Siderocalin (lipocalin 2), found in neutrophil granules, uterine secretions and secreted from epithelial cells in response to inflammation or tumorigenesis. is also an acute phase protein, with markedly elevated levels in the serum and the synovium during bacterial infection. Though implicated in diverse physiological processes, siderocalin's function was mysterious until our recent identification of specific, high-affinity ligands for this protein: bacterial phenolate-type ferric siderophores, such as enterochelin (aka enterobactin; KD = 0.4 nanomolar) and the mixed-type carboxymycobactin ferric siderophores. We therefore propose that siderocalin functions as an antibacterial agent, sequestering iron as ferric siderophore complexes, complementing the general anti-microbial iron-depletion strategy of the innate immune system. Supporting this hypothesis, we have found that siderocalin is a potent bacteriostatic agent in vitro under iron-limiting conditions and, when knocked-out, renders animals remarkably susceptible to bacterial infection. Siderocalin apparently uses a novel, degenerate recognition mechanism to cross-react with distinct types of siderophores, broadening the utility of the response. This functional hypothesis also rationalizes the association of some siderophores with virulence: by alternately utilizing siderophores with markedly reduced affinity for siderocalin, pathogens can escape siderocalin-mediated iron-deprivation. The goal of this project is to determine how siderophore-specific components of the innate immune response recognize microbial siderophores.
In Aim 1, we propose continuing crystallographic, mutagenesis and biophysical studies of siderocalin, and a panel of natural, synthetic and designed siderophores, to fully delineate how this protein recognizes siderophores. Siderocalin is a member of the diverse lipocalin protein family. We have also identified four distantly-related murine and avian lipocalins that either demonstrably bind alternate spectrums of bacterial siderophores or are predicted to on the basis of modeling studies.
In Aim 2, we propose analogous structural and biochemical studies of these proteins to similarly parse their recognition machinery. Ultimately, these studies will not only fully characterize this component of antibacterial immune surveillance, but will also allow continuing and future studies of virulence and pathogenesis - as well as defining the potential utility of these proteins as novel anti-bacterial therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI059432-01A1
Application #
6872755
Study Section
Special Emphasis Panel (ZRG1-IMM-F (07))
Program Officer
Winter, David B
Project Start
2005-03-01
Project End
2009-02-28
Budget Start
2005-03-01
Budget End
2006-02-28
Support Year
1
Fiscal Year
2005
Total Cost
$294,081
Indirect Cost
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
Correnti, Colin; Richardson, Vera; Sia, Allyson K et al. (2012) Siderocalin/Lcn2/NGAL/24p3 does not drive apoptosis through gentisic acid mediated iron withdrawal in hematopoietic cell lines. PLoS One 7:e43696
Correnti, Colin; Strong, Roland K (2012) Mammalian siderophores, siderophore-binding lipocalins, and the labile iron pool. J Biol Chem 287:13524-31
Correnti, Colin; Clifton, Matthew C; Abergel, Rebecca J et al. (2011) Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities. Structure 19:1796-806
Hoette, Trisha M; Clifton, Matthew C; Zawadzka, Anna M et al. (2011) Immune interference in Mycobacterium tuberculosis intracellular iron acquisition through siderocalin recognition of carboxymycobactins. ACS Chem Biol 6:1327-31
Bandaranayake, Ashok D; Correnti, Colin; Ryu, Byoung Y et al. (2011) Daedalus: a robust, turnkey platform for rapid production of decigram quantities of active recombinant proteins in human cell lines using novel lentiviral vectors. Nucleic Acids Res 39:e143
Halaas, Oyvind; Steigedal, Magnus; Haug, Markus et al. (2010) Intracellular Mycobacterium avium intersect transferrin in the Rab11(+) recycling endocytic pathway and avoid lipocalin 2 trafficking to the lysosomal pathway. J Infect Dis 201:783-92
Bao, Guanhu; Clifton, Matthew; Hoette, Trisha M et al. (2010) Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex. Nat Chem Biol 6:602-9
Abergel, Rebecca J; Clifton, Matthew C; Pizarro, Juan C et al. (2008) The siderocalin/enterobactin interaction: a link between mammalian immunity and bacterial iron transport. J Am Chem Soc 130:11524-34
Fischbach, Michael A; Lin, Hening; Zhou, Lu et al. (2006) The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2. Proc Natl Acad Sci U S A 103:16502-7
Abergel, Rebecca J; Wilson, Melissa K; Arceneaux, Jean E L et al. (2006) Anthrax pathogen evades the mammalian immune system through stealth siderophore production. Proc Natl Acad Sci U S A 103:18499-503

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