Biology of Int1p in Canadida albicans Fungemia

白色念珠菌真菌血症中 Int1p 的生物学

• 批准号: 6542867
• 负责人: MARGARET K HOSTETTER
• 金额: $ 32.7万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6542867
• 关键词: Candida albicans; MHC class II antigen; Mycoplasma; Saccharomyces cerevisiae; antigen presenting cell; candidiasis; clinical research; crystallization; cytokine; cytotoxic T lymphocyte; fungal genetics; gene deletion mutation; gene expression; genetically modified animals; helper T lymphocyte; heparin; host organism interaction; human genetic material tag; human subject; iatrogenic disease; laboratory mouse; leukocyte activation /transformation; mycosis; patient oriented research; protein structure function; proteolysis; superantigens; tissue /cell culture; virulence

Candida albicans fungemia leads to excess medical costs of 800 million dollars per year. Both neutropenic and non- neutropenic patients are affected; more than 85 percent harbor a central venous catheter, through which blood products, antibiotics, hyperalimentation fluids, and heparin are infused. Although more than 20 percent of infected patients die, predisposing factors other than neutropenia are poorly understood. The C. albicans gene INT1 links adhesion, morphogenesis and virulence in vitro and in vivo, but the mechanism of its virulence has not been elucidated. Preliminary data demonstrate Int1p-dependent superantigen-like effects. Both C. albicans and S. cerevisiae expressing Int1p activate CD4 T lymphocytes and expand the Vbeta2 subset; TNFalpha and IL-6 are released in response to C. albicans. Superantigen-like activity was originally localized to the first 434 amino acids of the Int1p amino terminus. This proposal focuses on Pep263, a polypeptide that is exposed and cleaved from the amino terminus of Int1p in the presence of heparin. 100 picomolar concentrations of purified, soluble Pep263 activate human and murine T lymphocytes and ex and T lymphocyte populations bearing Vbeta2, Vbeta17, and Vbeta22 subsets more rapidly and more potently than 105 C. albicans cells. In Specific Aim One we shall map Int1p domains essential for the expression and cleavage of Pep263 using INT1 mutants and human lymphocytes in vitro. The response of additional Vbeta subsets, the relative contributions of CD4/CD8 T lymphocytes, and their cytokine profiles will be determined. A newly discovered identity of Pep263 with the MHC Class II binding site of Mycoplasma arthritidis MAM, a well-known superantigen, will be exploited to identify relevant MHC Class II alleles on human and murine antigen-presenting cells. The effects of Pep263 and mutants lacking the MHC Class II binding site will be studied in mice transgenic for human HLA-DR and -DQ alleles. The crystal structure of Pep263 will be determined. In Specific Aim Two we shall test whether Kex2p or Int1p itself is the proprotein convertase that cleaves Pep263. Anti-proteases, heparin analogs, and antibodies to Pep263 will be studied as potential inhibitors. Direct and indirect mechanisms by which heparin accelerates cleavage of Pep263 will be analyzed using INT1-GFP constructs and INT1 mutants missing a putative heparin-binding site. These studies address the cellular, structural, and biochemical mechanisms underlying the heparin-accelerated generation of a C. albicans superantigen.

白色念珠菌真菌造真菌每年导致8亿美元的医疗费用过剩。 中性粒细胞减少和非中性粒细胞减少症患者均受到影响。超过85％的中央静脉导管含有中央静脉导管，并注入了血液产物，抗生素，高温液和肝素。尽管超过20％的受感染患者死亡，但中性粒细胞减少症以外的其他因素知之甚少。 白色念珠菌基因INT1在体外和体内连接粘附，形态发生和毒力，但是尚未阐明其毒力的机理。 初步数据证明了INT1P依赖性超抗原样效应。 表达INT1P的白色念珠菌和酿酒酵母都激活CD4 T淋巴细胞并扩大VBETA2子集。 TNFALPHA和IL-6是根据白色念珠菌响应的。 超抗原样活性最初定位于INT1P氨基末端的第一个434个氨基酸。 该提案的重点是PEP263，这是一种在存在肝素的情况下暴露并从INT1P的氨基末端裂解的多肽。 100纯净的可溶性PEP263激活人和鼠T淋巴细胞以及带有VBETA2，VBETA17和VBETA22亚群的EX和T淋巴细胞群体比105 C. C. albicans细胞更迅速，更有效。在特定目标中，我们将使用INT1突变体和人类淋巴细胞在体外绘制PEP263表达和裂解所必需的INT1P结构域。 将确定其他VBETA子集的响应，CD4/CD8 T淋巴细胞的相对贡献及其细胞因子谱。 将利用新发现的PEP263与MHC II类结合位点（一种众所周知的超抗原）的MHC II类结合位点，以识别有关人和鼠类抗原抗原呈现细胞的相关MHC II类等位基因。 将在人HLA -DR和-DQ等位基因的小鼠转基因中研究PEP263和缺乏MHC II类结合位点的突变体的影响。 将确定PEP263的晶体结构。 在特定的目标两个中，我们将测试Kex2p或INT1P本身是否是裂解Pep263的前蛋白转化酶。 将研究抗凝发，肝素类似物和对PEP263的抗体作为潜在的抑制剂。将使用INT1-GFP构建体和缺少假定的肝素结合位点的INT1突变体分析肝素加速PEP263裂解的直接和间接机制。 这些研究针对白色念珠菌超抗原的肝素加速产生的基础的细胞，结构和生化机制。