Development Of Tools For Molecular Study Of Cryptococcus

隐球菌分子研究工具的开发

• 批准号: 6808806
• 负责人: Kyung Kwon-Chung
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6808806
• 关键词: Cryptococcus neoformans; electroporation; fungal genetics; gene deletion mutation; gene expression; genetic manipulation; method development; molecular cloning; nucleic acid sequence; plasmids; polymerase chain reaction; transfection /expression vector; urease

Although there is much interest in Cryptococcal research, tools for genetic analysis in C. neoformans are less well developed than in Saccharomyces cerevisiae and Schizosaccharomyces pombe. By the end of 2003, the genomic sequence of C. neoformans will be finished and the function of genes can be analysed readily by using the sequence data as well as by gene disruptions. In order to effectively identify the genes disrupted randomly and elucidate their functions, a tagging system in deletion construct will be needed. There are several tagging systems that have been successfully used in bacteria as well as in fungi such as transposon or signature tagging methods (STM). These methods have not been successfully used in C. neoformans due to the lack of controlable transposon in C. neoformans as well as a lack of randomness in STM system. In 2003,we have investigated Agrobacterium mediated transformation system in C. neoformans in serotype D and found that single insertions occur randomly in most transformations. The frequency of transformation could be elavated significantly by using adenine auxotrophs. The Agrobacterium mediated transformation, therefore, will be most useful for gene disruptions at a large scale in C. neoformans. We also investigated the molecular bases of urease production in C. neoformans. Urease activity is one of the hallmarks of C. neoformans and clinical microbiology laboratories routinely use urease activity of yeasts as one of the diagnostic criteria for C. neoformans. Clinical isolates produce large amounts of urease and urease negative isolates causing infection in humans are extremely rare. Urease is a metalloenzyme that enables the hydrolysis of urea to ammonia and carbamate and under physiological conditions results in an increase in pH. The latter property is used as a detection tool for the tentative identification of C. neoformans. Such pH increases caused by urease, in other organisms, have been associated with survival in animal tissues and the cause of tissue damage induced by the production of ammonium hydroxide. Furthermore, urease is postulated to play a pivotal role in allowing C. neoformans to convert urea to usable nitrogen in its ecological niche. The exact role of urease in Cryptococcal pathogenesis, however, is unknown. In bacterial pathogenesis, the enzyme appears to alter host immune function by increasing microenvironmental pH at the infection site. Inhibition of urease activity has been reported to correlate with significant decreases in eliciting immune defense cells in the lungs of mice. Further investigation into the role of urease in crytptococcosis and other fungal infections is warranted even though urease-negative mutants are still pathogenic in animal models. We have recently obtained a urease-negative, serotype A isolate of C. neoformans from an immunocompromised patient. Using electroporative protocols we successfully complemented the urease-negative phenotype of the clinical isolate with a genomic library of the strain H-99, a clinical serotype A isolate of C. neoformans. Several thousand transformants were screened based on a selectable nutritional marker. An analysis of the episomes harbored by the tansformants identified a new gene of Cryptococcus neoformans that complemented the urease deficiency of the fungus. A sequence analysis of this gene, named CnSUDA, exhibited significant homology to a conserved family of suppressor proteins known to be involved in chromosome scaffolds facilitating mitotic segregation in other organisms. Preliminary data suggests that expression of SUDA allowed the secretion of urease by the organism. The interaction of scaffold proteins with histones in the nucleus is known to affect several functional aspects of cell physiology from structural aspects involving chromosomal condensation during cell division to gene regulation via transcriptional control. It is possible that SUDA may affect the structural integrity of the cell wall influencing secretion of urease.

尽管对隐球菌研究有很大的兴趣，但新近梭菌的遗传分析工具的发达工具不如酿酒酵母和scosacachomece pombe发达。到2003年底，将完成新梭菌的基因组序列，并且可以通过基因数据和基因破坏来轻松分析基因的功能。为了有效地确定被随机破坏并阐明其功能的基因，需要在删除构建体中的标记系统。在细菌以及真菌中成功使用了几种标记系统，例如转座子或签名标记方法（STM）。这些方法由于缺乏可控制的转座子以及Neoformans以及STM系统中缺乏随机性，因此未成功使用这些方法。在2003年，我们研究了血清型D中的Neoformans中的农杆菌介导的转化系统，发现在大多数转化中，单个插入都随机发生。通过使用腺嘌呤合子可观，可以显着阐明转化的频率。因此，农杆菌介导的转化对于新生梭菌中的大规模中的基因破坏最有用。我们还研究了Neoformans脲酶产生的分子碱基。尿素酶活性是Neoformans的标志之一，临床微生物实验室通常将酵母的尿素酶活性用作Neoformans的诊断标准之一。临床分离株产生大量的尿素酶，尿素酶阴性分离株引起人类感染非常罕见。脲酶是一种金属酶，可以使尿素水解为氨和氨基甲酸酯，并且在生理条件下会导致pH值增加。后一种特性用作暂定识别新梭菌的检测工具。在其他生物体中，由于尿素酶引起的这种pH值与动物组织中的存活以及氢氧化铵产生引起的组织损伤的原因有关。此外，假定尿素酶在允许Neoformans的尿素中发挥着关键作用，可以将尿素转化为其生态位中可用的氮。然而，尿素酶在隐球菌发病机理中的确切作用尚不清楚。在细菌发病机理中，该酶似乎通过增加感染部位的微环境pH来改变宿主免疫功能。据报道，尿素酶活性的抑制作用与引起小鼠肺中免疫防御细胞的显着降低相关。即使在动物模型中仍具有致病性，也有必要进一步研究尿素酶在裂孢菌病和其他真菌感染中的作用和其他真菌感染。 最近，我们获得了来自免疫功能低下的患者的尿素酶阴性A分离菌的血清型A分离。使用电抛剂方案，我们成功补充了临床分离株的尿素酶阴性表型，该菌株H-99是临床血清型A分离菌C. Neoformans。根据可选的营养标记筛选了几千个转化子。对革命剂所含有的偶发性的分析确定了一个新的加密环球球菌基因，该基因补充了真菌的尿素不足。 该基因的序列分析（称为CNSUDA）与已知参与染色体支架的保守抑制蛋白家族具有重要的同源性，从而促进了其他生物体中有丝分裂的隔离。初步数据表明，Suda的表达允许生物体分泌尿素。已知支架蛋白与组蛋白与组蛋白之间的相互作用已知会影响细胞生理的几个功能方面，从涉及细胞分裂期间染色体凝结到基因调节的结构方面通过转录控制。 SUDA可能会影响细胞壁影响尿素酶分泌的结构完整性。