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: 6743981
• 负责人: MARGARET K HOSTETTER
• 金额: $ 36.79万
• 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/6743981
• 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.

白色念珠菌菌血症导致每年超过8亿美元的医疗费用。 血供减少和非血供减少的患者都受到影响;超过85%的患者带有中心静脉导管，通过该导管输注血液制品、抗生素、高营养液和肝素。虽然超过20%的感染患者死亡，但对中性粒细胞减少症以外的诱发因素知之甚少。 梭白念珠菌INT 1基因在体内外与粘附、形态发生和毒力有关，但其毒力机制尚未阐明。 初步数据表明Int 1 p依赖性超抗原样效应。 两个C.白色念珠菌和S.表达Int 1 p的酿酒酵母激活CD 4 T淋巴细胞并扩增V β 2亚群; TNF α和IL-6响应于C.白色念珠菌 超抗原样活性最初定位于Int 1 p氨基末端的前434个氨基酸。 该提案的重点是Pep 263，这是一种在肝素存在下暴露并从Int 1 p的氨基末端裂解的多肽。 100皮摩尔浓度的纯化的可溶性Pep 263比105 ℃更迅速和更有效地激活携带V β 2、V β 17和V β 22亚群的人和鼠T淋巴细胞和ex和T淋巴细胞群。白色念珠菌细胞在《特定目标一》中，我们将使用INT 1突变体和体外人淋巴细胞来绘制对于Pep 263的表达和切割至关重要的Int 1 p结构域。 将确定其他V β亚群的应答、CD 4/CD 8 T淋巴细胞的相对贡献及其细胞因子谱。 一个新发现的身份Pep 263与MHC II类结合位点的关节炎支原体MAM，一个众所周知的超抗原，将被利用来确定相关的MHC II类等位基因的人类和小鼠抗原呈递细胞。 将在人类HLA-DR和-DQ等位基因转基因小鼠中研究Pep 263和缺乏MHC II类结合位点的突变体的作用。 确定Pep 263的晶体结构。 在具体目标2中，我们将测试Kex 2 p或Int 1 p本身是否是切割Pep 263的前蛋白转化酶。 抗蛋白酶、肝素类似物和Pep 263抗体将作为潜在的抑制剂进行研究。肝素加速Pep 263裂解的直接和间接机制将使用INT 1-GFP构建体和缺失假定肝素结合位点的INT 1突变体进行分析。 这些研究阐明了肝素加速C.白色念珠菌超抗原