Characterization of Cryptococcus neoformans variety gattii SOD2 reveals distinct roles of the two superoxide dismutases in fungal biology and virulence
Characterization of Cryptococcus neoformans variety gattii SOD2 reveals distinct roles of the two superoxide dismutases in fungal biology and virulence
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DOI：
10.1111/j.1365-2958.2005.04503.x
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发表时间：
2005-03-01
影响因子：
3.6
通讯作者：
Chaturvedi, S
中科院分区：
文献类型：
--
作者：
Narasipura, SD;Chaturvedi, V;Chaturvedi, S
我们研究了超氧化物歧化酶（SODs）的胶囊化酵母新生隐球菌（Cn）变种gattii的线粒体MnSOD（SOD 2）在真菌生物学和毒力的作用进行了分析。从Cn粘粒文库中克隆了SOD 2，通过同源重组构建了sod 2突变株和sod 2 + SOD 2重组株，并将sod 1突变株中的SOD 2替换为sod 2：：HYG等位基因，构建了两个sod 1 sod 2双突变株。SOD 2蛋白（SOD 2 p）编码225个氨基酸，与其它真菌SOD 2 ps的同源性为36 - 66%。SOD 2缺失使Cn在37 ℃、19 - 20%氧气（正常空气）中高度生长缺陷，并且在抗氧化剂抗坏血酸存在下将氧气限制为1.3%也可以逆转这种缺陷。sod 2突变体在37 ℃和30 ℃抗霉素A存在下积累了显著更多的活性氧（ROS），表明SOD 2 p是Cn对抗超氧阴离子（O-2（.-））的主要防御。在线粒体中。sod 2也非常容易受到氧化还原循环剂、高盐和营养限制的影响。sod 2突变体在鼻内感染的小鼠中是无毒的，而在静脉内感染的小鼠中其毒力明显减弱。sod 2突变株的毒力缺陷可能与其在高氧环境中的生长缺陷有关，而不是由于对吞噬细胞氧化杀伤的敏感性增加所致。sod 1 sod 2双突变体在小鼠体内无毒力。此外，sod 1 sod 2双突变体表现出显着减少其他已知的Cn毒力因子的活动，他们更容易受到中性粒细胞杀伤比sod 2单突变体。以前，我们报道过sod 1突变体在小鼠中的减毒是由于对吞噬细胞杀伤的敏感性增强，以及一些毒力因子活性的降低。因此，SOD 1 p和SOD 2 p在Cn var的生物学和毒力中发挥不同的作用。gattii通过独立的行动模式。
We studied superoxide dismutases (SODs) in the encapsulated yeast Cryptococcus neoformans (Cn) variety gattii to analyse the role of mitochondrial MnSOD (SOD2) in fungal biology and virulence. SOD2 was cloned from a Cn cosmid library, sod2 mutant and sod2+ SOD2 reconstituted strains were constructed by homologous recombination, and two sod1sod2 double mutants were constructed by replacing SOD2 in the sod1 mutant with the sod2::HYG allele. The SOD2 protein (SOD2p) encoded 225 amino acids, with 36 - 66% identity with other fungal SOD2ps. SOD2 deletion rendered Cn highly growth-defective at 37degreesC in 19 - 20% oxygen (normal air), and this defect was reversed by limiting oxygen to 1.3% as well in the presence of antioxidant, ascorbic acid. The sod2 mutant accumulated significantly more reactive oxygen species (ROS) at 37degreesC as well at 30degreesC in the presence of antimycin A, suggesting that SOD2p is the primary defence of Cn against the superoxide anion (O-2(.-)) in the mitochondria. The sod2 was also highly susceptible to redox-cycling agents, high salt and nutrient limitations. The sod2 mutant was avirulent in intranasally-infected mice and markedly attenuated in its virulence in intravenously infected mice. The virulence defect of sod2 mutant appeared related to its growth defects in high oxygen environment, but not resulting from increased sensitivity to oxidative killing by phagocytes. The sod1sod2 double mutants were avirulent in mice. Additionally, sod1sod2 double mutants showed a marked reduction in the activities of other known Cn virulence factors; and they were more susceptible to PMN killing than was the sod2 single mutant. Previously, we reported that the attenuation of sod1 mutant in mice was resulting from enhanced susceptibility to phagocyte killing, combined with a reduction in the activities of a number of virulence factors. Thus, SOD1p and SOD2p play distinct roles in the biology and virulence of Cn var. gattii via independent modes of action.