LACCASE REGULATION AND VIRULENCE IN CRYPTOCOCCUS

隐球菌的漆酶调节和毒力

• 批准号: 6510899
• 负责人: Peter Richard Williamson
• 金额: $ 30.54万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6510899
• 关键词: Cryptococcus neoformans; fungal genetics; gel mobility shift assay; gene expression; genetic enhancer element; genetic regulation; laboratory mouse; miscellaneous oxidoreductase; molecular cloning; posttranslational modifications; temperature; transcription factor; virulence

Description (Adapted from Abstract): Cryptococcus neoformans is a major pathogen in immunocompromised patients, causing life-threatening meningoencephalitis in approximately 6% of HIV positive individuals. In vitro melanin production has classically been associated with virulence in C. neoformans. During the previous funding period congenic knockout strains of C. neoformans establish the importance of the CNLAC1 gene in virulence and laccase-dependent catecholamine oxidation products have been identified in the brain of infected mice. Furthermore, a complex pattern of regulatory DNA-binding sites upstream of the CNLAC1 gene have been identified over been identified together with an unusual Sp1 enhancer site. Sp1 consensus sequences have been found in the other virulence genes such as Cap64 and Cap59 suggesting co-regulation of virulence. The hypothesis to be examined is that molecular regulators of CNLAC1 control virulence of Cryptococcus neoformans. The objectives of the proposed research are to identify and characterize genes involved in laccase expression to determine their role in virulence. Aim 1 proposes to identify and characterize important regulatory DNA-binding sites in the upstream region of CNLAC1. The plan is to use pVEW promoter plasmid and electromobility shift assays to determine significant enhancer and repressor regions under conditions of glucose repression and derepression as well as the host temperature of 37 C. Aim 2 proposes to identify and characterize genes involved in transcriptional enhancement of repression of CNLAC1 and determine their role in virulence. Genes will be cloned by homology to genes predicted by CNLAC1 DNA-binding sites and from laccase-deficient insertional mutants. For each gene the plan is to produce knockout and wild-type complemented strains of C. neoformans and test the effects of the gene on laccase production, capsule formation, secreted manno-proteins, urease activity and virulence. Aim 3 will identify and characterize genes involved in post-transcriptional modification of laccase from DL-1 mutants. Initially, the plans are to complement a vacuolar H+-ATPase on laccase secretion and assess for possible co-regulation of CNLAC1 and CNVPH1. It is anticipated that these studies will provide insights into regulation and virulence factors in C. neoformans that may be used to provide novel approaches to the treatment and control of cryptococcosis.

描述(摘自摘要)：新生隐球菌是免疫功能低下患者的主要病原体，在大约6%的HIV阳性患者中会导致危及生命的脑膜脑炎。在体外，黑色素的产生与新生葡萄球菌的毒力有经典的联系。在之前的资助期间，新生隐孢子虫的同源基因敲除菌株证实了CNLAC1基因在毒力中的重要性，并在感染小鼠的大脑中发现了依赖漆酶的儿茶酚胺氧化产物。此外，CNLAC1基因上游的调控DNA结合位点的复杂模式已被鉴定，并与一个不寻常的Sp1增强子位点一起被鉴定。在其他毒力基因如Cap64和Cap59中也发现了SP1的共同序列，这表明毒力的共同调节。需要检验的假设是，CNLAC1的分子调节器控制着新生隐球菌的毒力。这项研究的目的是鉴定和鉴定与漆酶表达有关的基因，以确定它们在毒力中的作用。目的1建议鉴定和鉴定CNLAC1上游区域的重要调控DNA结合位点。该计划利用pVEW启动子、质粒和电迁移率改变分析来确定在葡萄糖抑制和去抑制以及37℃的宿主温度条件下的重要增强子和抑制区。目的2建议鉴定和鉴定参与转录增强CNLAC1抑制的基因并确定它们在毒力中的作用。基因将通过与CNLAC1DNA结合位点预测的基因和漆酶缺失插入突变体的同源性来克隆。对于每个基因，计划生产敲除和野生型互补的新生葡萄球菌菌株，并测试该基因对漆酶产生、胶囊形成、分泌甘露糖蛋白、尿素酶活性和毒力的影响。目的3从DL-1突变株中鉴定与漆酶转录后修饰相关的基因。最初，这些计划是为了补充液泡H+-ATPase对漆酶分泌的作用，并评估CNLAC1和CNVPH1可能的共同调节。预计这些研究将提供对新生葡萄球菌的调节和毒力因素的见解，可用于为治疗和控制隐球菌病提供新的方法。