Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Lighting Up Iron Metabolism During Infection

By: Carol A. Rouzer, VICB Communications
Published:  December 22, 2017



A new bioluminescent probe of ferrous iron reveals changes in liver iron disposition during infection with Acenitobacter baumannii.


Due to its ability to cycle between ferric and ferrous states at redox potentials relevant to biological processes, iron serves as a cofactor for a large number of enzymes and transport proteins. However, its redox activity can also lead to toxicity if it is present at excessive concentrations. For these reasons, methods to assess and monitor iron status in vivo are of considerable interest. This is especially true for techniques that allow differentiation between ferric iron, which is the primary form found in storage pools, versus ferrous iron, which is present in metabolically active labile iron pools (LIPs). To address this need, Vanderbilt Institute of Chemical Biology member Eric Skaar, along with his collaborator Christopher Chang (University of California, Berkeley), report ICL-1 (iron-caged luciferin-1), a luminescent ferrous iron sensor for the detection of LIPs in vivo [A. T. Aron, M.C. Heffern, Z, R. Lonergan, et al. Proc. Natl. Acad. Sci. U.S.A., (2017) published online Nov 14, DOI: 10.1073/pnas.1708747114].

ICL-1 is a conjugate of D-aminoluciferin and 1,2,4-trioxolane, an iron-reactive endoperoxide. Reaction with ferrous iron releases free D-aminoluciferin, which can then serve as a substrate for the enzyme luciferase, thereby generating a bioluminescent signal (Figure 1). To verify that ICL-1 truly serves as a ferrous iron-specific probe, the investigators first incubated the molecule with varying concentrations of ferrous ammonium sulfate (FAS) in the presence of luciferase. They observed a bioluminescent signal that increased in intensity with FAS concentrations over a range of 25 to 100 μM. The signal increased maximum 7-fold or 30-fold when the reactions were carried out under aerobic or anaerobic conditions, respectively. Addition of the ferrous iron chelator bipyridine (BPY) blocked the signal, and no iron-dependent increase in signal was evident when ICL-1 was replaced with the iron-insensitive probe luciferin. These results confirmed that the luminescence generated from ICL-1 was dependent on the presence of ferrous iron. Further studies demonstrated that free copper could also produce increased luminescence in the presence of ICL-1 and luciferase. However, the greater than 10-fold higher concentrations of iron as compared to copper and the fact that most free copper is bound by glutathione or protein in vivo led the investigators to conclude that interference from copper would likely be minimal.



FIGURE 1. Schematic of the approach used to image labile ferrous iron stores. ICL-1 is injected into a mouse that expresses luciferase. ICL-1 reacts with ferrous iron in mouse tissues, releasing D-aminoluciferin, which serves as a luciferase substrate, producing bioluminescence. Figure reproduced with permission from A. T. Aron, M.C. Heffern, Z, R. Lonergan, et al. Proc. Natl. Acad. Sci. U.S.A., (2017) published online Nov 14, DOI: 10.1073/pnas.1708747114. Copyright 2017, A.T. Aron, et al.



To determine whether ICL-1 could be used to measure LIPs in biological systems, the investigators next tested its ability to detect ferrous iron in the PC3M-luc luciferase-expressing prostate cancer cell line. They were encouraged to find that ICL-1 is highly stable in cell culture medium and that they could measure a concentration-dependent increase in ICL-mediated bioluminescence by supplementing the cells with increasing concentrations of FAS. Again, addition of BPY blocked the luminescence signal (Figure 2). They obtained similar results from experiments with a number of other luciferase-expressing cell lines.




FIGURE 2. Luminescence signals from PC3M-luc cells treated with the indicated concentrations of ferrous ammonium sulfate (FAS) and/or bipyridine (BPY). Figure reproduced with permission from A. T. Aron, M.C. Heffern, Z, R. Lonergan, et al. Proc. Natl. Acad. Sci. U.S.A., (2017) published online Nov 14, DOI: 10.1073/pnas.1708747114. Copyright 2017, A.T. Aron, et al.



The researchers next evaluated the ability of ICL-1 to detect LIPs in vivo using transgenic mice that express luciferase globally under the actin promoter. Following injection of ICL-1, they detected bioluminescence from the mice that increased for a period of about 20 minutes and then dropped slowly thereafter for about 6 hours. The intensity of the signal increased with increasing dose of ICL-1. It was most intense in the region of the peritoneum, likely due to the high concentration of ferrous iron in the intestine. An interesting finding was that the signal intensity was higher in female than male mice, suggesting the possibility of a sex-based difference in iron pools. When mice were pre-injected with FAS, a 77% increase in ICL-1-dependent bioluminescence resulted, while a reduced signal was observed if the mice were pre-injected with BPY (Figure 3).



