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FIBRONECTIN RECEPTOR IN CANDIDA TROPICALIS

热带念珠菌中的纤连蛋白受体

基本信息

批准号：
6632085
负责人：
MARGARET K HOSTETTER
金额：
$ 31.91万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-03-01 至 2006-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6632085
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

项目摘要

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.

在念珠菌种类中，热带念珠菌的排名仅次于念珠菌