ORCID
0000-0002-1100-0458
Date of Award
5-8-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
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 enterobactin-sequestration 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 has greater antibiotic efficacy than the canonical enterobactin-sequestration mechanism. We hypothesized that this enterobactin-independent iron-sequestration mechanism extends Lcn2 antibiotic activity to enterobactin-null 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. These results implicate human metabolites in an efficacious, 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.
Language
English (en)
Chair and Committee
Jeffrey Henderson
Recommended Citation
Steinberg, Lindsey, "Antimicrobial Mechanisms of Human Lipocalin-2" (2024). Arts & Sciences Electronic Theses and Dissertations. 3062.
https://openscholarship.wustl.edu/art_sci_etds/3062