The allosteric behavior of Fur mediates oxidative stress signal transduction in Helicobacter pylori

Abstract

The microaerophilic gastric pathogen Helicobacter pylori is exposed to oxidative stress originating from the aerobic environment, the oxidative burst of phagocytes and the formation of reactive oxygen species, catalyzed by iron excess. Accordingly, the expression of genes involved in oxidative stress defense have been repeatedly linked to the ferric uptake regulator Fur. Moreover, mutations in the Fur protein affect the resistance to metronidazole, likely due to loss-of-function in the regulation of genes involved in redox control. Although many advances in the molecular understanding of HpFur function were made, little is known about the mechanisms that enable Fur to mediate the responses to oxidative stress. Here we show that iron-inducible, apo-Fur repressed genes, such as pfr and hydA, are induced shortly after oxidative stress, while their oxidative induction is lost in a fur knockout strain. On the contrary, holo-Fur repressed genes, such as frpB1 and fecA1, vary modestly in response to oxidative stress. This indicates that the oxidative stress signal specifically targets apo-Fur repressed genes, rather than impairing indiscriminately the regulatory function of Fur. Footprinting analyses showed that the oxidative signal strongly impairs the binding affinity of Fur toward apo-operators, while the binding toward holo-operators is less affected. Further evidence is presented that a reduced state of Fur is needed to maintain apo-repression, while oxidative conditions shift the preferred binding architecture of Fur toward the holo-operator binding conformation, even in the absence of iron. Together the results demonstrate that the allosteric regulation of Fur enables transduction of oxidative stress signals in H. pylori, supporting the concept that apo-Fur repressed genes can be considered oxidation inducible Fur regulatory targets. These findings may have important implications in the study of H. pylori treatment and resistance to antibiotics.

Keywords: allosteric regulation; antibiotic resistance; ferric uptake regulator; metal homeostasis; metalloproteins; oxidative stress; redox regulation; transcriptional regulation.

FIGURE 1

In vivo responses to oxidative stress of holo- and apo-Fur regulated genes. Transcript levels of apo-Fur (pfr, hydA); (A,B) and holo-Fur (frpB, fecA1); (C, D) repressed genes were assayed by RT-qPCR in wild-type and Δfur genetic backgrounds. Results are reported as the n-fold variation with respect to the untreated sample (white bars); light grey, dark grey and black bars correspond to treatments with 10 mM H₂O₂, 1 mM iron or 150 μM iron chelator, respectively, for 10 min. Data are reported in logarithmic scale; error bars indicate the standard deviations. The significance was calculated by a Student’s t-test. ns: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

DNase I protection patterns of Fur on the pfr promoter in reducing and oxidative conditions. DNase I footprinting assay of Fur protein on the Ppfr probe in presence of 150 μM of (NH₄)₂Fe(SO₄)₂ (A) or 150 μM Dipyridyl (B). A schematic representation of the promoter region is reported on the left side of the panel. Regions corresponding to Fur operator elements are indicated by boxes: black, holo-Fur operators; white, apo-Fur operators. Arrowheads indicate hypersensitivity bands to DNase I treatment. Black and white triangles indicate increasing concentrations of Fur protein, in the presence of iron and iron chelator, respectively. The redox condition of the assay is indicated on the top of the footprinting experiment: 5 mM H₂O₂ (oxidative), 1 mM DTT (mildly reducing), 5 mM DTT (reducing). Lane numbers 1 to 5: 0, 29, 58, 116, and 232 nM Fur dimer.

FIGURE 3

Differential Fur binding on the apo-repressed pfr promoter in response to hydrogen peroxide. (A) Considering the extreme affinity of the protein for the Ppfr OPI element, we performed a footprinting at lower Fur concentrations. The protein was preincubated with the Ppfr probe in binding buffer containing 1 mM DTT and 150 μM Dipyridyl for 10 min, then 5 mM H₂O₂ was added and the binding reaction was incubated for further 10 min (left panel); the control reaction (right panel) was treated with the same volume of water. Legends and symbols as in Figure 2. Lanes 1–5: 0, 0.3, 0.6, 3.3, and 8 nM Fur dimer, respectively. (B) EMSA performed on the pfr promoter probe with 1 mM DTT or 5 mM H₂O₂, in the presence of 150 μM Dipyridyl. Lanes 1–5: 0, 0.83, 1.7, 3.4, and 6.8 nM Fur dimer. A black arrowhead indicates the free probe, the white bar denotes the ladder generated by subsequent Fur binding events on the probe.

FIGURE 4

DNase I protection patterns of Fur on the frpB promoter in reducing and oxidative conditions. DNase I footprinting assay of Fur protein on the Ppfr probe in presence of 150 μM of (NH₄)₂Fe(SO₄)₂ (A) or 150 μM Dipyridyl (B). A schematic representation of the promoter region is reported on the left side of the panel. Regions corresponding to Fur operator elements are indicated by boxes: black, holo-Fur operators; white, apo-Fur operators. Arrowheads indicate hypersensitivity bands to DNase I digestion. Black and white triangles indicate increasing concentrations of Fur protein, in the presence of iron and iron chelator, respectively. The redox condition of the assay is indicated on the top of the footprinting experiment: 5 mM H₂O₂ (oxidative), 1 mM DTT (mildly reducing), 5 mM DTT (reducing). Lane numbers 1 to 5: 0, 29, 58, 116, and 232 nM Fur dimer.

FIGURE 5

Differential Fur binding to the fOPI holo-operator in response to hydrogen peroxide treatment. Differential protection at low Fur concentration; the protein was preincubated with the PfrpB probe in binding buffer containing 1 mM DTT and 150 μM of (NH₄)₂Fe(SO₄)₂ for 10 min, then 5 mM H₂O₂ was added and the binding reaction was incubated for further 10 min (left panel); the control reaction (right panel) was treated with the same volume of water. Legends and symbols as in Figure 4. Lanes 1–5: 0, 4, 8, 17, and 34 nM Fur dimer.

FIGURE 6

Model of Fur behavior in response to oxidative stress. apo-Fur conformation, light gray; holo-Fur conformation, dark gray. Fur represses iron uptake genes under iron-replete and oxidative conditions. apo-Fur targets (pfr) are only repressed under moderately reducing conditions. Upon an oxidative signal apo-Fur targets are induced, as a consequence of the allosteric behavior of Fur. Thereby, free intracellular iron can be scavenged by ferritins and metal-binding proteins, lowering the risk of iron-dependent oxidative damage that can be catalyzed by the high reactivity of this metal ion.