In human urinary tract infections, host cells release the antimicrobial protein siderocalin (SCN; also known as lipocalin-2, neutrophil gelatinase-associated lipocalin, or 24p3) into the urinary tract. By binding to ferric catechol complexes, SCN can sequester iron, a growth-limiting nutrient for most bacterial pathogens. Recent evidence links the antibacterial activity of SCN in human urine to iron sequestration and metabolomic variation between individuals. To determine whether these metabolomic associations correspond to functional Fe(III)-binding SCN ligands, we devised a biophysical protein binding screen to identify SCN ligands through direct analysis of human urine. This screen revealed a series of physiologic unconjugated urinary catechols that were able to function as SCN ligands of which pyrogallol in particular was positively associated with high urinary SCN activity. In a purified, defined culture system, these physiologic SCN ligands were sufficient to activate SCN antibacterial activity against Escherichia coli. In the presence of multiple SCN ligands, native mass spectrometry demonstrated that SCN may preferentially combine different ligands to coordinate iron, suggesting that availability of specific ligand combinations affects in vivo SCN antibacterial activity. These results support a mechanistic link between the human urinary metabolome and innate immune function.
Bibliographical noteFunding Information:
This work was supported in part by NIDDK, National Institutes of Health Grants R01DK099534, P50DK064540, and R56DK111930; National Center for Advancing Translational Sciences, National Institutes of Health Grant UL1TR000448; and the Longer Life Foundation (to J. P. H.). Mass spectrometry was supported by United States Public Health Service Grants P41-RR00954, P30-DK20579, P30-DK56341, and 8P41GM103422. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Supported by National Institutes of Health Grant T32GM007067-37 and a Monsanto Excellence Fund graduate fellowship. We thank Roland Strong and Jonathan Barasch for generously sharing siderocalin constructs and Lindsey Steinberg and Anne Robinson for careful reading of the manuscript.