approximately 30 genes, most of which have been previously annotated as acid stress associated. Finally, we investigate the role of Fur in acid-responsive modulation of gene expression and show that a large number of genes are aberrantly expressed in the Fur mutant specifically upon acid exposure. This fact likely explains the requirement for this regulator for growth and colonization in the stomach.

SUBMITTER: Gancz H

PROVIDER: S-EPMC1346641 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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Iron and pH homeostasis intersect at the level of Fur regulation in the gastric pathogen Helicobacter pylori.

Gancz Hanan H Censini Stefano S Merrell D Scott DS

Infection and immunity 20060101 1

Helicobacter pylori persistently colonizes the stomach of the majority of the world's population and is a tremendous medical burden due to its causal role in diverse gastric maladies. Since the stomach is a constantly changing environment, successful colonization of H. pylori within this niche requires regulation of bacterial gene expression to cope with the environmental fluctuations. In H. pylori, the ferric uptake regulator (Fur) has been shown to play an intricate role in adaptation of the b ...[more]

PMID: 16369017

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Project description:BackgroundHelicobacter pylori is a human stomach pathogen, naturally-competent for DNA uptake, and prone to homologous recombination. Extensive homoplasy (i.e., phylogenetically-unlinked identical variations) observed in H. pylori genes is considered a hallmark of such recombination. However, H. pylori also exhibits a high mutation rate. The relative adaptive role of homologous recombination and mutation in species diversity is a highly-debated issue in biology. Recombination results in homoplasy. While convergent mutation can also account for homoplasy, its contribution is thought to be minor. We demonstrate here that, contrary to dogma, convergent mutation is a key contributor to Helicobacter pylori homoplasy, potentially driven by adaptive evolution of proteins.ResultsOur present genome-wide analysis shows that homoplastic nonsynonymous (amino acid replacement) changes are not typically accompanied by homoplastic synonymous (silent) variations. Moreover, the majority of the codon positions with homoplastic nonsynonymous changes also contain different (i.e. non-homoplastic) nonsynonymous changes arising from mutation only. This indicates that, to a considerable extent, nonsynonymous homoplasy is due to convergent mutations. High mutation rate or limited availability of evolvable sites cannot explain this excessive convergence, as suggested by our simulation studies. Rather, the genes with convergent mutations are overrepresented in distinct functional categories, suggesting possible selective responses to conditions such as distinct micro-niches in single hosts, and to differences in host genotype, physiology, habitat and diet.ConclusionsWe propose that mutational convergence is a key player in H. pylori's adaptation and extraordinary persistence in human hosts. High frequency of mutational convergence could be due to saturation of evolvable sites capable of responding to selection pressures, while the number of mutable residues is far from saturation. We anticipate a similar scenario of mutational vs. recombinational genome dynamics or plasticity for other naturally competent microbes where strong positive selection could favor frequent convergent mutations in adaptive protein evolution.

Project description:Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen's ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host's stomach. Although Hp's glycans are linked to pathogenesis, Hp's glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp's glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp's azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.

Dataset Information

Iron and pH homeostasis intersect at the level of Fur regulation in the gastric pathogen Helicobacter pylori.

Publications

Iron and pH homeostasis intersect at the level of Fur regulation in the gastric pathogen Helicobacter pylori.

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