Functions of Cryptococcus neoformans mating type loci

新型隐球菌交配型位点的功能

• 批准号: 7058258
• 负责人: JOSEPH HEITMAN
• 金额: $ 37.6万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-15 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7058258
• 关键词: Basidiomycetes; Cryptococcus neoformans; evolution; fungal genetics; gene mutation; genetic strain; laboratory mouse; laboratory rabbit; life cycle; molecular cloning; nucleic acid sequence; polymerase chain reaction; reproduction; southern blotting; virulence

Cryptococcus neoformans is a fungal pathogen that infects the human brain. The organism has a defined sexual cycle involving haploid MATalpha and MATa strains, and the MATalpha locus has been linked to virulence and differentiation. Most environmental and clinical isolates are MATalpha mating type, and MATalpha strains are more virulent than congenic MATa strains. MATalpha strains also haploid fruit and produce filaments and infectious basidiospores when nitrogen limited. Thus, the structure and function of the mating type loci and their links to virulence and ecology are of considerable interest. Moore and Edman used difference cloning to identify part of the MATalpha locus encoding a mating pheromone. Later, Wickes and colleagues identified a Ste12 homolog that is MATalpha-specific but not encoded by the known MATalpha region. STE12alpha is required for virulence of a congenic lab adapted serotype D strain but is dispensable for virulence of a pathogenic serotype A clinical isolate. To establish functions of the mating type loci, we propose to clone, sequence, and mutate the MAT loci from three divergent C. neoformans varieties. MATalpha and MATa strains of each variety were known or identified in our lab and MATalpha and MATa specific genes were cloned. Six BAC libraries were constructed to isolate clones spanning each MAT locus. Sequencing of the serotype A and D MATalpha and MATa loci is in progress. These loci are unusually large (greater than 100 kb) with more than a dozen genes encoding three pheromones and a pheromone receptor, homologs of Ste20, Stel1, Ste12, and a Zn2+ finger protein. Other genes encode a myosin, a favoprotein, and a translation factor. Our studies reveal the MATalpha and MATa loci encode diverged alleles of the same genes and have extensively rearranged during evolution. Mutation of individual MATalpha locus genes confers defects in mating, fruiting, and virulence. A haploid strain lacking a 50 kb region of the MATalpha locus was inviable, indicating one or more genes in the region is essential. A diploid a/delta strain deleted for this part of the MATalpha locus was still self-filamentous like an a/alpha diploid, and a/a and alpha/alpha diploids were not self- filamentous, excluding a ploidy-dependent model. Our preliminary results support a locus-dependent model. We have discovered a novel, linked region of the MATalpha locus encoding a homeodomain homolog (Hdp1alpha) and a candidate transcriptional regulator (Rum1alpha), and we will establish the functions of these genes. We also propose to develop DNA based methods to test if MAT locus alterations occur naturally and affect fertility or virulence. These studies will provide insights into the role of MAT loci and sexual cycles in fungal differentiation and virulence.

新型隐球菌是一种感染人类大脑的真菌病原体。 该生物体具有确定的性周期，涉及单倍体MAT α和MAT α菌株，MAT α基因座与毒力和分化有关。 大多数环境和临床分离株是MAT α交配型，MAT α菌株比同类MATa菌株毒力更强。当氮限制时，MAT α菌株也产生单倍体果实并产生细丝和感染性担孢子。 因此，交配型基因座的结构和功能及其与毒力和生态学的联系是相当感兴趣的。 摩尔和埃德曼使用差异克隆来鉴定编码交配信息素的MAT α基因座的一部分。 后来，Wickes及其同事鉴定了一种Ste 12同源物，它是MAT α特异性的，但不是由已知的MAT α区域编码的。 STE 12 α是同类实验室适应血清型D菌株毒力所必需的，但对致病性血清型A临床分离株的毒力不敏感。为了建立交配型基因座的功能，我们建议从三个不同的C.新变型 本实验室已知或鉴定了每个品种的MAT α和MAT a菌株，并克隆了MAT α和MAT a特异性基因。 构建六个BAC文库以分离跨越每个MAT基因座的克隆。 血清型A和D MAT α和MAT a基因座的测序正在进行中。 这些基因座是非常大的（大于100 kb）与十几个基因编码三个信息素和信息素受体，同源的Ste 20，Stel 1，Ste 12，和锌2+指蛋白。其他基因编码肌球蛋白、favoprotein和翻译因子。 我们的研究揭示了MATalpha和MATa基因座编码相同基因的分歧等位基因，并在进化过程中广泛重排。 个别MAT α基因座基因的突变导致交配、结实和毒力缺陷。缺少MAT α基因座的50 kb区域的单倍体菌株是不存活的，表明该区域中的一个或多个基因是必需的。 缺失MAT α基因座的这一部分的二倍体α/δ菌株仍然像α/α二倍体一样是自丝状的，并且α/α和α/α二倍体不是自丝状的，排除了倍性依赖性模型。 我们的初步结果支持一个位点依赖模型。 我们已经发现了一个新的，连接区的MAT α基因座编码同源结构域同源物（Hdp 1alpha）和候选转录调节因子（Rum 1alpha），我们将建立这些基因的功能。我们还建议开发基于DNA的方法来测试MAT基因座的改变是否自然发生并影响生育力或毒力。这些研究将提供MAT位点和性周期在真菌分化和毒力中的作用的见解。