Microbial siderophore-specific innate immune responses

描述（由申请人提供）：脂钙素（脂钙素2），发现于中性粒细胞颗粒，子宫分泌物和上皮细胞在炎症或肿瘤发生反应中分泌。也是一种急性期蛋白，在细菌感染期间血清和滑膜中的水平明显升高。虽然与多种生理过程有关，但铁铁蛋白的功能一直很神秘，直到我们最近发现了这种蛋白质的特异性、高亲和力配体：细菌酚型铁铁载体，如肠螯合蛋白（又名肠obactin； KD = 0.4纳摩尔）和混合型羧基mycoactin铁铁载体。因此，我们提出铁苷作为一种抗菌剂，将铁作为铁载体络合物隔离，补充了先天免疫系统的一般抗微生物铁耗尽策略。为了支持这一假设，我们发现铁苷在体外限铁条件下是一种有效的抑菌剂，当敲除时，使动物对细菌感染非常敏感。铁苷酸显然使用一种新颖的、退化的识别机制与不同类型的铁载体交叉反应，扩大了反应的效用。这种功能假说也合理化了一些铁载体与毒力的关联：通过交替利用对铁铁钙素亲和力显著降低的铁载体，病原体可以逃避铁铁钙素介导的缺铁。该项目的目标是确定先天免疫反应的铁载体特异性成分如何识别微生物铁载体。在目标1中，我们建议继续进行铁蛋白蛋白的晶体学、诱变和生物物理研究，以及一组天然的、合成的和设计的铁载体，以充分描述这种蛋白质如何识别铁载体。铁钙蛋白是多种脂钙蛋白家族的一员。我们还确定了四种亲缘关系较远的小鼠和鸟类脂钙蛋白，它们要么明显结合细菌铁载体的交替光谱，要么在建模研究的基础上预测。在Aim 2中，我们提出了类似的结构和生化研究，以类似地解析这些蛋白质的识别机制。最终，这些研究不仅将充分表征抗菌免疫监视的这一组成部分，而且将允许继续和未来的毒力和发病机制的研究，以及定义这些蛋白质作为新型抗菌治疗的潜在效用。

DESCRIPTION (provided by applicant): 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.