Role of Mg2+ and pH in the modification of Salmonella lipid A after endocytosis by macrophage tumour cells
Role of Mg2+ and pH in the modification of Salmonella lipid A after endocytosis by macrophage tumour cells
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DOI：
10.1111/j.1365-2958.2004.04409.x
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发表时间：
2005-01-01
影响因子：
3.6
通讯作者：
Raetz, CRH
中科院分区：
文献类型：
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作者：
Gibbons, HS;Kalb, SR;Raetz, CRH
鼠伤寒沙门氏菌的脂质 A 被额外的酰基和/或极性取代基共价修饰，以响应 PhoP/PhoQ 和/或 PmrA/PmrB 信号系统的激活，这些信号系统分别由低 Mg2+ 浓度和弱酸性 pH 下的生长诱导。尽管这些条件被认为存在于巨噬细胞吞噬溶酶体内，但没有提出内吞作用后脂质 A 修饰的直接证据。为了解决这个问题，我们在 P-32(i) 存在的情况下在 RAW264.7 细胞内培养鼠伤寒沙门氏菌，然后分离标记的脂质 A 级分，发现该级分被磷酸乙醇胺、氨基阿拉伯糖、2-羟基肉豆蔻酸酯和/或棕榈酸酯部分广泛衍生化。在组织培养基中生长的鼠伤寒沙门氏菌合成的脂质 A 分子缺乏所有这些取代基，但 2-羟基肉豆蔻酸链除外，该链仍然存在。使用限定的基本培养基来模拟胞内 pH 值和内体的 Mg2+ 浓度，我们发现在酸性、低 Mg2+ 培养基中生长的鼠伤寒沙门氏菌的脂质 A 与从 RAW264.7 细胞内化的细菌中分离出的脂质 A 非常相似。鼠伤寒沙门氏菌脂质 A 修饰的一个子集是由单独的低 Mg2+ 诱导的。大肠杆菌 K-12 W3110 修饰其脂质 A 分子以响应酸性但不低 Mg2+ 条件下的生长。在高 Mg2+、弱碱性培养基中生长会抑制大多数脂质 A 修饰，鼠伤寒沙门氏菌中的 2-羟基肉豆蔻酸除外。虽然lpxO转录受到低Mg2+生长的刺激，但鼠伤寒沙门氏菌中含有2-羟基肉豆蔻酸酯的脂质A的生物合成不依赖于PhoP/PhoQ和PmrA/PmrB。我们的标记方法应该适用于不同细菌内吞作用诱导的脂质 A 修饰的研究。
Lipid A of Salmonella typhimurium is covalently modified with additional acyl and/or polar substituents in response to activation of the PhoP/PhoQ and/or PmrA/PmrB signalling systems, which are induced by growth at low Mg2+ concentrations and mild acid pH respectively. Although these conditions are thought to exist within macrophage phagolysosomes, no direct evidence for lipid A modification after endocytosis has been presented. To address this issue, we grew S. typhimurium inside RAW264.7 cells in the presence of P-32(i), and then isolated the labelled lipid A fraction, which was found to be extensively derivatized with phosphoethanolamine, aminoarabinose, 2-hydroxymyristate and/or palmitate moieties. S. typhimurium grown in tissue culture medium synthesized lipid A molecules lacking all these substituents with the exception of the 2-hydroxymyristate chain, which was still present. Using defined minimal media to simulate the intracellular pH and Mg2+ concentrations of endosomes, we found that lipid A of S. typhimurium grown in an acidic, low-Mg2+ medium closely resembled lipid A isolated from bacteria internalized by RAW264.7 cells. A subset of S. typhimurium lipid A modifications were induced by low Mg2+ alone. Escherichia coli K-12 W3110 modified its lipid A molecules in response to growth under acidic but not low-Mg2+ conditions. Growth in a high-Mg2+, mildly alkaline medium resulted in suppression of most lipid A modifications with the exception of the 2-hydroxymyristate in S. typhimurium. Although lpxO transcription was stimulated by growth on low Mg2+, the biosynthesis of lipid A species containing 2-hydroxymyristate was independent of PhoP/PhoQ and PmrA/PmrB in S. typhimurium. Our labelling methods should be applicable to studies of lipid A modifications induced by endocytosis of diverse bacteria.