Functional Characterization of Salmonella Typhimurium Encoded YciF, a Domain of Unknown Function (DUF892) Family Protein, and Its Role in Protection during Bile and Oxidative Stress

Abstract

YciF (STM14_2092) is a member of the domain of unknown function (DUF892) family. It is an uncharacterized protein involved in stress responses in Salmonella Typhimurium. In this study, we investigated the significance of YciF and its DUF892 domain during bile and oxidative stress responses of S. Typhimurium. Purified wild-type YciF forms higher order oligomers, binds to iron, and displays ferroxidase activity. Studies on the site-specific mutants revealed that the ferroxidase activity of YciF is dependent on the two metal binding sites present within the DUF892 domain. Transcriptional analysis displayed that the ΔcspE strain, which has compromised expression of YciF, encounters iron toxicity due to dysregulation of iron homeostasis in the presence of bile. Utilizing this observation, we demonstrate that the bile mediated iron toxicity in ΔcspE causes lethality, primarily through the generation of reactive oxygen species (ROS). Expression of wild-type YciF, but not the three mutants of the DUF892 domain, in ΔcspE alleviate ROS in the presence of bile. Our results establish the role of YciF as a ferroxidase that can sequester excess iron in the cellular milieu to counter ROS-associated cell death. This is the first report of biochemical and functional characterization of a member of the DUF892 family. IMPORTANCE The DUF892 domain has a wide taxonomic distribution encompassing several bacterial pathogens. This domain belongs to the ferritin-like superfamily; however, it has not been biochemically and functionally characterized. This is the first report of characterization of a member of this family. In this study, we demonstrate that S. Typhimurium YciF is an iron binding protein with ferroxidase activity, which is dependent on the metal binding sites present within the DUF892 domain. YciF combats iron toxicity and oxidative damage caused due to exposure to bile. The functional characterization of YciF delineates the significance of the DUF892 domain in bacteria. In addition, our studies on S. Typhimurium bile stress response divulged the importance of comprehensive iron homeostasis and ROS in bacteria.

Keywords: DUF892 member; Salmonella; YciF; bile; biochemistry; metal binding; reactive oxygen species; stress proteins; stress response.

FIG 1

(A) YciF contains a single ferritin-like domain, DUF892, that spans almost the entire length of the protein. (B) Multiple sequence alignment using clustal omega shows that YciF is highly conserved across Enterobacteriaceae. The residues of DUF892 domain involved in metal ion-coordination are highlighted in turquoise. (C) Comparison of diiron centers of ribonucleotide reductase (PDB.id: 1PFR), frog M-ferritin (PDB. Id: 1MFR), and YciF (PDB.id: 4ERU). E143 and E58 are the single bidentate residues for YciF and frog M-ferritin, respectively, whereas ribonucleotide reductase has two carboxylate bridging residues (E115 and E238).

FIG 2

S. Typhimurium YciF and its metal binding sites mutants form higher order oligomers. (A) 12.5% SDS-PAGE profile of Strep II tagged YciF, Q54A, E113Q, and E143D. Monomeric YciF and its mutants have a molecular weight of approximately 20 kDa. (B) 4% to15% gradient native PAGE profile of YciF, Q54A, E113Q, and E143D. (C, D) Analytical size exclusion chromatography to determine the apparent molecular weight of YciF and its mutants. Purified proteins were subjected to Superdex S-200 column. Molecular weight standard was run on the same column maintaining similar elution conditions. The table inside represents the molecular mass calculated from linear regression equation.

FIG 3

S. Typhimurium YciF binds to iron and displays ferroxidase activity. (A) Thermal shift assay was performed to determine the native ligand. 10 μg YciF was incubated with 200 μM (NH₄)₂Fe(SO₄)₂, MgSO₄, ZnCl₂, and EDTA. The table represents the melting temperature (Tm). (B) Comparison of iron binding of YciF and its metal binding sites mutants. Purified proteins were incubated with 200 μM (NH₄)₂Fe(SO₄)₂ and subjected to native PAGE followed by staining with Ferene-S and Coomassie brilliant blue. BSA was used as negative control. (C) Determination of ferroxidase activity of YciF and the mutants at different concentrations of (NH₄)₂Fe(SO₄)₂. Formation of ferric ion was measured at 310 nm. (D) Kinetic comparison of ferroxidase activity of YciF, Q54A, E113Q, E143D. (NH₄)₂Fe(SO₄)₂ was added at different concentrations and absorbance at 310 nm was measured for 600 s at 30-s interval. Values were blanked against no protein control to correct for auto-oxidation of ferrous ion. Graphs were plotted using nonlinear regression. The data are representative of three independent protein preparations.

