Calprotectin and metal withholding during Candida albicans invasion of the kidney

白色念珠菌侵入肾脏期间钙卫蛋白和金属扣留

• 批准号: 9121135
• 负责人: Angelique Nicole Besold
• 金额: $ 5.48万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9121135
• 关键词: Abscess; Antifungal Agents; Area; Aspartic Acid; Binding; Binding Sites; Candida; Candida albicans; Complement; Disseminated candidiasis; Environment; Exhibits; Future; Gene Expression; Genes; Goals; Growth; Harvest; Histidine; Histologic; Homeostasis; Human; Immune response; Immunity; Immunohistochemistry; In Vitro; Inductively Coupled Plasma Mass Spectrometry; Infection; Ions; Kidney; Laboratories; Lesion; Leukocyte L1 Antigen Complex; Life; Mediating; Messenger RNA; Metals; Mus; Nutrient; Nutritional; Organ; Organism; Oxidative Stress; Play; Process; Proteins; Public Health; RNA analysis; Role; Series; Serum; Side; Site; Staining method; Stains; Starvation; Stress; Superoxide Dismutase; System; Testing; Toxic effect; Up-Regulation; Wild Type Mouse; Yeasts; Zinc deficiency; antimicrobial; antioxidant enzyme; biological adaptation to stress; combat; fight against; fighting; insight; kidney infection; laser capture microdissection; mouse model; mutant; neutrophil; pathogen; prevent; public health relevance; response; uptake

 DESCRIPTION (provided by applicant): Calprotectin (CP) is an abundant protein in neutrophils that is known to sequester Zn and Mn from pathogens using two distinct metal sites in a process known as nutritional immunity. CP has potent anti-microbial activity against many pathogens, including the fungal pathogen Candida albicans. However, the mechanism by which CP exerts its anti-candida activity is unknown. Recent studies from the Culotta lab have shown that, in a murine model of disseminated candidiasis, C. albicans lesions in the kidneys are associated with profound clearing of Zn, suggestive of Zn withholding from the pathogen, possibly by CP. Surprisingly, Cu may also be withheld from C. albicans during kidney infection as the yeast present in these lesions exhibit mRNA markers of Cu starvation. There is no known role for CP in withholding Cu, but biochemically CP has the capacity to bind Cu. Thus, I began to investigate if CP could be responsible for Cu and/or Zn sequestration from C. albicans. Using laboratory yeast cultures, I have shown that CP can sequester Zn and Cu away from C. albicans and Cu sequestration requires metal binding residues in CP that are distinct from the Zn binding site. I have also shown that C. albicans upregulates Cu or Zn uptake genes in response to the metal limiting environment imposed by CP. Given these results, I hypothesize that CP aids in the fight against C. albicans during kidney infection by sequestering multiple metal ions, including Cu, away from the pathogen. To investigate this, I will carry out the following aims. Aim 1: To understand the impact of CP on metal accumulation and metal stress responses in cultures of C. albicans. To gain a better understanding of the mechanism by which CP can sequester Cu versus Zn, I will test the ability of various site specific metal binding mutants of CP to inhibit C. albicans growth in culture and to withhold Cu and Zn from this pathogen. Yeast gene expression will also be analyzed to define any Cu and Zn starvation stress responses as well as any oxidative stress that occurs in C. albicans when CP attacks this pathogen. Aim 2: To understand the connection between CP and metal homeostasis in C. albicans during kidney infection. After establishing a role of CP in sequestering both Zn and Cu away from C. albicans in vitro, I will investigate if CP is responsible for the decrease in kidney Cu and Zn observed in the aforementioned murine model of disseminated candidiasis. First, kidneys from infected mice will be examined histologically to tract neutrophil and CP recruitment to sites of fungal lesions. Laser capture microdissection will be used to isolate fungal lesions in the kidney for the analysis of metals and fungal mRNA. By this approach, we can define local changes in Cu and Zn (and other metals) and will identify any fungal stress responses by examining markers of Cu and Zn uptake as well as oxidative stress. These analyses will be carried out in both WT and in CP-deficient mice to determine if CP is responsible for metal withholding from C. albicans during fungal invasion of the kidney. Together, these studies will provide insight into how CP combats C. albicans during the innate immune response, including an unprecedented role for Cu sequestration by CP in nutritional immunity.

 描述（由申请人提供）：钙卫蛋白（CP）是中性粒细胞中丰富的蛋白质，已知其在称为营养免疫的过程中利用两个不同的金属位点从病原体中隔离锌和锰。 CP 对许多病原体具有有效的抗菌活性，包括真菌病原体白色念珠菌。然而，CP 发挥其抗念珠菌活性的机制尚不清楚。 Culotta 实验室最近的研究表明，在播散性念珠菌病的小鼠模型中，肾脏中的白色念珠菌病变与 Zn 的深度清除有关，这表明 CP 可能会从病原体中扣留 Zn。令人惊讶的是，在肾脏感染期间，白色念珠菌中的铜也可能被抑制，因为这些病变中存在的酵母表现出铜饥饿的 mRNA 标记。 CP 在抑制 Cu 方面的作用尚不清楚，但生化上 CP 具有结合 Cu 的能力。因此，我开始研究 CP 是否能够从白色念珠菌中螯合铜和/或锌。使用实验室酵母培养物，我已经证明 CP 可以将 Zn 和 Cu 隔离于白色念珠菌，并且 Cu 隔离需要 CP 中与 Zn 结合位点不同的金属结合残基。我还表明，白色念珠菌会上调 Cu 或 Zn 摄取基因，以响应 CP 施加的金属限制环境。鉴于这些结果，我推测 CP 通过隔离多种金属离子（包括 Cu）远离病原体，有助于在肾脏感染期间对抗白色念珠菌。为了调查这一点，我将实现以下目标。目标 1：了解 CP 对白色念珠菌培养物中金属积累和金属应激反应的影响。为了更好地了解 CP 螯合铜与锌的机制，我将测试 CP 的各种位点特异性金属结合突变体抑制白色念珠菌在培养物中生长以及从该病原体中抑制铜和锌的能力。还将分析酵母基因表达，以确定任何铜和锌饥饿应激反应，以及当 CP 攻击白色念珠菌时该病原体时发生的任何氧化应激。目标 2：了解肾脏感染期间白色念珠菌 CP 与金属稳态之间的联系。在确定 CP 在体外隔离白色念珠菌中的 Zn 和 Cu 的作用后，我将研究 CP 是否是在上述播散性念珠菌病小鼠模型中观察到的肾脏 Cu 和 Zn 减少的原因。首先，对受感染小鼠的肾脏进行组织学检查，以引导中性粒细胞和 CP 募集到真菌病变部位。激光捕获显微切割将用于分离真菌病变 肾脏用于金属和真菌 mRNA 的分析。通过这种方法，我们可以定义铜和锌（以及其他金属）的局部变化，并通过检查铜和锌吸收标记以及氧化应激来识别任何真菌应激反应。这些分析将在 WT 和 CP 缺陷小鼠中进行，以确定 CP 是否是真菌侵入肾脏期间从白色念珠菌中扣留金属的原因。总之，这些研究将深入了解 CP 如何在先天免疫反应期间对抗白色念珠菌，包括 CP 螯合铜在营养免疫中发挥的前所未有的作用。