新生隐球菌半胱氨酸转运蛋白Mup1鉴定及其表达调控研究

Identification and transcriptional regulation of l-cysteine transporter Mup1 in Cryptococcus neoformans

摘要:

【目的】 鉴定新生隐球菌(Cryptococcus neoformans)的半胱氨酸转运蛋白及其对致病性的影响。【方法】 构建候选基因敲除株,检测突变株以半胱氨酸为唯一硫源的生长情况;检测半胱氨酸转运蛋白Mup1对新生隐球菌毒力因子表达和不同胁迫条件下生长的影响;通过新生隐球菌大蜡螟(Galleria mellonella)和小鼠感染模型分析Mup1对致病性的影响;通过转录组分析和酵母单杂交研究硫代谢核心转录因子Cys3与Mup1的调控关系。【结果】 Mup1具有转运半胱氨酸、胱氨酸、胱硫醚和同型半胱氨酸的能力。基因MUP1缺失不影响毒力因子表达和细胞对应激的反应。大蜡螟和小鼠隐球菌感染模型表明Mup1对新生隐球菌的致病性无显著影响。转录组分析和酵母单杂交实验显示Cys3可能间接调控MUP1的转录。【结论】 新生隐球菌Mup1具有转运半胱氨酸、胱氨酸、胱硫醚和同型半胱氨酸的功能,但不影响致病性,基因转录可能受Cys3的间接调控。

Abstract:

[Objective] To identify the l-cysteine transporter of Cryptococcus neoformans and study the effect of the transporter on the fungal pathogenicity. [Methods] We constructed the candidate gene-deleted strains, and examined the growth of the mutants with l-cysteine as the sole sulfur source. We then tested the effect of the l-cysteine transporter Mup1 on the expression of virulence factors and the growth of C. neoformans under different stress conditions. Further, we used the Galleria mellonella infection model and the mouse infection model to explore the effect of MUP1 on the pathogenicity of C. neoformans. RNA-Seq and yeast one-hybrid assay were employed to investigate the regulatory relationship between the master transcription factor Cys3 and Mup1. [Results] Mup1 could transport l-cysteine, l-cystine, dl-cystathionine, and l-homocysteine. The deletion of gene MUP1 did not affect the expression of virulence factors or the cellular response to stress. Mup1 had no significant effect on the pathogenicity of C. neoformans in G. mellonella and mice. Cys3 might indirectly regulate the transcription of MUP1. [Conclusion] Mup1 is the transporter of l-cysteine, l-cystine, dl-cystathionine, and l-homocysteine in C. neoformans. It does not affect the pathogenicity of C. neoformans and may be indirectly regulated by Cys3.