Novel chimeric spermidine synthase-saccharopine dehydrogenase gene (SPE3-LYS9) in the human pathogen Cryptococcus neoformans
Novel chimeric spermidine synthase-saccharopine dehydrogenase gene (SPE3-LYS9) in the human pathogen Cryptococcus neoformans
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DOI：
10.1128/ec.3.3.752-763.2004
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发表时间：
2004-06-01
期刊：
影响因子：
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通讯作者：
McCusker, JH
中科院分区：
文献类型：
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作者：
Kingsbury, JM;Yang, ZG;McCusker, JH
新型隐球菌LYS 9基因（编码蔗糖脱氢酶）被克隆，并发现与SPE 3（编码亚精胺合酶）的进化保守嵌合体的一部分。构建了spe 3-lys 9、spe 3-LYS 9和SPE 3-lys 9突变体，它们分别是赖氨酸和亚精胺、亚精胺和赖氨酸营养缺陷型的。因此，SPE 3-LYS 9编码功能性亚精胺合酶和糖嘌呤脱氢酶基因产物。与酿酒酵母spe 3突变株相比，精胺不能满足新生隐球菌spe 3-LYS 9突变株的多胺营养缺陷型。spe 3-LYS 9突变体的体外表型包括减少的荚膜和黑色素产生和生长速率，而SPE 3-lys 9突变体在30 ℃下生长缓慢，在丰富的培养基中对温度敏感，并且在赖氨酸饥饿时死亡。与体外的蔗糖脱氢酶和亚精胺合酶的重要性一致，spe 3-lys 9突变体是无毒的，不能在体内生存，这两个功能单独贡献的毒力。SPE 3-LYS 9的mRNA水平表现出受外源亚精胺或赖氨酸或饥饿亚精胺或赖氨酸的影响的证据很少，因此，任何调节都可能是转录后的。在S.酿酒酵母C.新变型SPE 3-LYS 9 cDNA互补lys 9突变体，但不互补spe 3突变体。而在S.酿酒酵母的截短的基因产物，仅由C. neoformans SPE 3，补充了SPE 3突变体，提示可能的调控模式。因此，我们确定并描述了一种新的嵌合SPE 3-LYS 9基因，它可能连接亚精胺和赖氨酸的生物合成在C neoformans。
The Cryptococcus neoformans LYS9 gene (encoding saccharopine dehydrogenase) was cloned and found to be part of an evolutionarily conserved chimera with SPE3 (encoding spermidine synthase). spe3-lys9, spe3-LYS9, and SPE3-lys9 mutants were constructed, and these were auxotrophic for lysine and spermidine, spermidine, and lysine, respectively. Thus, SPE3-LYS9 encodes functional spermidine synthase and saccharopine dehydrogenase gene products. In contrast to Saccharomyces cerevisiae spe3 mutants, the polyamine auxotrophy of C neoformans spe3-LYS9 mutants was not satisfied by spermine. In vitro phenotypes of spe3-LYS9 mutants included reduced capsule and melanin production and growth rate, while SPE3-lys9 mutants grew slowly at 30degreesC, were temperature sensitive in rich medium, and died upon lysine starvation. Consistent with the importance of saccharopine dehydrogenase and spermidine synthase in vitro, spe3-lys9 mutants were avirulent and unable to survive in vivo and both functions individually contributed to virulence. SPE3-LYS9 mRNA levels showed little evidence of being influenced by exogenous spermidine or lysine or starvation for spermidine or lysine; thus, any regulation is likely to be posttranscriptional. Expression in S. cerevisiae of the full-length C. neoformans SPE3-LYS9 cDNA complemented a lys9 mutant but not a spe3 mutant. However, expression in S. cerevisiae of a truncated gene product, consisting of only C. neoformans SPE3, complemented a spe3 mutant, suggesting possible modes of regulation. Therefore, we identified and describe a novel chimeric SPE3-LYS9 gene, which may link spermidine and lysine biosynthesis in C neoformans.