FIGURE 3. Luminescence signal from luciferase-expressing mice following injection of ICL-1 with or without ferrous ammonium sulfate (FAC) and/or bipyridine (BPY) as indicated. Figure reproduced with permission from A. T. Aron, M.C. Heffern, Z, R. Lonergan, et al. Proc. Natl. Acad. Sci. U.S.A., (2017) published online Nov 14, DOI: 10.1073/pnas.1708747114. Copyright 2017, A.T. Aron, et al.


Having verified the ability of ICL-1 to detect LIPs in an intact organism, the investigators next wanted to use the probe to evaluate changes in iron metabolism resulting from a disease state. For these studies, they chose a mouse model of systemic Acinetobacter baumannii infection. A. baumannii is a Gram-negative bacterium that causes serious infections in patients with compromised immune systems. It is a growing clinical problem, particularly as antibiotic resistance among pathogens continues to increase. As is the case for all bacteria, A. baumannii requires iron for survival, and it has developed mechanisms to secure adequate iron from the host during infection. In response, host organisms have evolved mechanisms to sequester iron from pathogenic bacteria, a process referred to as nutritional immunity. Thus, the researchers hypothesized that mice would respond to an acute A. baumannii infection with a change in the disposition of LIPs. To test this hypothesis, the researchers infected male luciferase-expressing mice with A. baumannii via a retro-orbital injection. After 24 hours they administered ICL-1 and imaged the mice for bioluminescence. They then sacrificed the mice to measure the quantities of bacteria in lungs, heart, kidneys, and liver. The latter measurements confirmed the presence of A. baumannii in all organs, confirming systemic infection. The bioluminescence data demonstrated an overall increase in signal from the infected mice in addition to a shift in localization from the peritoneum to the vicinity of the liver, lungs, and heart. The intensity of the signal correlated with the level of bacteria in the organs of the mice. Inductively-coupled plasma mass spectrometry (ICP-MS) of samples from liver, lungs, and heart revealed no significant difference in the levels of iron in the lungs and heart between infected and uninfected mice. However, the livers of the infected mice exhibited a marked increase in iron as compared to those of the control mice. Laser ablation ICP-MS of liver slices confirmed the presence of the excess iron and showed that it was concentrated primarily at the periphery of the infected livers.

Figure 4. Luminescence signals from luciferase-expressing mice that were mock infected or infected with A. baumannii as indicated. Figure reproduced with permission from A. T. Aron, M.C. Heffern, Z, R. Lonergan, et al. Proc. Natl. Acad. Sci. U.S.A., (2017) published online Nov 14, DOI: 10.1073/pnas.1708747114. Copyright 2017, A.T. Aron, et al.



The ICL-1 data indicated that mice respond to A. baumannii infection by concentrating iron in the liver. To better understand the mechanism by which this occurs, the researchers examined liver homogenates from infected and control mice for the expression levels of multiple genes that encode proteins relevant to the regulation of iron metabolism. The results revealed that in the liver, A. baumannii infection leads to a decrease in expression of the FPN iron exporter accompanied by a mild elevation of expression of hepcidin, a hormone that stimulates the degradation of FPN. Expression of the transferrin receptor was unaffected by the infection, while that of the DMT1 divalent metal transporter was reduced. The overall level of transferrin in the serum of infected mice was also lower than that of controls. Expression of LCN2, a glycoprotein that sequesters bacterial iron-binding siderophores was elevated in response to infection. LCN2 also plays a role in the import of transferrin- and nontransferrin-bound iron. Expression of the ferritin subunit FHC increased in response to infection, whereas that of FLC decreased. These changes typically accompany rapid iron uptake. Furthermore, the data revealed a decrease in expression of IRP1 and IRP2 in infected mice relative to those of controls. These proteins repress translation through the iron-responsive element of the ferritin mRNA. Overall serum ferritin levels increased in response to infection, consistent with prior reports.

The data clearly demonstrate the utility of ICL-1 as a probe of LIPs in vivo. Unfortunately, this probe cannot immediately be translated for clinical use because it requires the presence of luciferase for luminescence generation. However, the availability of luciferase-expressing mice will enable many interesting future studies of iron dynamics in animal models, such as the one reported here for A. baumannii infection.



ViewProceedings of the National Academy of Sciences article: In vivo bioluminescence imaging of labile iron accumulation in a murine model of Acinetobacter baumannii infection





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