Yersiniabactin reduces the respiratory oxidative stress response of innate immune cells

Abstract

Enterobacteriaceae that contain the High Pathogenicity Island (HPI), which encodes the siderophore yersiniabactin, display increased virulence. This increased virulence may be explained by the increased iron scavenging of the bacteria, which would both enhance bacterial growth and limit the availability of iron to cells of the innate immune system, which require iron to catalyze the Haber-Weiss reaction that produces hydroxyl radicals. In this study, we show that yersiniabactin increases bacterial growth when iron-saturated lactoferrin is the main iron source. This suggests that yersiniabactin provides bacteria with additional iron from saturated lactoferrin during infection. Furthermore, the production of ROS by polymorphonuclear leukocytes, monocytes, and a mouse macrophage cell line is blocked by yersiniabactin, as yersiniabactin reduces iron availability to the cells. Importantly, iron functions as a catalyst during the Haber-Weiss reaction, which generates hydroxyl radicals. While the physiologic role of the Haber-Weiss reaction in the production of hydroxyl radicals has been controversial, the siderophores yersiniabactin, aerobactin, and deferoxamine and the iron-chelator deferiprone also reduce ROS production in activated innate immune cells. This suggests that this reaction takes place under physiological conditions. Of the tested iron chelators, yersiniabactin was the most effective in reducing the ROS production in the tested innate immune cells. The likely decreased bacterial killing by innate immune cells resulting from the reduced production of hydroxyl radicals may explain why the HPI-containing Enterobacteriaceae are more virulent. This model centered on the reduced killing capacity of innate immune cells, which is indirectly caused by yersiniabactin, is in agreement with the observation that the highly pathogenic group of Yersinia is more lethal than the weakly pathogenic and the non-pathogenic group.

Figure 1. In the irp2 knockout strain the production of yersiniabactin is blocked.

Yersiniabactin production was tested using a GFP reporter assay with the knockout strain WA-CS irp1::Kan^r containing the pCJG3.3N plasmid as the reporter strain to detect yersiniabactin in supernatants. The number of bacteria versus the measured arbitrary amount of fluorescence from 50,000 counted bacteria is shown in panel A. A) Supernatants from the irp2 knockout (E4Δirp2) cultures (grey) induced a lower amount of GFP compared to supernatants from the wild-type (WT) cultures (white). This indicates that production of yersiniabactin was blocked in the irp2 knockout. The experiments were performed in duplicate. B) The GFP reporter assay is dependent on yersiniabactin production, and production of yersiniabactin in the irp2 knockout is blocked. Yersiniabactin production is only seen in the wild-type (WT) cells cultured in iron-depleted medium (NBD), while the irp2 knockout (E4Δirp2) strain cultured in NBD and iron-containing medium (NB), as well as the wild-type strain grown in NB, did not produce yersiniabactin. Three different experiments were performed, with each sample analyzed in duplicate. C) The expression of HMWP1 and HMWP2 is disrupted by the insertion of a kanamycin resistance gene into irp2 using the Tagetron Knockout System. The irp2 knockout (E4Δirp2) strain was not able to produce HMWP1 and HMWP2 when cultured in NBD. M: marker; lane 1: wild-type strain E4; lane 2–5: irp2 gene knockouts created using the wild-type strain E4; lane 6 wild-type EHOS strain 03-702; lane 7: wild-type EHOS strain 03-819 served as HPI-positive control because the HMWP2 of this strain was confirmed by Edman degradation.

Figure 2. Growth curves of the EHOS E4 isolate and its corresponding irp2 knockout strain.

The results are presented as the mean of three experiments performed in duplicate on different days. Cells were grown in: A) nutrient broth treated with 1% Chelex; B) nutrient broth treated with 1% Chelex and supplemented with 80% iron-saturated lactoferrin (∼6.2 µM, Fe-LF) or unsaturated lactoferrin (∼6.4 µM,0-LF); C) nutrient broth treated with 1% Chelex and supplemented with 17 µM holo-transferrin or 17 µM apo-transferrin; D) nutrient broth treated with 1% Chelex and supplemented with 10 µM or 30 µM hemin.

