新生隐球菌RBP1基因克隆与功能分析

Cloning and functional analysis of RBP1 gene in Cryptococcus neoformans

新生隐球菌是自然界广泛存在的具荚膜的酵母型病原真菌,能侵染人类中枢神经系统引起真菌性脑膜炎,每年导致全球大约18万人死亡。本研究在前期隐球菌交配表达谱的基础上,选择一上调表达的RNA结合蛋白基因(CNAG_04772),进行克隆和功能分析。结果表明该基因全长2 247bp,cDNA全长1 518bp,编码505个氨基酸组成的蛋白,含有2个RNA识别基序RRM1和RRM2,命名为RBP1。基因表达模式分析表明RBP1在隐球菌酵母细胞、担子以及担孢子阶段都有表达,交配菌丝阶段不表达;亚细胞定位分析表明Rbp1蛋白定位于隐球菌的细胞核和细胞质中。与野生型菌株H99相比,敲除突变体菌株能够交配并产生双核菌丝,但丧失产生担孢子的能力,而互补菌株与野生型菌株H99间无显著差异。致病力测定结果显示,敲除突变体菌株致病性显著降低。

Cryptococcus neoformans is a major encapsulated yeast pathogen that often infects the human central nervous system（CNS） to cause meningitis, resulting in over 180 000 deaths worldwide annually. According to the whole genome-wide expression profiles during mating stage of C. neoformans, the CNAG_04772 gene that encodes an RNA binding protein was upregulated during mating process. Here we report the cloning and functional an alyses of the CNAG_04772 gene. Sequence analysis showed that CNAG_04772, named RBP1, has a full length of 2 247 bp. The cDNA coding region of RBP1 contains 1 518 bp of nucleotides that encode a protein consisted of 505 amino acids containing two RNA recognit ion motifs, RRM1 and RRM2. Gene expression pattern analysis showed that RBP1 expresses in the developmental stages such as yeast cell, basidium and basidiospores, but not in mating hyphae. Subcellular localization analysis indicates that Rbp1 is targeted t o cytoplasm and nucleus. Rbp1 was found to be essential for fungal sexual reproduction, as basidiospore production was blocked in bilateral mating between rbp1Δ mutants, even though normal dikaryotic hyphae were observed during mating. Rbp1 is also importa nt for fungal virulence, as virulence of rbp1Δ mutants was significantly attenuated in a murine systemic-infection model.