Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans.
Effect of the laccase gene CNLAC1, on virulence of Cryptococcus neoformans.
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DOI：
10.1084/jem.184.2.377
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发表时间：
1996-08-01
期刊：
影响因子：
--
通讯作者：
Williamson PR
中科院分区：
文献类型：
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作者：
Salas SD;Bennett JE;Kwon-Chung KJ;Perfect JR;Williamson PR
为了在分子基础上评估新型隐球菌产生的黑色素与毒力之间的关系，我们提出了以下问题：(a) 新型隐球菌的漆酶结构基因 CNLAC1 在体内表达吗？ (b) 能否通过与 CNLAC1 互补来恢复 cnlac1 (Mel-) 突变体的小鼠毒力？ (c) CNLAC1 的靶向基因删除是否会降低小鼠的毒力？当隐球菌漆酶催化某些芳香族化合物（包括左旋多巴）氧化成醌，然后聚合成黑色素时，就会产生黑色素。为了评估 CNLAC1 转录，从感染兔子脑脊液中的新型隐球菌中提取 RNA。逆转录聚合酶链反应检测到CNLAC1转录物，表明感染宿主中可能产生漆酶。为了评估 CNLAC1 缺失对毒力的影响，通过破坏该基因的 5' 端获得 Mel 突变体 (10S)。在与亲本品系进行多次回交以消除转化过程中引入的意外遗传缺陷后，对 Mel- 后代进行了测试，发现对小鼠的毒性比 Mel+ 后代低得多。另一种 Mel- 菌株 (mel2)，从 J.C. Edman（加州大学旧金山分校，CA）获得，产生 CNLAC1 转录本，但没有检测到黑色素。该突变体的表征揭示了 CNLAC1 中的碱基替换，该替换将假定的铜结合位点中的组氨酸更改为酪氨酸。当通过定点诱变将该碱基变化引入CNLAC1时，它不再将mel2转化为Mel+，表明该组氨酸在漆酶活性中的重要性。 mel2 衍生突变体与 CNLAC1 的互补恢复了 Mel+ 表型并增加了毒力。这些结果支持 CNLAC1 基因产物在毒力中发挥作用的概念。
To assess the relationship between melanin production by Cryptococcus neoformans and virulence on a molecular basis, we asked: (a) is CNLAC1, the laccase structural gene of C. neoformans, expressed in vivo?; (b) can mouse virulence be restored to cnlac1 (Mel-) mutants by complementation with CNLAC1?; and (c) will targeted gene deletion of CNLAC1 decrease virulence for mice? Melanin is produced when cryptococcal laccase catalyzes the oxidation of certain aromatic compounds, including L-dopa, to quinones, which then polymerize to melanin. To assess CNLAC1 transcription, RNA was extracted from C. neoformans in cerebrospinal fluid of infected rabbits. Reverse transcriptase-polymerase chain reaction detected CNLAC1 transcript, indicating that laccase may be produced in the infected host. To assess the effect of CNLAC1 deletion on virulence, a Mel- mutant (10S) was obtained by disruption of the 5' end of the gene. After multiple backcrosses with a parental strain to remove unintended genetic defects introduced by the transformation process, a Mel- progeny was tested and found to be much less virulent for mice than a Mel+ progeny. Another Mel- strain (mel2), obtained from J.C. Edman (University of California at San Francisco, CA), produced CNLAC1 transcript but no detectable melanin. Characterization of this mutant revealed a base substitution in CNLAC1 that changed a histidine to tyrosine in a putative copper- binding site. When this base change was introduced into CNLAC1 by site- directed mutagenesis, it no longer transformed mel2 to Mel+, indicating the importance of this histidine in laccase activity. Complementation of a mel2-derived mutant with CNLAC1 restored the Mel+ phenotype and increased virulence. These results support the concept that the CNLAC1 gene product has a role in virulence.