MOLECULAR BASIS OF VIRULENCE FACTORS OF ORAL FUNGAL SP.

描述（由申请人提供）： 这是K22学者发展和教师过渡奖申请的修订，代表玛丽安Jabra-Rizk提交，他目前是MD牙科学院U诊断科学和病理学系的博士后研究员。一个为期2年的学者发展阶段和三年的教师过渡赢得了在医学博士大学的中心，涉及共同导师和顾问在约翰霍普金斯，弗吉尼亚州和维尔茨堡大学的U，并将建立博士Jabra-Rizk作为一个独立的专家在口腔真菌疾病。在医学博士的U咨询委员会将评估她的进展和援助的教师过渡。在学者发展过程中要检验的假设是C。dubliniensis是一种表现出恒定的细胞表面疏水性的物种，具有与C.白色念珠菌CSH 1基因，其编码参与细胞粘附的疏水细胞壁蛋白，但在MNN甘露糖基化基因家族和其他推定的毒力基因的表达方面不同。计划有两个目标：1。(a)确定C的存在。白色念珠菌CSH 1和CaMNN 9基因或其同源基因。使用基于白色念珠菌CSIII和CaMNN 9基因序列设计的PCR引物对都柏林念珠菌进行PCR扩增。 这些将随后使用C. dubliniensis CDS 6基因组DNA和探针杂交。梭将使用最新技术（ura 3 amxotrophic C.（B）通过评估C. dubliniensis突变体中的URA-blaster来确定CdCSh 1基因的破坏对粘附的影响。与野生型相比，Dubliniensis敲除克隆的细胞表面疏水性7对纤连蛋白的粘附、对具核梭杆菌和合并的人颊上皮细胞的粘附。（c）测定巨噬细胞吞噬37 C生长的C.白念珠菌、37 C生长的CdCSH 1突变体和野生型C. dubliniensis和C.白念珠菌mnn 9突变体，以确定细胞壁甘露聚糖侧链的结构变化对C. dubliniensis和C.白色念珠菌逃避宿主细胞吞噬作用。2.基于cDNA微阵列基因序列的C。白念珠菌基因组中，我们将确定CSH 1和CaMNN 9基因在亲水性（37 C-生长的C. 白色念珠菌）和疏水性（250 ℃生长的C.白色念珠菌、25和37 C生长的C. dubliniensis和C. albicans突变体、A9 V10和Camnn 9）酵母细胞以及C.在Aim 1中产生的都柏林菌CdCSH 1突变体。Jabra-Rizic博士随后将利用这些信息和产生的突变体来研究C. dubliniensis和C.白念珠菌在学院过渡阶段使用DNA微阵列。她还将分析热休克蛋白90基因和其他糖基化和热休克蛋白基因在C.都柏林的。此外，还获得了一株C.产生白色念珠菌并评估其耐热性和巨噬细胞的吞噬作用。长期计划包括使用产生的突变体来研究念珠菌甘露糖蛋白特异性宿主免疫调节，例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.