FIG 4

qRT-PCR analysis of genes responsible for iron balance in S. Typhimurium and intracellular iron estimation reveal iron disbalance in ΔcspE in the presence of bile. (A) Genes encoding ferri-siderophore transporters: fepA, fhuC are responsible for uptake of Fe3+ while sitA and feoB are involved in Fe²⁺ uptake. (B) Genes encoding enzymes responsible for intracellular release of iron from siderophores. (C) Genes regulating intracellular iron level: ftnB and dps are iron storage proteins whereas fur is a negative regulator of iron uptake. Cq value of WT strain grown in LB was used to normalize Cq values of all the panels. Data shown as mean ± SEM and is representative of six independent experiments. (D) Quantitation of intracellular iron levels in WT and ΔcspE strains upon bile treatment. Data shown as mean ± SEM and is representative of four independent experiments. P values for qRT-PCR were measured by one-way ANOVA using Sidak's multiple-comparison test. For intracellular iron estimation, P value was calculated using two-way ANOVA using Sidak's multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

YciF combats bile mediated oxidative stress. (A) S. Typhimurium WT and ΔcspE strains overexpressing YciF were grown in absence or presence of 3% bile for 6 h. Cells were stained with DCFDA and analyzed by flow cytometry. (B) The percentage DCFDA positive cells based on flow cytometry data. (C) Quantitation of intracellular ROS levels upon 3% bile treatment in strains overexpressing YciF and the metal binding sites mutants using DCFDA staining. Fluorescence was measured using excitation and emission wavelength of 485 nm and 535 nm, respectively. (D) Growth of strains overexpressing YciF and the mutants in absence or presence of 3% bile determined by measuring O.D. at 600 nm. Cells were plated on LB agar post bile treatment and colonies were counted. Data shown as mean ± SEM and is representative of four independent experiments. P values were analyzed by two-way ANOVA using Sidak's multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 6

Iron chelation reduces ROS and increases the growth of ΔcspE strain in the presence of bile. S. Typhimurium WT and ΔcspE were grown in LB alone and LB containing 200 μM bipyridyl/3% bile/3% bile + 200 μM bipyridyl for 6 h. For H₂O₂ treatment, the mentioned strains were grown for 3 h. (A) O.D. was measured at 600 nm and cells were plated on LB agar following 3% bile treatment and colonies were counted. (B) WT and ΔcspE strains were spotted onto LB agar plates after 6 h of 3% bile treatment. (C) Cellular ROS levels in absence or presence of 2,2-bipyridyl following 3% bile treatment was quantitated using DCFDA staining. Fluorescence was determined at excitation and emission wavelength of 485 nm and 535 nm, respectively. (D) O.D. was measured at 600 nm after H₂O₂ treatment. Data shown as mean ± SEM and is representative of four independent experiments. P values were measured by two-way ANOVA using Sidak's multiple-comparison test. **, P < 0.01; ****, P < 0.0001.

FIG 7

Schematic explaining the role of YciF during bile stress. Bile is known to cause an increase in oxidative stress. This, combined with H₂O₂ produced endogenously due to aerobic metabolism, will undergo Fenton reaction, upon iron excess causing further sustained increase in ROS. Thus, the ΔcspE strain that shows disruption of iron homeostasis upon bile treatment, is likely to be more susceptible to Fenton reaction compared to the wild-type strain. Higher ROS leads to cell death that can be mitigated by overexpression of YciF which binds to iron and has ferroxidase activity to help the ΔcspE strain detoxify the excess iron. Image was constructed using Biorender.com.