Random and site‐specific mutagenesis of the Helicobacter pylori ferric uptake regulator provides insight into Fur structure–function relationships

Abstract

Summary The ferric uptake regulator (Fur) of Helicobacter pylori is a global regulator that is important for colonization and survival within the gastric mucosa. H. pylori Fur is unique in its ability to activate and repress gene expression in both the iron-bound (Fe-Fur) and apo forms (apo-Fur). In the current study we combined random and site-specific mutagenesis to identify amino acid residues important for both Fe-Fur and apo-Fur function. We identified 25 mutations that affected Fe-Fur repression and 23 mutations that affected apo-Fur repression, as determined by transcriptional analyses of the Fe-Fur target gene amiE, and the apo-Fur target gene, pfr. In addition, eight of these mutations also significantly affected levels of Fur in the cell. Based on regulatory phenotypes, we selected several representative mutations to characterize further. Of those selected, we purified the wildtype (HpFurWT) and three mutant Fur proteins (HpFurE5A, HpFurA92T, and HpFurH134Y), which represent mutations in the N-terminal extension, the regulatory metal binding site (S2) and the structural metal binding site (S3), respectively. Purified proteins were evaluated for secondary structure by circular dichroism spectroscopy, iron-binding by atomic absorption spectrophotometry, oligomerization in iron-substituted and apo conditions by in vitro cross-linking assays, and DNA binding to Fe-Fur and apo-Fur target sequences by fluorescence anisotropy. The results showed that the N-terminal, S2, and S3 regions play distinct roles in terms of Fur structure-function relationships. Overall, these studies provide novel information regarding the role of these residues in Fur function, and provide mechanistic insight into how H. pylori Fur regulates gene expression in both the iron-bound and apo forms of the protein.

Helicobacter pylori Adaptation In Vivo in Response to a High-Salt Diet

“…The Fur R88H and P45T mutations are each located close to Fur amino acid residues important for iron coordination of Fur (E90 and H42, respectively) (55), and therefore, we speculate that these mutations might affect the ability of Fur to regulate its target genes. Consistent with this hypothesis, several mutations close to Fur R88 (e.g., R87Q, Y89C, and E90Q) are known to impair Fur regulatory activity (56). Although substitution mutations at the Fur R88 position are not found commonly in H. pylori isolates from humans, an R88H mutation identical to the mutation observed in the present study has been detected in at least three H. pylori isolates from humans (57).…”

Pathogenesis of Helicobacter pylori Infection

“…Gilbreath et al. characterized Fur by targeted and random mutagenesis and found novel structural features which enable the protein to exert its function in its iron‐bound and unloaded conformation. In addition to playing with host iron supplies, H. pylori is auxotrophic for cholesterol which it takes up from human cells, making use again of host supplies for its own good.…”

“…The Fur regulator appears to play an important role in environmental adaptation of H. pylori in vivo, including the bacterial response to local iron availability, and backing up the interplay of virulence factors such as VacA and the cagPAI with iron. Gilbreath et al [35] characterized Fur by targeted and random mutagenesis and found novel structural features which enable the protein to exert its function in its ironbound and unloaded conformation. In addition to playing with host iron supplies, H. pylori is auxotrophic for cholesterol which it takes up from human cells, making use again of host supplies for its own good.…”

Functional insights into the interplay between DNA interaction and metal coordination in ferric uptake regulators

“…Combined with the crystal structure of CjFurZn, these observations suggest that the S3 site plays two important functions in CjFur biology, in contributing to S2 metalation and in correctly aligning the DNA binding domain of CjFur for the control of apo-regulated genes. Previous studies documented a similar interplay in Bacillus subtilis and Helicobacter pylori Fur showing that mutation of the S3 site impacts the incorporation of metals in both metal binding sites 39 , 40 . However, we note that BsFur mutants in S2/S3 were unaffected in DNA binding under saturating manganese conditions while CjFur ΔS2 and CjFur ΔS3 mutants showed defects even under 500 molar excess of manganese.…”