Swansea University Medical School, Swansea University, Swansea, UK.
School of Chemistry, Cardiff University, Cardiff, UK.
FASEB J. 2021 Jun;35(6):e21640. doi: 10.1096/fj.202100164R.
Certain species of pathogenic bacteria damage tissues by secreting cholesterol-dependent cytolysins, which form pores in the plasma membranes of animal cells. However, reducing cholesterol protects cells against these cytolysins. As the first committed step of cholesterol biosynthesis is catalyzed by squalene synthase, we explored whether inhibiting this enzyme protected cells against cholesterol-dependent cytolysins. We first synthesized 22 different nitrogen-containing bisphosphonate molecules that were designed to inhibit squalene synthase. Squalene synthase inhibition was quantified using a cell-free enzyme assay, and validated by computer modeling of bisphosphonate molecules binding to squalene synthase. The bisphosphonates were then screened for their ability to protect HeLa cells against the damage caused by the cholesterol-dependent cytolysin, pyolysin. The most effective bisphosphonate reduced pyolysin-induced leakage of lactate dehydrogenase into cell supernatants by >80%, and reduced pyolysin-induced cytolysis from >75% to <25%. In addition, this bisphosphonate reduced pyolysin-induced leakage of potassium from cells, limited changes in the cytoskeleton, prevented mitogen-activated protein kinases cell stress responses, and reduced cellular cholesterol. The bisphosphonate also protected cells against another cholesterol-dependent cytolysin, streptolysin O, and protected lung epithelial cells and primary dermal fibroblasts against cytolysis. Our findings imply that treatment with bisphosphonates that inhibit squalene synthase might help protect tissues against pathogenic bacteria that secrete cholesterol-dependent cytolysins.
某些致病细菌通过分泌胆固醇依赖性细胞溶素来破坏组织，这些细胞溶素在动物细胞的质膜上形成孔。然而，降低胆固醇可以保护细胞免受这些细胞溶素的伤害。由于胆固醇生物合成的第一步是由鲨烯合酶催化的，我们探讨了抑制这种酶是否可以保护细胞免受胆固醇依赖性细胞溶素的侵害。我们首先合成了 22 种不同的含氮双膦酸盐分子，这些分子旨在抑制鲨烯合酶。使用无细胞酶测定法定量鲨烯合酶抑制作用，并通过双膦酸盐分子与鲨烯合酶结合的计算机建模进行验证。然后筛选这些双膦酸盐分子保护 HeLa 细胞免受胆固醇依赖性细胞溶素——溶胞素损害的能力。最有效的双膦酸盐将溶胞素诱导的乳酸脱氢酶漏出到细胞上清液中的比例降低了>80%，并将溶胞素诱导的细胞溶解从>75%降低到<25%。此外，这种双膦酸盐还减少了溶胞素诱导的钾从细胞中漏出，限制了细胞骨架的变化，防止了丝裂原激活的蛋白激酶细胞应激反应，并降低了细胞内胆固醇。双膦酸盐还可以保护细胞免受另一种胆固醇依赖性细胞溶素——链球菌溶血素 O 的侵害，并保护肺上皮细胞和原代真皮成纤维细胞免受细胞溶解。我们的研究结果表明，用抑制鲨烯合酶的双膦酸盐治疗可能有助于保护组织免受分泌胆固醇依赖性细胞溶素的致病细菌的侵害。
