MOLECULAR MARKER FOR INVASIVE CANDIDA ALBICANS

机会致病菌白色念珠菌粘附于上皮， 在侵入血流之前在粘膜屏障处复制。 我们 最近已经鉴定了一种“整合素样蛋白”， 在抗原性、结构和功能上与α亚基相关 β 2整合素α M（Mac-1或CD 11b/CD 18）和α X（p150，95或 CD 11 c/CD 18）--白细胞粘附糖蛋白家族的两个成员。 这种蛋白的主要功能是促进上皮细胞和 通过识别蛋白质中的RGD序列， iC 3b和纤维蛋白原作为粘附基质。 为实现上一个五年期供资的具体目标， 期间，我们现在已经克隆了一个基因（α INT 1）编码一种新的表面 蛋白质C.并分析了其结构。 像 α-亚基的β 2整联蛋白，α INT 1基因产物含有 iC 3b和纤维蛋白原的配体结合位点，两个EF-手二价阳离子 结合位点、疏水跨膜结构域和酪氨酸残基 在胞质尾部。 α INT 1也编码其自身的RGD三肽。 S.具有α INT 1编码序列的酿酒酵母 在半乳糖诱导型启动子的控制下， 在单倍体酵母中形成。 在Specific Aim One中，一系列缺失突变体缺乏 配体结合位点，一个或两个二价阳离子结合位点，RGD 序列，或跨膜结构域和/或胞质尾区将被 在大肠杆菌中表达coli或S.酿酒酵母并分析功能缺陷 在6项试验中：a）与上皮单层的粘附; B）iC 3 b结合 ELISA; c）凝集; d）S.酿酒酵母;和 e/f）单克隆和多克隆抗体的结合。 场发射 扫描电子显微镜将用于确认表面表达 in C.白色念珠菌芽生孢子和定位细胞内水库， 非表面携带的缺失突变体。 在具体目标2中， 原核生物中模拟纤维蛋白原结合位点的α INt 1 p结构域 将使用缺失突变体绘制相关区域， 纤维蛋白原（Fbg）的定义为纯化的Fbg，分离的 Fbg的γ链、D/E片段和内部肽。 具体目标 三，杂合和纯合无效突变体在手将用于 研究α-INT 1在C.白色念珠菌 在具体目标4中，我们将测试α Int 1 p可能的相互作用 与S.酿酒酵母， 并与人类β亚基使用基因敲除，酵母双杂交 系统和CHO细胞转染。 上述研究应 提供了关于α Int 1 p在 粘附、侵入和形态发生过程。白色念珠菌和 从而为念珠菌感染的控制提出新的策略。

The opportunistic pathogen Candida albicans adheres to epithelium and replicates at the mucosal barrier prior to invading the bloodstream. We have recently characterized an "integrin-like protein" that is antigenically, structurally, and functionally related to the alpha subunits of the beta2 integrins alphaM (Mac-1 or CD11b/CD18) and alphaX (p150,95 or CD11c/CD18)--two members of the leukocyte adhesion glycoprotein family. A major function of this protein is the facilitation of epithelial and endothelial adhesion by recognition of RGD sequences in proteins such as iC3b and fibrinogen that serve as substrates for adhesion. In attainment of the specific aims of the previous five-year funding period, we have now cloned a gene (alpha INT1) encoding a novel surface protein in C. albicans and have analyzed its structure. Like the alpha-subunits of the beta2 integrins, the alpha INT1 gene product contains a ligand binding site for iC3b and fibrinogen, two EF-hand divalent cation binding sites, a hydrophobic transmembrane domain, and a tyrosine residue in the cytoplasmic tail. Alpha INT1 also encodes its own RGD tripeptide. Transformation of S. cerevisiae with the coding sequence of alpha INT1 under the control of a galactose-inducible promoter induces germ tube formation in haploid yeast. In Specific Aim One, a series of deletion mutants lacking the ligand-binding site, one or both divalent cation binding sites, the RGD sequence, or the transmembrane domain an/or cytoplasmic tail will be expressed in E. coli or S. cerevisiae and analyzed for functional deficits in six assays: a) adhesion to epithelial monolayers; b) iC3b-binding ELISA; c) agglutination; d) formation of germ tubes in S. cerevisiae; and e/f) binding of monoclonal and polyclonal antibodies. Field emission scanning electron microscopy will be utilized to confirm surface expression in C. albicans blastospores and to localize intracellular reservoirs for deletion mutants that are not surface-borne. In Specific Aim Two, those domains of alpha INt1p that mimic fibrinogen-binding sites in prokaryotes will be mapped wih the use of deletion mutants, and the relevant regions of fibrinogen (Fbg) will be defined with purified Fbg, isolated gamma-chain, D/E fragments, and internal peptides of Fbg. In specific Aim Three, heterozygous and homozygous null mutants in hand will be used to study the role of alpha INT1 in adhesion and morphogenesis in C. albicans. In Specific Aim Four, we will test the possible interaction of alpha Int1p with the G alpha subunit and 3 cytoskeletal proteins from S. cerevisiae, and with human beta-subunits using gene knockouts, the yeast two-hybrid system, and CHO cell transfection. The studies outlined above should provide novel information regarding the role of alpha Int1p in the processes of adhesion, invasion, and morphogenesis in C. albicans and thereby bring forward new strategies for the control of candidal infection.