人类致病菌新型隐球酵母Snf1/AMPK 蛋白激酶调节细胞壁的完整性

Snf1/AMPK protein kinase modulates cell wall integrity in the human pathogenic yeast Cryptococcus neoformans

摘要:

摘要：【目的】Snf1/AMPK在真核生物中是重要的且高度保守的一类蛋白激酶。在新型隐球酵母中，SNF1 基因在调节致病因子的生物合成和细胞毒力方面具有重要作用。本文进一步报道了该基因在维持细胞壁完整方面的新功能，这一功能在其他微生物中未见报道。【方法】利用荧光增白剂染料（Calcofluor white dye）染色,荧光显微观察细胞分离、胞壁完整性；利用恒定流速和压力水流冲击菌落,测定细胞黏附琼脂糖表面能力；在含有十二烷基硫酸钠（Sodium dodecyl sulfate，SDS），刚果红（Congo red）染料和增白剂（Fluorescent Brightener 28）的培养基上观察突变株的生长情况，以验证细胞壁完整性。【结果】SNF1 基因突变菌株对细胞壁抑制剂SDS等敏感，表明细胞壁完整性的损坏；在葡萄糖固体培养基上表现为细胞与琼脂间的黏附力丧失；在热击压力下，该菌株不能正常生长，而这种生长缺陷能够被渗透平衡抑制。【结论】新型隐球酵母SNF1 基因对于维持细胞壁完整性是非常重要的，并且影响细胞与琼脂间黏附作用以及细胞对抗热的能力。

Abstract:

Abstract: [Objective] The Snf1/AMPK family of protein kinases is highly conserved among eukaryotes. Our previous study showed that Cryptococcus neoformans SNF1 played critical roles in the production of virulence factors and virulence itself. In this paper, we report a novel function of SNF1 in cell wall integrity. [Methods] We used Calcofluor white staining epifluorescence microscopy to evaluate the cell wall integrity and cell segregation; tap water with constant flow rate and pressure to wash yeast colonies to evaluate cell-to-agar adhesion capability; growth on Sodium dodecyl sulfate (SDS), Congo red and Fluorescent Brightener 28-containing agar to examine cell wall integrity. [Results] The disruption mutant of SNF1 was sensitive to SDS and Congo red, suggesting impairments in the cell wall. The mutant cells showed abnormal separation, defects in adhesion to agar surface, and growth defects at high temperature which could be suppressed by osmotic stabilization. [Conclusion] C. neoformans SNF1 was essential for cell wall integrity that was likely responsible for normal adhesion of the cells to agar and resistance to heat.