IDENTIFYING CANDIDA GENE IN THRUSH USING IVIAT

描述：（逐字摘要）申请人在体内使用了一种新的 微生物表达技术，称为体内诱导抗原技术 （IVIAT）研究口咽念珠菌病（OPC）的发病机制， 艾滋病毒感染者。IVIAT使用血清中的抗白色念珠菌抗体 的HIV感染的患者，以确定在体内表达的抗原，由C. 白色念珠菌，但在常规体外生长过程中不表达。初步 研究，他们已经证实IVIAT可以识别C. 白色念珠菌在这项提案中，他们建议修改IVIAT以识别抗原 表达的C.白念珠菌在OPC期间表达，但在 口腔粘膜中C.白色念珠菌或在体外生长期间。他们 推测C.白色念珠菌仅 在OPC过程中是重要的毒力因子，识别这些抗原将 提供了深入了解的机制，其中C。白色念珠菌是由 无害的微生物转化为入侵性病原体此应用程序有 五个具体目标。在第一个具体目标中，两个单独的合并批次的 血清，一份来自HIV感染的OPC患者，另一份来自HIV感染的 定植的患者，将被体外生长的 临床C.白色念珠菌分离物，以去除与抗原反应的所有抗体 体外表达。吸附的血清将用于鉴别筛选 梭菌白色念珠菌基因组表达文库。在具体目标2中，质粒DNA 将从与OPC血清反应但不与 定殖血清和开放阅读框架（ORF）负责 将测定血清反应性。在具体目标3中，他们将确认， IVIAT抗原不被C.体外培养白色念珠菌。具体目标 4、克隆C。白念珠菌基因编码IVIAT抗原，并纯化 这些基因表达的蛋白质。最后，在具体目标5中，他们将确认 IVIAT抗原存在于鹅口疮样品中， HIV感染者光镜免疫组化使用抗体 对纯化的蛋白质产生的反应。他们希望这项研究能够确定 新猫栉头蚤白念珠菌的毒力因子，增加我们对体液免疫的了解 响应OPC，并导致潜在的应用，药物，疫苗或 诊断测试开发根据该项目的经验，IVIAT 应易于适用于研究其他念珠菌感染和其他真菌感染 病原体IVIAT相对于现有技术的潜在优势包括： 使用人免疫应答来鉴定体内表达的基因， 比动物模型，它的相对技术简单， 不同类型C.白色念珠菌感染 人类

DESCRIPTION: (abstract verbatim) The applicants have used a novel in vivo microbial expression technology called In Vivo Induced Antigen Technology (IVIAT) to study the pathogenesis of oropharyngeal candidiasis (OPC) in HIV-infected patients. IVIAT uses anti-Candida albicans antibodies in the sera of HIV-infected patients to identify antigens that are expressed in vivo by C. albicans but are not expressed during routine in vitro growth. In preliminary studies, they have confirmed that IVIAT can identify virulence factors for C. albicans. In this proposal, they propose to modify IVIAT to identify antigens expressed by C. albicans during OPC but not expressed during either colonization of the oral mucosa by C. albicans or during in vitro growth. They hypothesize that some of the antigens expressed by C. albicans exclusively during OPC are important virulence factors, and identifying these antigens will provide insight into the mechanisms by which C. albicans is transformed from a harmless commensal organism into an invasive pathogen. This application has five specific aims. In the first specific aim, two separate pooled batches of sera, one from HIV-infected patients with OPC and the other from HIV-infected patients with colonization, will be exhaustively adsorbed with in vitro grown clinical C. albicans isolates to remove all antibodies that react with antigens expressed in vitro. The adsorbed sera will be used for differential screening of a C. albicans genomic expression library. In specific aim 2, the plasmid DNA will be purified from clones that are reactive with the OPC sera but not with the colonization sera and the open reading frames (ORFs) responsible for the serum reactivity will be determined. In specific aim 3, they will confirm that the IVIAT antigens are not expressed by C. albicans in vitro. In specific aim 4, they will clone C. albicans genes encoding IVIAT antigens and purify the proteins expressed by these genes. Lastly, in specific aim 5, they will confirm that the IVIAT antigens are present within thrush samples recovered from HIV-infected patients by light microscopic immunohistochemistry using antibody raised against the purified proteins. They hope that this study will identify new C. albicans virulence factors, increase our understanding of the humoral response to OPC, and lead to potential applications for drug, vaccine or diagnostic test development. With the experience from this project, IVIAT should be readily adaptable to study other candidal infections and other fungal pathogens. Potential advantages of IVIAT over existing technologies include the use of the human immune response to identify in vivo expressed genes rather than animal models, its relative technical simplicity, and its ability to study differential gene expression in different types of C. albicans infections in humans.