Modulation of urinary siderophores by the diet, gut microbiota and inflammation in mice

Abstract

Mammalian siderophores are believed to play a critical role in maintaining iron homeostasis. However, the properties and functions of mammalian siderophores have not been fully clarified. In this study, we have employed Chrome Azurol S (CAS) assay which is a well-established method for bacterial siderophores study, to detect and quantify mammalian siderophores in urine samples. Our study demonstrates that siderophores in urine can be altered by diet, gut microbiota and inflammation. C57BL/6 mice, fed on plant-based chow diets which contain numerous phytochemicals, have more siderophores in the urine compared to those fed on purified diets. Urinary siderophores were upregulated in iron overload conditions, but not altered by other tested nutrients status. Further, germ-free mice displayed 50% reduced urinary siderophores, in comparison to conventional mice, indicating microbiota biotransformation is critical in generating or stimulating host metabolism to create more siderophores. Altered urinary siderophores levels during inflammation suggest that host health conditions influence systemic siderophores level. This is the first report to measure urinary siderophores as a whole, describing how siderophores levels are modulated under different physiological conditions. We believe that our study opens up a new field in mammalian siderophores research and the technique we used in a novel manner has the potential to be applied to clinical purpose.

Bacterial Siderophores Hijack Neutrophil Functions

“…The use of Ent in the modified assay yielded higher MFI readings compared to DFO (Fig. 6B); the higher sensitivity with Ent may be due to its superior affinity and kinetics in chelating iron than DFO that is known to be slow iron chelator (33). Although the level of cytosolic LIP in Lcn2-deficient BMDNs was still detected as two-fold higher than WT BMDNs in this modified assay, our use of Ent provided several key mechanistic insights.…”

Gut Microbiota and Iron: The Crucial Actors in Health and Disease

“…Additionally, iron transporters such as DMT1 have been shown to express in the apical surfaces of the mammalian proximal colon, indicating an involvement of the host in exacerbating the iron availability in the bacteria-dense large intestine [65,66]. Iron speciation and the potential presence of lactoferrin, also known as lactotransferrin, lipocalin-2 (only expressed at low level in healthy host) and as-yet unidentified defence proteins in colonic mucosa might contribute to the limitation of iron at this site, which enables gut microbes to synthesize siderophores, the small, high-affinity iron-chelating compounds, for their needs under the circumstances of limited amount of iron in their surrounding environment (Figure 2) [67].…”

Enterobactin induces the chemokine, interleukin-8, from intestinal epithelia by chelating intracellular iron

“…Ent pre-saturated with FeCl 3 at a 1:1 ratio drastically diminished the halo appearance on CAS plate, indicating that Ent binding to an equimolar concentration of iron is sufficient to prevent further iron chelation ( Figure 1(b )). To quantify this observation, we performed a more sensitive CAS liquid assay 30 with Ent in the presence or absence of equimolar FeCl 3 . As expected, iron-bound Ent did not display any iron-chelating activity ( Figure 1(c )).…”

“…CAS agar plates and liquid reagents were prepared as previously described by Schwyn and Neilands. 30,67 The principle of the assay is that CAS remains blue when complexed with iron, but changes to orange when the iron is chelated by siderophores. Equal concentrations of Ent (0.25--5 mM) were incubated on a CAS agar plate and monitored over 0-12 h for the formation of orange halo.…”

“…Percent iron chelation was calculated using 0-100 µM of pyrocatechol as a positive control as described previously. 30…”