A proteomics approach to Candida albicans biofilm

描述(由申请人提供)： 这项研究将主要在西班牙马德里大学与Cesar Nombela博士合作进行，作为NIH赠款R01 DE14029的延伸。父母资助和这项FIRCA申请的假设是，基因表达的变化伴随着白色念珠菌从自由生活的浮游细胞转变为生物膜的固着成员。父母赠款的重点是单一和混合物种生物膜和从口腔中回收的生物体的表达谱(微阵列)(目标1和2)，以及通过产生缺陷突变菌株来分析选定的受调控基因(目标3)。这个FIRCA项目是一个蛋白质组项目，目的是检查蛋白质的表达，并确定浮游生物和固着生物之间的差异。Nombela和他的同事已经建立了一种蛋白质组学方法的可行性，研究了白色念珠菌酵母细胞和生殖管的细胞壁亚组分中的蛋白质，并使用了质量分光光度(MS)技术(MALDI-TOF)来鉴定酿酒酵母和白念珠菌的细胞壁蛋白质。将检查细胞壁和细胞质提取物，如果来自亲本赠款的表达分析表明，在时间允许的情况下，可能还会检查其他亚细胞组分。在目标1中，生物膜生物提取物的轮廓将与酵母细胞和生殖管的轮廓进行比较。这与家长拨款的第一个目标相辅相成。在目标2中，检查将扩大到从选定的突变菌株中提取的物质。这补充了父母拨款的第三个目标，即构建突变菌株并检查生物膜的形成和全球基因表达。蛋白质将通过2D-PAGE和图谱进行分离，以确定蛋白质丰度的定性和定量差异。浮游生物和生物膜生物或野生型和突变菌株之间的不同蛋白质将通过质谱学方法进行鉴定，优先考虑AIM 1图谱的差异。

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.