Kraus Peter R, Nichols Connie B, Heitman Joseph
Department of Molecular Genetics and Microbiology, 322 CARL Building, Box 3546, Research Drive, Duke University Medical Center, Durham, NC 27710, USA.
Eukaryot Cell. 2005 Jun;4(6):1079-87. doi: 10.1128/EC.4.6.1079-1087.2005.
The function of calcium as a signaling molecule is conserved in eukaryotes from fungi to humans. Previous studies have identified the calcium-activated phosphatase calcineurin as a critical factor in governing growth of the human pathogenic fungus Cryptococcus neoformans at mammalian body temperature. Here, we employed insertional mutagenesis to identify new genes required for growth at 37 degrees C. One insertion mutant, cam1-ts, that displayed a growth defect at 37 degrees C and hypersensitivity to the calcineurin inhibitor FK506 at 25 degrees C was isolated. Both phenotypes were linked to the dominant marker in genetic crosses, and molecular analysis revealed that the insertion occurred in the 3' untranslated region of the gene encoding the calcineurin activator calmodulin (CAM1) and impairs growth at 37 degrees C by significantly reducing calmodulin mRNA abundance. The CAM1 gene was demonstrated to be essential using genetic analysis of a CAM1/cam1Delta diploid strain. In the absence of calcineurin function, the cam1-ts mutant displayed a severe morphological defect with impaired bud formation. Expression of a calmodulin-independent calcineurin mutant did not suppress the growth defect of the cam1-ts mutant at 37 degrees C, indicating that calmodulin promotes growth at high temperature via calcineurin-dependent and -independent pathways. In addition, a Ca2+-binding-defective allele of CAM1 complemented the 37 degrees C growth defect, FK506 hypersensitivity, and morphogenesis defect of the cam1-ts mutant. Our findings reveal that calmodulin performs Ca2+- and calcineurin-independent and -dependent roles in controlling C. neoformans morphogenesis and high-temperature growth.
钙作为信号分子的功能在从真菌到人类的真核生物中是保守的。先前的研究已确定钙激活磷酸酶钙调神经磷酸酶是控制人类致病真菌新型隐球菌在哺乳动物体温下生长的关键因素。在此，我们采用插入诱变来鉴定在37℃生长所需的新基因。分离出一个插入突变体cam1-ts，它在37℃表现出生长缺陷，在25℃对钙调神经磷酸酶抑制剂FK506高度敏感。这两种表型在遗传杂交中都与显性标记相关联，分子分析表明插入发生在编码钙调神经磷酸酶激活剂钙调蛋白（CAM1）的基因的3'非翻译区，并通过显著降低钙调蛋白mRNA丰度损害37℃下的生长。通过对CAM1/cam1Delta二倍体菌株的遗传分析证明CAM1基因是必需的。在没有钙调神经磷酸酶功能的情况下，cam1-ts突变体表现出严重的形态缺陷，芽形成受损。不依赖钙调蛋白的钙调神经磷酸酶突变体的表达不能抑制cam1-ts突变体在37℃的生长缺陷，表明钙调蛋白通过依赖和不依赖钙调神经磷酸酶的途径促进高温下的生长。此外，CAM1的一个Ca2+结合缺陷等位基因补充了cam1-ts突变体的37℃生长缺陷、FK506超敏反应和形态发生缺陷。我们的研究结果表明，钙调蛋白在控制新型隐球菌形态发生和高温生长中发挥着不依赖和依赖Ca2+及钙调神经磷酸酶的作用。
