In contrast with earlier assumptions, which classified human tear lipocalin (Tlc) as an outlier member of the lipocalin protein family, the 1.8-Å resolution crystal structure of the recombinant apoprotein confirms the typical eight-stranded antiparallel ␤-barrel architecture with an ␣-helix attached to it. The fold of Tlc most closely resembles the bovine dander allergen Bos d 2, a well characterized prototypic lipocalin, but also reveals similarity with ␤-lactoglobulin. However, compared with other lipocalin structures Tlc exhibits an extremely wide ligand pocket, whose entrance is formed by four partially disordered loops. The cavity deeply extends into the ␤-barrel structure, where it ends in two distinct lobes. This unusual structural feature explains the known promiscuity of Tlc for various ligands, with chemical structures ranging from lipids and retinoids to the macrocyclic antibiotic rifampin and even to microbial siderophores. Notably, earlier findings of biological activity as a thiol protease inhibitor have no correspondence in the three-dimensional structure of Tlc, rather it appears that its proteolytic fragments could be responsible for this phenomenon. Hence, the present structural analysis sheds new light on the ligand binding activity of this functionally obscure but abundant human lipocalin.

Human tear lipocalin (TL; also known as Lcn1) is a secretory protein present in large amounts in fluids that cover epithelial surfaces such as tears and respiratory secretions. It is supposed to act as a physiological scavenger of hydrophobic, potentially harmful molecules, but there is evidence that it also inhibits bacterial growth. In the present study, we reconsidered the possibility that TL might interfere with microbial growth by scavenging of siderophores, as described for human neutrophil gelatinase-associated lipocalin (NGAL). Indeed, our experiments revealed that TL binds to microbial siderophores with high affinities. In contrast to NGAL, which was shown to have some specificity for bacterial catecholate-type siderophores, TL binds to a broad array of siderophores, including bacterial catecholate-type enterobactin and hydroxamate-type desferrioxamine B, and all major classes of fungal siderophores. By adding exogenous TL, bacterial and fungal growth could be inhibited under iron-limiting conditions. Thus, TL might be a novel member of the innate immune system especially involved in mucosal defense against fungal infections.

Filamentous fungi are an important cause of pulmonary and systemic morbidity and mortality, and also cause corneal blindness and visual impairment worldwide. Utilizing in vitro neutrophil killing assays and a model of fungal infection of the cornea, we demonstrated that Dectin-1 dependent IL-6 production regulates expression of iron chelators, heme and siderophore binding proteins and hepcidin in infected mice. In addition, we show that human neutrophils synthesize lipocalin-1, which sequesters fungal siderophores, and that topical lipocalin-1 or lactoferrin restricts fungal growth in vivo. Conversely, we show that exogenous iron or the xenosiderophore deferroxamine enhances fungal growth in infected mice. By examining mutant Aspergillus and Fusarium strains, we found that fungal transcriptional responses to low iron levels and extracellular siderophores are essential for fungal growth during infection. Further, we showed that targeting fungal iron acquisition or siderophore biosynthesis by topical application of iron chelators or statins reduces fungal growth in the cornea by 60% and that dual therapy with the iron chelator deferiprone and statins further restricts fungal growth by 75%. Together, these studies identify specific host iron-chelating and fungal iron-acquisition mediators that regulate fungal growth, and demonstrate that therapeutic inhibition of fungal iron acquisition can be utilized to treat topical fungal infections.