A proteomics approach to Candida albicans biofilm

描述（由申请人提供）： 本研究将主要在西班牙马德里孔普卢顿大学与塞萨尔诺贝拉博士合作完成，作为NIH资助R 01 DE 14029的延伸。父母补助金和FIRCA申请的假设是，基因表达的改变伴随着白色念珠菌从自由生活的嗜酸性细胞到生物膜的固着成员的变化。母基金的重点是在单一和混合物种生物膜和从口腔中回收的生物体中进行表达谱分析（微阵列）（目标1和2），并通过产生缺陷突变株（目标3）分析选定的调控基因。这个FIRCA项目是一个蛋白质组学项目，旨在研究蛋白质表达并确定固着生物和固着生物之间的差异。Nombela及其同事通过研究C.白色念珠菌酵母细胞和芽管，并已使用质谱分光光度（MS）技术（MALDI-TOF）鉴定来自酿酒酵母和C.白色念珠菌细胞质将检查细胞壁和细胞质提取物，如果通过来自亲本授权的表达分析指示，则在时间允许的情况下可以检查其他亚细胞级分。在目标1中，将生物膜生物体提取物的概况与酵母细胞和芽管的概况进行比较。这是对父母补助金第一个目标的补充。在目标2中，检查将扩展到选定突变菌株的提取物。这补充了母基金的第三个目标，即构建突变菌株并检查生物膜形成和全局基因表达。将通过2D-PAGE分离蛋白质，并比较图谱，以确定蛋白质丰度的定性和定量差异。将通过质谱方法鉴定嗜酸性和生物膜生物体或野生型和突变菌株之间存在差异的蛋白质，优先考虑Aim 1谱的差异。

DESCRIPTION (provided by applicant): This research will be done primarily in Spain at Universidad de Complutense Madrid in collaboration with Dr. Cesar Nombela as an extension of NIH grant R01 DE14029. The hypothesis of the parent grant and this FIRCA application is that alterations in gene expression accompany the change of Candida albicans from a free living planktonic cell to a sessile member of a biofilm. The parent grant focuses on expression profiling (microarrays) in mono- and mixed species biofilm and in organisms recovered from the oral cavity (Aims 1 and 2) and analysis of selected regulated genes through generation of deficient mutant strains (Aim 3). This FIRCA project is a proteomic project to examine protein expression and identify differences between planktonic and sessile organisms. Nombela and colleagues have established the feasibility of a proteomics approach with studies of proteins in cell wall subfractions of C. albicans yeast cells and germ tubes and have used mass spectrophotometric (MS) techniques (MALDI-TOF) for identification of cell wall proteins from Saccharomyces cerevisiae and C. albicans cytoplasm Cell wall and cytoplasmic extracts will be examined and, time permitting other subcellular fractions may be examined if indicated by expression analysis derived from the parent grant. In Aim 1, the profiles of extracts from biofilm organisms will be compared to that of yeast cells and germ tubes. This complements the first aim of the parent grant. In Aim 2, the examination will be extended to extracts from selected mutant strains. This complements the third aim of the parent grant in which the mutant strains are constructed and examined for biofilm formation and global gene expression. Proteins will be separated by 2D-PAGE and profiles compared to identify qualitative and quantitative differences in protein abundance. Proteins that differ between planktonic and biofilm organisms or wild type and mutant strain will be identified by mass spectrometric methods with priority given to differences in Aim 1 profiles.