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.

 说明(申请人提供)：钙保护素(CP)是中性粒细胞中的一种丰富的蛋白质，已知在营养免疫过程中通过两个不同的金属位置将锌和锰从病原体中隔离出来。CP对许多病原体具有很强的抗微生物活性，包括真菌病原体白色念珠菌。然而，CP发挥抗念珠菌活性的机制尚不清楚。库洛塔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锌的深刻清除有关，这表明锌可能通过CP抑制了病原体。令人惊讶的是，在肾脏感染期间，铜也可能被白念珠菌抑制，因为这些病变中存在的酵母显示出铜饥饿的mRNA标记。目前尚不清楚CP对铜的抑制作用，但从生化角度看，CP具有结合铜的能力。因此，我开始研究铜绿假单胞菌是否可能对白色念珠菌的铜和/或锌的隔离负责。使用实验室酵母培养物，我已经证明了CP可以将锌和铜从白色念珠菌中隔离出来，而铜的隔离需要CP中与锌结合部位不同的金属结合残留物。我还展示了白色念珠菌上调铜或锌摄取基因，以响应CP施加的金属限制环境。鉴于这些结果，我假设CP通过隔离多种金属离子，包括铜，远离病原体，在肾脏感染期间帮助抗击白色念珠菌。为了研究这一点，我将实现以下目标。目的1：了解铜绿假单胞菌对白念珠菌培养中金属积累和金属应激反应的影响。为了更好地了解铜绿假单胞菌隔离铜锌的机制，我将测试铜绿假单胞菌的各种定点金属结合突变体在培养过程中抑制白色念珠菌生长的能力，以及抑制这种病原菌的铜和锌的能力。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攻击白念珠菌时发生的任何氧化应激。目的：了解白色念珠菌肾脏感染过程中CP与金属动态平衡的关系。在建立了CP在体外将锌和铜从白色念珠菌中隔离出来的作用后，我将研究CP是否与上述播散性念珠菌病小鼠模型中观察到的肾脏铜和锌的减少有关。首先，将对感染小鼠的肾脏进行组织学检查，以将中性粒细胞和CP募集到真菌损伤的部位。激光捕获显微切割将用于分离真菌病变 肾脏用于分析金属和真菌的mRNA。通过这种方法，我们可以确定铜和锌(和其他金属)的局部变化，并将通过检测铜和锌吸收的标记以及氧化应激来识别任何真菌的应激反应。这些分析将在WT和CP缺陷小鼠身上进行，以确定CP是否对真菌入侵肾脏期间白色念珠菌的金属滞留负责。总之，这些研究将为CP如何在先天性免疫反应中对抗白色念珠菌提供洞察力，包括CP在营养免疫中对铜的封存具有前所未有的作用。