FIBRONECTIN RECEPTOR IN CANDIDA TROPICALIS

在念珠菌种类中，热带假丝酵母菌仅次于假丝酵母菌 白念珠菌在免疫低下人群中的致残率和死亡率 宿主，特别是接受癌症化疗的患者或 患有真菌性心内膜炎。预防念珠菌感染的任何策略 应该包括这两个物种，这两个物种加起来占更多 超过75%的心脏感染。然而，与白色念珠菌不同的是，C. 热带菌不形成细丝(例如，芽管、假菌丝或 真菌丝)在大多数与临床环境相关的条件下。 因此，了解念珠菌的毒力必须 不仅阐述了白色念珠菌的形态发生，而且还探讨了这些机制 被非二形性念珠菌物种用来附着和入侵他们的 人类的主人。 识别细胞外基质纤维连接蛋白(FN)是一种手段 热带毛滴虫通过它附着在人的上皮细胞上；此外， 纯化的FN可通过多克隆抑制热带毛滴虫的黏附 FN的抗体，以及包含RGD序列和 FN中的侧翼残基。我们现在已经从C. 热带的Kd值约为2.3×10-9M，并具有 分离出新的内部多肽序列。在具体目标一中，我们将 使用这些新的肽序列作为简并的基础 利用寡核苷酸识别FN受体编码基因 热带毛虫基因组DNA文库的筛选。我们将分析 热带毛滴虫患者分离株Fn受体的保守性 其他病原真菌经Southern印迹或聚合酶链式反应。以特定的目标 第二，我们将使用体外试验和等基因突变来定义 FN受体的定位及其基因产物的功能 热带毛滴虫在FN上的粘附性和侵袭性及其结合部位 分子。在具体目标三中，我们将使用表面生物素化和 免疫沉淀法鉴定C。 可能与FN受体相互作用的热带生物。以特定的目标 四、白念珠菌基因产物Int1p和Fn的共表达 白念珠菌和热带念珠菌中的受体以确定对 形态发生和致病力。在具体的目标五中，我们将使用我们的 小鼠的同基因突变体、重组体和共表达变体 了解静脉感染模型和免疫反应是否 Fn受体是热带毛滴虫的毒力因子。如果不是 杂合子或纯合子缺失突变体被证明较少 我们将分析C3和Fn的作用 作为调理素抗念珠菌吞噬作用。这些实验应该能够 美国将在热带毛滴虫中定义一种新的表面蛋白，它在 体外黏附和侵袭及其在肿瘤发病机制中的作用 活着。在热带毛滴虫中鉴定的表面蛋白(S)应该能够 美国将开发创新的预防和治疗模式 应对占念珠菌75%以上的两个物种 感染。

Among Candida species, Candida tropicalis ranks second only to Candida albicans as a cause or morbidity and mortality among immunocompromised hosts, especially in patients undergoing cancer chemotherapy or those with fungal endocarditis. Any strategy to prevent candidal infections should include both of these species, which together account for more than 75 percent of cadidal infections. However, unlike C. albicans C. tropicalis does not form filaments (e.g. germ tubes, pseudohyphae, or true hyphae) under most conditions relevant to the clinical setting. Therefore, an understanding of virulence in candidal species must address not only morphogenesis in C. albicans but also those mechanisms used by non-dimorphic candidal species to attach to and invade their human hosts. Recognition of the extracellular matrix fibronectin (FN) is one means by which C. tropicalis attaches to human epithelial cells; moreover, adhesion of C. tropicalis can be inhibited by purified FN, by polyclonal antibodies to FN, and by peptides encompassing the RGD sequence and flanking residues in FN. We have now purified a FN receptor from C. tropicalis, have calculated its KD at approximately 2.3x10-9 M and have isolated novel internal peptide sequences. In Specific Aim One, we will use these novel peptide sequences as the basis for degenerate oligonucleotides to identify the gene encoding the FN receptor by screening a library of C. tropicalis genomic DNA. We will analyze the conservation of the FN receptor in patient isolates of C. tropicalis and other pathogenic fungi by Southern blotting or PCR. In Specific Aim Two, we will use in vitro assays and isogenic mutants to define the localization of the FN receptor, the functions of the gene product in adhesion and invasion, and the binding site for C. tropicalis on the FN molecule. In Specific Aim Three, we will use surface biotinylation and immunoprecipitation to characterize other surface proteins in C. tropicalis that may interact with the FN receptor. In Specific Aim Four, we will co-express the C. albicans gene product Int1p and the FN receptor in both C. albicans and C. tropicalis to determine effects on morphogenesis and virulence. In Specific Aim Five, we will use our isogenic mutants, reintegrants, and co-expression variants in a murine model of intravenous infection and immune response to understand whether the FN receptor is a virulence factor for C. tropicalis. If either the heterozygote or the homozygous deletion mutant proves to be less virulent than the parent strain, we will analyze the role of C3 and FN as opsonins for candidal phagocytosis. These experiments should enable us to define a novel surface protein in C. tropicalis, its roles in adhesion and invasion in vitro, and its contribution to pathogenesis in vivo. The surface protein(s) identified in C. tropicalis should enable us to develop innovative preventive and therapeutic modalities to address two species that account for more than 75 percent of candidal infections.