极长链脂肪酸的合成缺陷对酵母细胞膜的稳定性和多烯类药物敏感性的影响

Effects of synthetic defects of very long-chain fatty acids on cell membrane stability and sensitivity of polyenes in yeast

摘要:

【背景】脂肪酸延长酶家族参与脂肪酸代谢具有真核生物的高度保守性，且与膜脂的代谢密切相关。但细胞极长链脂肪酸(Very long-chain fatty acid，VLCFA)的合成缺陷对膜的稳定性及多烯类药物的敏感性影响并不完全明晰。【目的】探究细胞VLCFA延长酶ELO1、ELO2和ELO3的作用及功能。【方法】研究脂肪酸延长酶缺陷型elo1?、elo2?和elo3?对多烯类药物两性霉素B (Amphotericin B，AmB)、制霉菌素(Nystatin，Ny)及唑类硝酸益康唑(Econazole nitrate，Eco)的响应，检测不同酵母细胞的麦角固醇，检测其对Na+的响应及胞内钠钾离子水平。【结果】发现细胞VLCFA延长酶ELO2和ELO3缺陷后对AmB高度敏感；VLCFA延长酶缺陷突变株elo2?和elo3?对其它多烯类药物Ny及唑类药物Eco也十分敏感；细胞膜不饱和脂肪酸增加也会改变膜的稳定性，实验结果表明外源油酸(Oleic acid，OLA)增加了elo2?和elo3?突变体的AmB敏感性；相对野生型BY4741和elo1?，缺陷菌株elo2?和elo3?中麦角固醇的含量有显著下降；钠钾离子平衡是维护细胞正常生理的必要条件，也是检测细胞膜稳定性的重要参数，发现VLCFA的合成缺陷菌株对高浓度的NaCl比野生型菌株更敏感，使用ICP-AES检测不同浓度AmB胁迫下细胞内钠钾离子水平，也显示VLCFA延长酶缺陷菌株中，钠水平表现出上升趋势，并且细胞内钾含量明显降低。【结论】细胞VLCFA的合成缺陷会导致细胞膜更脆弱、稳定性下降，从而提高真菌对多烯类药物的敏感性，也表明脂肪酸延长酶是潜在的抗真菌治疗靶点。

Abstract:

[Background] Very long-chain fatty acids (VLCFA) are involved in the synthesis of membrane lipid sphingolipids and closely related to the metabolism of membrane lipid ergosterol and phospholipids. Defects in the synthesis of VLCFA in cells can damage the stability of cell membranes and thus enhance the fungal susceptibility to polyene-based drugs. However, the effect of the synthesis defects of VLCFA on the membrane stability and the sensitivity to polyene drugs is not entirely clear. [Objective] The role and function of the cell long-chain fatty acid elongases ELO1, ELO2 and ELO3. [Methods] The response of mutants with the synthesis defects of VLCFA, elo1?, elo2? and elo3?, to amphotericin B (AmB), one of the polyene drugs, and other drugs, such as nystatin (Ny) and econazole nitrate (Eco) was investigated. The ergosterol contents of different yeast cells were detected, and its response to Na+ and intracellular sodium and potassium levels was examined. [Results] We found that elo2? and elo3? mutants were highly sensitive to AmB. In addition, these two mutants were also sensitive to other drugs, such as Ny and Eco. Generally increased unsaturated fatty acids affect membrane stability. Our results showed that exogenous oleic acid (OLA) increased the sensitivity of elo2? and elo3? mutants to AmB. Compared with those of the wild-type and elo1? mutant, the ergosterol contents of the defective strains elo2? and elo3? decreased significantly. Sodium-potassium ion balance was a necessary condition for maintaining the normal physiology of cells, and was also an important parameter for measuring cell membrane stability. We found that defective strains were more sensitive to high concentrations of NaCl than wild-type strains. The levels of sodium and potassium in cells under different concentrations of AmB were detected by ICP-AES. The data showed that sodium levels were elevated in VLCFA synthesis-deficient strains compared with the wild-type strain. The potassium content in mutants was decreased significantly. [Conclusion] The synthetic defects of VLCFA could lead to the more fragile cell membrane and lower stability, thus increasing the sensitivity of fungi to polyenes. It suggests that fatty acid elongase could be the target of potential antifungal therapy.