MOLECULAR GENETICS, BIOCHEMISTRY AND THERAPY OF CANDIDA ALBICANS

白色念珠菌的分子遗传学、生物化学和治疗

• 批准号: 2566847
• 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/2566847
• 关键词: Candida albicans; Cryptococcus neoformans; Saccharomyces cerevisiae; candidiasis; cerebrospinal fluid; dihydroxyphenylalanine; enzyme activity; fungal genetics; gene deletion mutation; genetic strain; hydrogen peroxide; laboratory mouse; laboratory rabbit; melanins; microorganism metabolism; monophenol monooxygenase; mutant; neutrophil; nitrogen oxides; phenols; polymerase chain reaction; site directed mutagenesis; virulence

Laccase of Cryptococcus neoformans is encoded by the gene CNLAC1 and causes certain aminophenols and diphenols to be converted to melanin. Laccase activity was shown to increase virulence of this fungus for mice. Nearly isogenic strains were obtained which differed only at the CNLAC1 locus. Two melanin negative strains were studied, one which had been mutagenized and had a base substitution at condon 164 which changed histidine to tyrosine in a putative copper binding site. Site directed mutagenesis showed that this base substitution led to loss of laccase activity even though normal amounts of message were synthesized. Another melanin negative mutant had a 4 kb deletion beginning upstream to base 68 of the ORF, created by gene replacement. These melanin negative strains were compared to nearly isogenic strains which contained functioning copies of CNLAC1 supplied by transformation and nonhomologous integration. The melanin negative mutants were significantly less virulent for mice than the melanin positive strains. The greater virulence of melanin positive strains could not be explained by melanin protecting against oxidative attack. Melanin producing strains grown on L-DOPA medium were not protected from killing by hydrogen peroxide, oxides of nitrogen or human neutrophils. Cryptococci obtained from cerebrospinal fluid of infected rabbits were shown to produce CNLAC1 transcript on reverse transcriptase-polymerase chain reaction. Considering that L-DOPA is present in the central nervouse system, it seems likely that the effect of CNLAC1 on virulence is related to melanin formation, but the mechanism by which melanin affects virulence is unknown. Cryptococcal laccase has been expressed in Saccharomyces cerevisiae and Pichia pastoris so that antibody may be raised to this antigen and used for intracellular localization studies. Amounts of secreted laccase remain low, indicating a need for an improved expression system.

新型隐球菌的漆酶由基因CNLAC 1编码， 导致某些氨基酚和二酚转化为黑色素。 漆酶活性显示出增加该真菌对小鼠的毒力。 获得了仅在CNLAC 1上不同的近等基因菌株 基因座 研究了两种黑色素阴性菌株，其中一种已被 突变并在密码子164处发生碱基置换， 组氨酸到酪氨酸在推定的铜结合位点。 定点 突变表明，这种碱基取代导致漆酶的损失 即使合成了正常数量的消息， 另一 黑色素阴性突变体具有从碱基上游开始的4kb缺失 68的ORF，由基因替换产生。 这些黑色素阴性 菌株进行了比较，近等基因菌株，其中包含 转化和非同源提供CNLAC 1的功能拷贝 一体化 黑色素阴性突变体明显少于 对小鼠的毒性高于黑色素阳性菌株。 越大 黑色素不能解释黑色素阳性菌株的毒力 保护免受氧化攻击。 黑色素产生菌株生长在 L-DOPA培养基不能防止过氧化氢杀死， 氮氧化物或人嗜中性粒细胞。 隐球菌来自 感染家兔脑脊液中产生CNLAC 1 逆转录聚合酶链反应。 考虑到左旋多巴存在于中枢神经系统， CNLAC 1对毒力的影响似乎与黑色素有关 形成，但黑色素影响毒力的机制是 未知 隐球菌漆酶已在酿酒酵母中表达， 巴斯德毕赤酵母（Pichia pastoris），从而可以产生针对该抗原的抗体并使用 用于细胞内定位研究。 漆酶分泌量 仍然很低，表明需要改进的表达系统。