Program: Biochemistry Biophysics and Structural Biology
Current advisor: Jeffrey P. Henderson, MD, PhD
Undergraduate university: Washington University
Lipocalin-2 (Lcn2, also known as siderocalin or neutrophil gelatinase-associated lipocalin) is an innate immune protein released by neutrophils and epithelial cells at sites of infection. It is understood to limit bacterial growth by restricting access to iron, an essential nutrient, but it does not avidly bind iron in isolation. At least two iron restricting Lcn2 mechanisms are described in the literature. In the prototypical mechanism, Lcn2 binds and prevents uptake of enterobactin, a high affinity iron chelator secreted by E. coli and closely related Enterobacterales. Sequestration of this bacterial siderophore prevents nutritional iron delivery to these pathogens. However, the narrow species specificity of this mechanism is unusual for an innate immune response. In a recently proposed alternative mechanism, Lcn2 binds ferric complexes of human diet-derived catecholate metabolites, sequestering iron away from bacterial access in a siderophore-independent manner. This thesis is designed to characterize and compare these two mechanisms of Lcn2 antimicrobial function.
We first developed and employed native mass spectrometry, covalent labeling, and protein digestion techniques to probe Lcn2-enterobactin and Lcn2-monocatechol binding. We found that Lcn2 can bind both ferric and aferric enterobactin, potentially sequestering this siderophore even before it has scavenged iron. We further demonstrate site-specific covalent labeling for use in future studies of the binding site preferences of monocatechol Lcn2 ligands.
Next, we developed an experimental culture system to directly compare the enterobactinsequestration and iron-sequestration modes of Lcn2 activity. Using enterobactin-producing model uropathogenic E. coli strains, we observed that physiologically plausible catechol
metabolite concentrations significantly increase the antibiotic potency of Lcn2. This suggests that the Lcn2 iron-sequestration mechanism is more potently antibiotic than the canonical enterobactin-sequestration mechanism. We hypothesized that this enterobactin-independent ironsequestration mechanism extends Lcn2 antibiotic activity to enterobactin- pathogens. We tested this hypothesis with several strains of Acinetobacter baumanii, a non-enterobactin producing, non-Enterobacterales species associated with significant antibiotic resistance. We observed profound inhibition of A. baumanii only when Lcn2 was combined with catechol metabolites.
Finally, we use preliminary data and methods developed to enable future investigation of the interactions between non-enterobactin siderophores and Lcn2. Specifically, we ask whether modified versions of enterobactin called salmochelins can disrupt certain Lcn2-iron-monocatechol complexes.
These results implicate human metabolites in a potent, broad-spectrum antimicrobial Lcn2 mechanism that inhibits multiple medically significant pathogens. The catechol metabolite dependence of this activity suggests that dietary catechol supplementation could improve host resistance to infection. This represents a plausible, non-antibiotic strategy for prevention or treatment of clinical infections by antibiotic resistant bacteria.
Guo C, Steinberg LK, Cheng M, Song JH, Henderson JP, Gross ML. 2020 Site-Specific Siderocalin Binding to Ferric and Ferric-Free Enterobactin As Revealed by Mass Spectrometry. ACS Chem Biol, 15(5):1154-1160.
Guo C, Steinberg LK, Henderson JP, Gross ML. 2020 Organic Solvents for Enhanced Proteolysis of Stable Proteins for Hydrogen-Deuterium Exchange Mass Spectrometry. Anal Chem, 92(17):11553-11557.