Cholesterol-dependent cytolysins (CDCs) are the distinct class of β-barrel pore-forming toxins (β-PFTs) that attack eukaryotic cell membranes, and form large, oligomeric, transmembrane β-barrel pores. Listeriolysin O (LLO) is a prominent member in the CDC family. As documented for the other CDCs, membrane cholesterol is essential for the pore-forming functionality of LLO. However, it remains obscure how exactly cholesterol facilitates its pore formation. Here, we show that cholesterol promotes both membrane-binding and oligomerization of LLO. We demonstrate cholesterol not only facilitates membrane-binding, it also enhances the saturation threshold of LLO-membrane association, and alteration of the cholesterol-recognition motif in the LLO mutant (LLOT515G-L516G) compromises its pore-forming efficacy. Interestingly, such defect of LLOT515G-L516G could be rescued in the presence of higher membrane cholesterol levels, suggesting cholesterol can augment the pore-forming efficacy of LLO even in the absence of a direct toxin-cholesterol interaction. Furthermore, we find the membrane-binding and pore-forming abilities of LLOT515G-L516G, but not those of LLO, correlate with the cholesterol-dependent rigidity/ordering of the membrane lipid bilayer. Our data further suggest that the line tension derived from the lipid phase heterogeneity of the cholesterol-containing membranes could play a pivotal role in LLO function, particularly in the absence of cholesterol binding. Therefore, in addition to its receptor-like role, we conclude cholesterol can further facilitate the pore-forming, membrane-damaging functionality of LLO by asserting the optimal physicochemical environment in membranes. To the best of our knowledge, this aspect of the cholesterol-mediated regulation of the CDC mode of action has not been appreciated thus far.

中文翻译：

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胆固醇依赖性相互作用和胆固醇介导的脂相异质性在调节李斯特氏菌溶血素 O 孔形成中的纠缠作用。


胆固醇依赖性溶细胞素 (CDC) 是一类独特的 β-桶成孔毒素 (β-PFT)，它攻击真核细胞膜，并形成大的寡聚跨膜 β-桶孔。李斯特菌溶血素 O (LLO) 是 CDC 家族的重要成员。正如其他 CDC 所记录的那样，膜胆固醇对于 LLO 的成孔功能至关重要。然而，胆固醇究竟如何促进其毛孔形成仍不清楚。在这里，我们发现胆固醇促进 LLO 的膜结合和寡聚化。我们证明胆固醇不仅促进膜结合，还提高 LLO 膜结合的饱和阈值，并且 LLO 突变体 (LLOT515G-L516G) 中胆固醇识别基序的改变会损害其成孔功效。有趣的是，LLOT515G-L516G 的这种缺陷可以在膜胆固醇水平较高的情况下得到挽救，这表明即使在没有直接毒素-胆固醇相互作用的情况下，胆固醇也可以增强 LLO 的成孔功效。此外，我们发现 LLOT515G-L516G 的膜结合和成孔能力（而非 LLO）与膜脂双层的胆固醇依赖性刚性/有序性相关。我们的数据进一步表明，源自含胆固醇膜的脂相异质性的线张力可能在 LLO 功能中发挥关键作用，特别是在没有胆固醇结合的情况下。因此，除了其类似受体的作用外，我们得出结论，胆固醇可以通过维持膜中的最佳理化环境，进一步促进 LLO 的成孔、膜损伤功能。 据我们所知，胆固醇介导的 CDC 作用模式调节的这一方面迄今为止尚未得到重视。