Biology of Int1p in Canadida albicans Fungemia

白色念珠菌菌血症每年导致8亿美元的额外医疗费用。中性粒细胞减少症和非中性粒细胞减少症患者均受影响；85%以上的医院有中心静脉导管，血液制品、抗生素、高营养液体和肝素都是通过它输注的。尽管超过20%的感染者死亡，但除中性粒细胞减少症外的诱发因素尚不清楚。白色念珠菌基因INT1与体外和体内的粘附、形态发生和毒力有关，但其毒力机制尚未阐明。初步数据显示了依赖于int1p的超级抗原样效应。表达Int1p的白色念珠菌和酿酒念珠菌均能激活CD4 T淋巴细胞并扩增vbet2亚群；TNFalpha和IL-6是对白色念珠菌的反应。超级抗原样活性最初定位于Int1p氨基末端的前434个氨基酸。这一建议的重点是Pep263，这是一种在肝素存在下从Int1p的氨基端暴露和切割的多肽。100皮摩尔浓度的纯化、可溶性Pep263比105白念珠菌细胞更迅速、更有效地激活人和小鼠T淋巴细胞以及携带Vbeta2、Vbeta17和Vbeta22亚群的ex和T淋巴细胞群。在特异性目的一中，我们将利用INT1突变体和人淋巴细胞在体外绘制Pep263表达和切割所必需的Int1p结构域。其他Vbeta亚群的反应、CD4/CD8 T淋巴细胞的相对贡献以及它们的细胞因子谱将被确定。新发现的Pep263与关节炎支原体MAM（一种众所周知的超抗原）MHC II类结合位点的同一性，将被用于鉴定人和小鼠抗原呈递细胞上相关的MHC II类等位基因。将研究Pep263和缺乏MHC II类结合位点的突变体对人HLA-DR和-DQ等位基因转基因小鼠的影响。将测定Pep263的晶体结构。在特异性目标2中，我们将测试Kex2p或Int1p本身是否是切割Pep263的蛋白转化酶。将研究抗蛋白酶、肝素类似物和Pep263抗体作为潜在的抑制剂。肝素加速Pep263裂解的直接和间接机制将使用INT1- gfp构建体和缺少肝素结合位点的INT1突变体进行分析。这些研究解决了肝素加速生成白色念珠菌超抗原的细胞、结构和生化机制。

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.