STUDIES ON PATHOGENIC FUNGI

病原真菌的研究

• 批准号: 5200553
• 负责人: J BENNETT
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5200553
• 关键词: Cryptococcus neoformans; carbohydrate biosynthesis; cell transformation; enzyme activity; fungal antigens; fungal genetics; gene deletion mutation; genetic mapping; genetic transcription; human subject; laboratory mouse; mannose; microorganism metabolism; nucleic acid sequence; oxidoreductase; protein purification; protein sequence; virulence

An isolate of Cryptococcus neoformans in which the structural gene for diphenol oxidase (CNLAC1) has been dirupted was crossed with a wild type and the F1 progency studied for virulence. A F1 strain which produced diphenol oxidase (DPO) killed mice whereas a strain which did not produce DPO did not cause lethal infection. However, complementation of the CNLAC1 deletant with CNLAC1 produced a strain which was not lethal for mice, even though the isolate produced DPO. All these strains were the same mating type (a) and had the uracil auxotrophy of the CNLAC1 deletant restored by either backcrossing or transformation. The deletant was found to be missing not only the 5' end of the gene and the URA5 gene used for positive selection, but also approximately 4kb of upstream sequences. Although no transcript has been detected from the area of the 5' deleted sequences as yet, it is possible that sequences coding for regulatory or structural genes are present in this area. The upstream deletion might have decreased virulence apart from disrupted CNLAC1 gene. Another experiemnt which supports the nonessentiality of the CNLAC1 gene for virulence is that a mutant (mel2) obtained from J.C. Edman did not produce DPO but was able to kill mice. When transformed with CNLAC1 the isolate made DPO but did not have a substantial increase in virulence. CNLAC1 was used to integratively transform Pichia pastoris. The expressed and secreted protein was partially purified on phenylsepharose and DEAE chromatography. The expressed protein was contained in a fraction containing at least two proteins, based on agar gel precipitin bands. The mixture was heavily glycosylated, containing 41% mannose. Deglycosylated enzyme was inactive. On TSK 4000 HPLC chromatography, DPO enzymatic activity was approximately 100-130 kd and had a pI of less than 4.5 on isoelectric focusing. When compared with a DPO transcript size of 1.3-1.8 kb, it seems likely that DPO is being expressed as a heavily mannosylated protein with a heterodisperse molecular weight.

一种新型隐球菌的分离株， 二酚氧化酶（CNLAC 1）已中断与野生型杂交 并对F1代进行了毒力测定。 一种F1菌株， 二酚氧化酶（DPO）杀死小鼠，而不产生 DPO未引起致死性感染。 然而， CNLAC 1缺失株与CNLAC 1产生了一种菌株， 小鼠，即使分离物产生DPO。 所有这些菌株 相同的交配型（a），具有CNLAC 1缺失体的尿嘧啶营养缺陷型 通过回交或转化恢复。 这个罪犯 发现不仅缺失基因的5'端和URA 5基因， 用于阳性选择，但也可以使用约4kb的上游 序列的 虽然没有从该地区检测到转录本， 5'缺失的序列，有可能编码 调控或结构基因存在于该区域中。 上游 除CNLAC 1基因被破坏外，缺失可能降低了毒力。 另一个支持CNLAC 1基因非必需性的实验 从J.C.埃德曼没有 产生DPO但能杀死老鼠。 当用CNLAC 1转化时， 分离物产生DPO，但毒力没有显著增加。 CNLAC 1用于整合转化巴斯德毕赤酵母。 的 表达和分泌的蛋白在苯基琼脂糖凝胶上部分纯化 和DEAE层析。 表达的蛋白质包含在一个 至少含有两种蛋白质的组分，基于琼脂凝胶沉淀素 乐队. 该混合物是高度糖基化的，含有41%的甘露糖。 去糖基化酶无活性。 在TSK 4000 HPLC色谱上，DPO 酶活性约为100-130 kd，pI小于 4.5等电聚焦 与DPO转录本大小相比， 1.3-1.8 kb，DPO似乎很可能被表达为一个重 具有异分散分子量的甘露糖基化蛋白质。