新型隐球酵母氯离子通道在阳离子内稳态中的趋异作用

Divergent roles of CLC1 in cation homeostasis in Cryptococcus neoformans

新型隐球酵母Cryptococcus neoformans有两个变种(varieties),即grubii和neoformans。目前研究最多的两个菌株H99(血清型A)和JEC21(血清型D)分别代表这两个变种,两者的毒性差别显著,为研究新型隐球酵母菌株间毒性的进化提供了良好模型。通过比较JEC21的clc1-突变体Tx1与早先鉴定的H99clc1-菌株Mlac3发现,JEC21CLC1同样决定铜离子的吸收。Tx1中丧失的漆酶活力可以通过外源Cu2+的加入得以恢复,而漆酶基因LAC1的转录与野生型相比没有明显变化,此外铜离子转运蛋白基因CTR4在Tx1中的转录水平有着明显的上调,并且不能被外源铜离子抑制--这说明Tx1细胞中缺乏铜离子。另外实验发现,Tx1对1mol/L Na+或者K+十分敏感,生长受到明显的抑制,而Mlac3则不受影响。同时,与Mlac3不同,Tx1在pH8.0的条件下仍然能够正常生长。实验表明,CLC1在新型隐球酵母不同变种中对于Cu2+存在相同调节作用,但是对于Na+、K+、H+的调节作用在JEC21与H99菌株间存在差异。这个工作对于解释H99和JEC21间的毒力差异提供了证据。

Cryptococcus neoformans has two varieties,var.grubii and var.neoformans.Two well-studied strains H99（serotype A） and JEC21（serotype D） are representatives of var.grubii and var.neoformans respectively.The virulence of the two strains is different which provides an ideal model for researching the evolution of virulence.By comparing the JEC21-derived clc1-mutant strain Tx1 and previously reported H99-derived clc1-mutant strain Mlac3,it was found that CLC1 also participated in copper uptake in JEC21.The demolished laccase activity in Tx1 strain was restored with exogenous copper,while the transcription of the laccase gene LAC1 was at a comparable level to JEC21.Compared with the wild-type strain,the transcription of the copper transporter gene CTR4 was highly upregulated in Tx1 which was not repressed by exogenous copper obviously.These data revealed a lack of intracellular copper in Tx1 cells.In contrast,Tx1 was shown to be hypersensitive to 1mol/L Na＋/K＋,while Mlac3 was not.Tx1 was viable under pH8.0 condition,whereas Mlac3 was sensitive to pH8.0.The results demonstrated that CLC1 shared a common role in Cu2＋ homeostasis,but divergent roles in regulating Na＋,K＋ and H＋ between H99 and JEC21.This work may provide support to the virulence difference between H99 and JEC21.