FIBRONECTIN RECEPTOR IN CANDIDA TROPICALIS

热带念珠菌中的纤连蛋白受体

• 批准号: 6163991
• 负责人: MARGARET K HOSTETTER
• 金额: $ 22.54万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6163991
• 关键词: Candida; Saccharomyces cerevisiae; antibody; fibronectins; fungal proteins; gene expression; human tissue; immunoprecipitation; laboratory mouse; mutant; oligonucleotides; protein localization; protein sequence; receptor expression; southern blotting; virulence

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.

在念珠菌属中，热带念珠菌仅次于念珠菌属 白色念珠菌作为免疫功能低下患者发病率和死亡率的原因 宿主，特别是在接受癌症化疗的患者或那些 真菌性心内膜炎 任何预防念珠菌感染的策略 应该包括这两个物种，它们加在一起 超过75%的感染率。 然而，与C.白色念珠菌 tropicalis不形成细丝（例如芽管，假菌丝，或 真菌丝）。 因此，了解念珠菌的毒力必须 不仅解决了C.白色念珠菌以及这些机制 由非二型念珠菌物种用来附着和侵入它们的 人类宿主 细胞外基质纤连蛋白（FN）的识别是一种手段， 其中C. Tropicalis附着于人上皮细胞;此外， C.附着力纯化的FN、多克隆FN和多克隆FN均能抑制Tropicalis的表达。 FN的抗体，以及包含RGD序列的肽， FN中的侧翼残基。 我们已经从C. tropicalis，计算出其KD约为2.3x10-9 M， 分离的新的内部肽序列。 在具体目标1中，我们将 使用这些新的肽序列作为简并的基础 寡核苷酸来鉴定编码FN受体的基因， 筛选C.热带假单胞菌基因组DNA 我们将分析 FN受体在C.热带和 其它病原真菌的DNA印迹或PCR检测。 具体目标 第二，我们将使用体外试验和同基因突变体来确定 FN受体的定位，基因产物在 粘附和侵袭，以及C. FN上的热带 分子。 在具体目标三中，我们将使用表面生物素化， 免疫沉淀法来表征C. 可能与FN受体相互作用。 具体目标 第四，我们将共同表达C。白色念珠菌基因产物Int 1 p和FN C.白色念珠菌和C.确定对 形态发生和毒力。 在具体目标五中，我们将使用我们的 小鼠中的同基因突变体、再整合体和共表达变体 静脉感染和免疫反应的模型，以了解是否 FN受体是C.热带植物如果或者 杂合子或纯合子缺失突变体被证明较少 毒力强于亲本菌株，我们将分析C3和FN的作用 作为念珠菌吞噬作用的调理素。 这些实验应该能够 我们在C. tropicalis，其作用 粘附和侵袭，以及其在肿瘤发病中的作用。 vivo.在C. Tropicalis应该能够 我们将开发创新的预防和治疗方法， 解决两个物种，占75%以上的念珠菌， 感染.