MOLECULAR BASIS OF VIRULENCE FACTORS OF ORAL FUNGAL SP.

描述(由申请人提供)： 这是代表Mary Ann Jabra-Rizk提交的K22学者发展和教师过渡奖申请的修订版，Mary Ann Jabra-Rizk目前是MD牙学院诊断科学和病理学系的博士后研究员。两年的学者发展阶段和三年的教师过渡阶段将集中在MD大学，由约翰·霍普金斯大学、弗吉尼亚大学和维尔茨堡大学的共同导师和顾问参与，并将使Jabra-Rizk博士成为口腔真菌疾病的独立专家。MD大学的一个咨询委员会将评估她在教师过渡方面的进展和帮助。在学者发展过程中需要检验的假设是，都柏林隐孢子虫是一种具有恒定细胞表面疏水性的物种，与白色念珠菌CSHL基因具有同源性，该基因编码参与细胞黏附的疏水细胞壁蛋白，但在MNN甘露糖化家族基因和其他可能的毒力基因的表达上有所不同。(A)根据白念珠菌CSIII和CaMNN9基因序列设计聚合酶链式反应(PCR)引物，确定白念珠菌CSHL和CaMNN9基因及其同源基因在都柏林念珠菌中的存在。随后将使用都柏林弯孢杆菌CDS6基因组DNA和探针杂交来扩增和克隆这些基因。将使用基因中断技术中的最新技术(URA-blaster中的ura3氨基营养的杜氏隐孢子虫突变体)产生CSH 1同源基因的都柏林隐孢子虫敲除(B)通过评估都柏林隐孢子虫敲除克隆的细胞表面疏水性7对纤维连接蛋白的粘附性、对梭杆菌与核梭杆菌的粘附性以及对人颊上皮细胞的粘附性来确定CDCSh1基因的破坏对粘附性的影响，与野生型相比(C)确定巨噬细胞吞噬37C培养的白色念珠菌的能力的差异，37C培养的CDCSH1突变体、野生型都柏林假丝酵母菌和白念珠菌mnn9突变体，以确定细胞壁甘露聚糖侧链结构变化对都柏林假丝酵母菌和白念珠菌逃避宿主细胞吞噬能力的影响。2.基于白念珠菌基因组中基因的cDNA微阵列序列，我们将确定亲水性(37C生长的白念珠菌)和疏水性(250C生长的白念珠菌，25和37C生长的都柏林假丝酵母菌和白念珠菌突变体，A9V10和Camnn9)酵母细胞以及在AIM 1中产生的杜氏假丝酵母菌CDCSH1突变体之间CSHL和CaMNN9基因的差异表达水平。Jabra-Rizic博士随后将利用所产生的信息和突变体，利用DNA微阵列研究杜氏假丝酵母菌和白念珠菌在教师过渡阶段的全基因组差异。她还将分析HSP90基因和其他糖基化和热休克蛋白基因在都柏林弯曲霉中表达水平的差异。此外，还将产生白色念珠菌的HSP90敲除突变体，并评估其耐热性和巨噬细胞的吞噬功能。长期计划包括使用产生的突变株来研究念珠菌甘露糖蛋白特异的宿主免疫调节，如细胞因子和趋化因子的刺激。

DESCRIPTION (provided by applicant): This is a Revision of a K22 Scholar Development and Faculty Transition Award application, submitted on behalf of Mary Ann Jabra-Rizk who is currently a Post-Doctoral Fellow in the Department of Diagnostic Sciences and Pathology at the U of MD Dental School. A 2 year Scholar Development phase and three years of Faculty Transition win be centered at U of MD involving co-mentors and consultants at Johns Hopkins, the U of VA and the U of Wurzburg and will establish Dr. Jabra-Rizk as an independent expert in oral fungal diseases. An Advisory Committee at the U of MD will evaluate her progress and aid in the Faculty Transition. The hypothesis to be tested during the Scholar Development is that C. dubliniensis is a species that exhibits constant cell surface hydrophobicity, possessing a homologue to the C. albicans CSHl gene which encodes a hydrophobic cell wall protein involved in cell adherence but differs in the expression of the MNN mannosylation family of genes and other putative virulence genes. Two aims are planned: 1. (a) Determine the presence of the C. albicans CSHl and CaMNN9 genes or homologues of the genes in C. dubliniensis using PCR primers designed based on the C albicans CSIII and CaMNN9 gene sequences. These will be subsequently amplified and cloned using C. dubliniensis CDS6 genomic DNA and probe hybridization. A C. dubliniensis knockout of the CSH 1 homologue gene will be generated using the latest technique (ura3 amxotrophic C. dubliniensis mutants in the URA-blaster) in gene disruption technique (b) Determine the effect of the disruption of the CdCShl gene on adherence by assessing the C. dubliniensis knockout clone for cell surface hydrophobicity7 adhesion to fibronectin, adherence to Fusobacterium nucleatum and pooled human buccal epithelial cells, in comparison to the wild type (c) Determine differences in the ability of macrophages to phagocytize 37C-grown C. albicans, 37C grown CdCSH1 mutant and wild type C. dubliniensis and the C. albicans mnn9 mutant in order to determine the effect of structural changes in the side chains of cell wall mannan on the ability of C. dubliniensis and C. albicans to evade host cell phagocytosis. 2. Based on cDNA microarray sequences of genes in the C. albicans genome, we will determine levels of differential expression of the CSHl and CaMNN9 genes between hydrophilic (37C-grown C. albicans) and hydrophobic (250C-grown C. albicans, 25 and 37C-grown C. dubliniensis and C. albicans mutants, A9V10 and Camnn9) yeast cells, as well as the C. dubliniensis CdCSHl mutant generated in Aim 1. Dr. Jabra-Rizic will subsequently utilize the information and mutants generated to investigate whole genome differences between C. dubliniensis and C. albicans in the Faculty Transition phase using DNA microarrays. She will also analyze differences in the levels of expression of hsp90 gene and other glycosylation and heat shock proteins genes in C. dubliniensis. In addition, a hsp90 knock out mutant of C. albicans will be generated and assessed for thermotolerance and phagcytosis by macrophages. Long range plans include using the mutants generated to study Candida mannoprotein-specific host immunomodulation, such as stimulation of cytokines and chemokines.