Biology of Int1p in Canadida albicans Fungemia

白色念珠菌菌血症导致每年8亿美元的超额医疗费用。中性粒细胞减少和非中性粒细胞减少的患者都会受到影响；超过85%的患者拥有中心静脉导管，血液产品、抗生素、高营养液和肝素都可以通过中心静脉导管输注。尽管超过20%的感染患者死亡，但除了中性粒细胞减少外，其他诱因还知之甚少。白念珠菌INT1基因在体内外将黏附、形态发生和毒力联系在一起，但其毒力机制尚不清楚。初步数据显示Int1p依赖的超抗原样效应。表达Int1p的白念珠菌和酿酒酵母都能激活CD4T淋巴细胞并扩大Vbeta2亚群；白念珠菌可释放TNFpha和IL-6。超抗原样活性最初定位于Int1p氨基末端的前434个氨基酸。这项提案的重点是Pep263，这是一种在肝素存在下暴露并从Int1p的氨基末端切割出来的多肽。100皮摩尔浓度的纯化的可溶性Pep263比105个白色念珠菌细胞更快和更有效地激活携带Vbeta2、Vbeta17和Vbeta22亚群的人和小鼠T淋巴细胞以及EX和T淋巴细胞群体。在特定的目标一，我们将使用INT1突变体和人淋巴细胞在体外定位对于Pep263的表达和切割至关重要的Int1p结构域。其他Vbeta亚群的反应、CD4/CD8T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与已知的超抗原关节炎支原体MAM的MHC Class II结合位点的同源性，将被用来鉴定人和小鼠抗原提呈细胞上相关的MHC Class II等位基因。Pep263和缺乏MHC Class II结合位点的突变体将在转人类HLA-DR和-DQ等位基因的小鼠中进行研究。将确定Pep263的晶体结构。在特定的目标二中，我们将测试Kex2p或Int1p本身是否是裂解Pep263的前蛋白转换酶。抗蛋白水解酶、肝素类似物和Pep263抗体将作为潜在的抑制剂进行研究。肝素加速Pep263切割的直接和间接机制将使用INT1-GFP结构和INT1突变体缺失推测的肝素结合位点来分析。这些研究涉及肝素加速产生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.