Figure 3. ROS production by PMNs is reduced by yersiniabactin.

To determine the effect of yersiniabactin on the ROS production of PMNs, PMNs were pre-incubated with yersiniabactin. PMNs were then activated by PMA, and the production of ROS was measured using luminol. A) Absolute ROS production measured in arbitrary fluorescence units (A.U.) of PMNs after pre-incubation with yersiniabactin or iron-saturated yersiniabactin or the controls (PMNs that were only pre-incubated in RPMI/HSA medium and PMNs that were pre-incubated with yersiniabactin but not stimulated with PMA). B) The yersiniabactin-mediated inhibition of ROS production by PMNs is concentration dependent. Red line: the concentration-dependent decrease in production of ROS following treatment with yersiniabactin. Blue line: no decrease in ROS production following treatment with different concentrations of iron-saturated yersiniabactin. Black dotted line: negative control (PMNs incubated with yersiniabactin, but not stimulated with PMA); ***p value<0.0005, **p value<0.005, *p value<0.05. C) Treatment of PMNs with unsaturated lactoferrin or holo-transferrin partly inhibits the yersiniabactin-mediated inhibition of ROS production. Black bars: relative ROS production following treatment with yersiniabactin; gray bars: relative ROS production following treatment with yersiniabactin and unsaturated lactoferrin; white bars: relative ROS production following treatment with yersiniabactin and holo-transferrin. ^aConcentration of the iron chelator. D) Treatment of PMNs with saturated lactoferrin or apo-transferrin partly inhibits the yersiniabactin-mediated inhibition of ROS production. Black bars: relative ROS production following treatment with yersiniabactin; gray bars: relative ROS production following treatment with yersiniabactin and saturated lactoferrin; white bars: relative ROS production following treatment with yersiniabactin and apo-transferrin. All experiments were replicated three times, and each sample was analyzed in duplicate.

Figure 4. Other iron chelators also reduce ROS production by PMNs.

Along with yersiniabactin, aerobactin, deferoxamine, and deferiprone also reduce ROS production by PMNs. To determine the effect of other iron-binding molecules on ROS production by PMNs, PMNs were pre-incubated with aerobactin, deferoxamine, deferiprone, or yersiniabactin. The pre-treated PMNs were then activated using PMA, and the production of ROS was measured using luminol. Red line: the concentration-dependent decrease in ROS production following yersiniabactin treatment. Gray line: the decrease in ROS production following treatment with aerobactin. Yellow line: the concentration-dependent decrease in ROS production following deferiprone treatment. Green line: the concentration-dependent decrease in ROS production following deferoxamine treatment. The level of ROS produced by stimulated PMNs without additives was set as 100%. All experiments were replicated three times, and each sample was analyzed in duplicate.

Figure 5. ROS production by PBMC-derived monocytes and in the J774A.1 cell line (mouse macrophages) is also reduced by pre-incubation with iron-binding molecules.

A) ROS production in PBMC-derived monocytes pre-incubated with yersiniabactin or iron-saturated yersiniabactin. Black bars: PBMCs pre-incubated with 90 µM yersiniabactin and subsequently activated with PMA. Gray bar: PBMCs pre-incubated with 90 µM iron-saturated yersiniabactin and subsequently activated with PMA. White bar: no compounds added. ^aBar: Negative control: 90 µM yersiniabactin was added but the cells were not stimulated with PMA; **p value = 0.0053, *p value = 0.02. B) Yersiniabactin, aerobactin, deferoxamine, and deferiprone all inhibit ROS production in the J774A.1cell line (mouse macrophages) in a concentration-dependent manner, but yersiniabactin is the most potent in reducing ROS production. Red line: concentration-dependent decrease in ROS production following treatment with yersiniabactin. Blue line: concentration-dependent decrease in ROS production following treatment with iron-saturated yersiniabactin. Gray line: concentration-dependent decrease in ROS production following treatment with aerobactin. Yellow line: concentration-dependent decrease in ROS production following treatment with deferiprone. Green line: concentration-dependent decrease in ROS production following treatment with deferoxamine. The level produced by stimulated PMNs without additives was set as 100%. All experiments were replicated three times, and each sample was analyzed in duplicate.