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The Crystal Structure of the C-Terminal Domain of the Salmonella enterica PduO Protein: An Old Fold with a New Heme-Binding Mode.

ABSTRACT: The two-domain protein PduO, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica is an ATP:Cob(I)alamin adenosyltransferase, but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) is, however, unknown. In this study, comparative growth assays with a set of Salmonella mutant strains showed that this domain is necessary for effective in vivo catabolism of 1,2-propanediol. It was also shown that isolated, recombinantly-expressed PduOC binds heme in vivo. The structure of PduOC co-crystallized with heme was solved (1.9 Å resolution) showing an octameric assembly with four heme moieities. The four heme groups are highly solvent-exposed and the heme iron is hexa-coordinated with bis-His ligation by histidines from different monomers. Static light scattering confirmed the octameric assembly in solution, but a mutation of the heme-coordinating histidine caused dissociation into dimers. Isothermal titration calorimetry using the PduOC apoprotein showed strong heme binding (K d = 1.6 × 10(-7) M). Biochemical experiments showed that the absence of the C-terminal domain in PduO did not affect adenosyltransferase activity in vitro. The evidence suggests that PduOC:heme plays an important role in the set of cobalamin transformations required for effective catabolism of 1,2-propanediol. Salmonella PduO is one of the rare proteins which binds the redox-active metabolites heme and cobalamin, and the heme-binding mode of the C-terminal domain differs from that in other members of this protein family.

SUBMITTER: Ortiz de Orue Lucana D

PROVIDER: S-EPMC4923194 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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The Crystal Structure of the C-Terminal Domain of the Salmonella enterica PduO Protein: An Old Fold with a New Heme-Binding Mode.

Ortiz de Orué Lucana Darío D Hickey Neal N Hensel Michael M Klare Johann P JP Geremia Silvano S Tiufiakova Tatiana T Torda Andrew E AE

Frontiers in microbiology 20160628

The two-domain protein PduO, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica is an ATP:Cob(I)alamin adenosyltransferase, but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) is, however, unknown. In this study, comparative growth assays with a set of Salmonella mutant strains showed that this domain is necessary for effective in vivo catabolism of 1,2-propanediol. It was also shown that isolated, re ...[more]

PMID: 27446048

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Project description:An essential feature of the pathogenesis of the Salmonella enterica serovar Enteritidis wild type (WT) is its ability to survive under diverse microenvironmental stress conditions, such as encountering antimicrobial peptides (AMPs) or glucose and micronutrient starvation. These stress factors trigger virulence genes carried on Salmonella pathogenicity islands (SPIs) and determine the efficiency of enteric infection. Although the oligosaccharide/oligonucleotide binding-fold (OB-fold) family of proteins has been identified as an important stress response and virulence determinant, functional information on members of this family is currently limited. In this study, we decipher the role of YdeI, which belongs to OB-fold family of proteins, in stress response and virulence of S Enteritidis. When ydeI was deleted, the ΔydeI mutant showed reduced survival during exposure to AMPs or glucose and Mg2+ starvation stress compared to the WT. Green fluorescent protein (GFP) reporter and quantitative real-time PCR (qRT-PCR) assays showed ydeI was transcriptionally regulated by PhoP, which is a major regulator of stress and virulence. Furthermore, the ΔydeI mutant displayed ∼89% reduced invasion into HCT116 cells, ∼15-fold-reduced intramacrophage survival, and downregulation of several SPI-1 and SPI-2 genes encoding the type 3 secretion system apparatus and effector proteins. The mutant showed attenuated virulence compared to the WT, confirmed by its reduced bacterial counts in feces, mesenteric lymph node (mLN), spleen, and liver of C57BL/6 mice. qRT-PCR analyses of the ΔydeI mutant displayed differential expression of 45 PhoP-regulated genes, which were majorly involved in metabolism, transport, membrane remodeling, and drug resistance under different stress conditions. YdeI is, therefore, an important protein that modulates S Enteritidis virulence and adaptation to stress during infection.IMPORTANCE S Enteritidis during its life cycle encounters diverse stress factors inside the host. These intracellular conditions allow activation of specialized secretion systems to cause infection. We report a conserved membrane protein, YdeI, and elucidate its role in protection against various intracellular stress conditions. A key aspect of the study of a pathogen's stress response mechanism is its clinical relevance during host-pathogen interaction. Bacterial adaptation to stress plays a vital role in evolution of a pathogen's resistance to therapeutic agents. Therefore, investigation of the role of YdeI is vital for understanding the molecular basis of regulation of Salmonella pathogenesis. In conclusion, our findings may contribute to finding potential targets to develop new intervention strategies for treatment and prevention of enteric diseases.

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Dataset Information

The Crystal Structure of the C-Terminal Domain of the Salmonella enterica PduO Protein: An Old Fold with a New Heme-Binding Mode.

Publications

The Crystal Structure of the C-Terminal Domain of the Salmonella enterica PduO Protein: An Old Fold with a New Heme-Binding Mode.

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