Department of Infectious Diseases, Genentech, Inc., South San Francisco, California, USA.
Department of Structural Biology, Genentech, Inc., South San Francisco, California, USA.
Antimicrob Agents Chemother. 2018 Mar 27;62(4). doi: 10.1128/AAC.02151-17. Print 2018 Apr.
The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral β-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of growth followed by selection of compounds that induced the extracytoplasmic σ stress response. Mutations in , , and conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolC), which confers high-level resistance to G0507 but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.
外膜是革兰氏阴性细菌的重要结构组成部分，由脂蛋白、脂多糖、磷脂和整合的β-桶膜蛋白组成。一种称为 Lol 系统的专用机制可确保脂蛋白从内膜正确转运到外膜。LolCDE ABC 转运体是内膜成分，对细菌的生存至关重要。在这里，我们报告了一种新型吡咯并嘧啶二酮化合物 G0507，它是在生长抑制剂表型筛选后，通过选择诱导细胞外 σ 应激反应的化合物而被鉴定出来的。突变 、 和 赋予了 G0507 的抗性，表明 LolCDE 是其分子靶标。用 G0507 处理 细胞会导致完全加工的 Lpp（一种外膜脂蛋白）在内膜中积累。使用纯化的蛋白复合物，我们发现 G0507 与 LolCDE 结合并刺激其 ATP 酶活性。G0507 仍然与 LolCDE 结合，LolCDE 中存在 LolC 的 Q258K 取代（LolC），这赋予了 G0507 的高水平抗性，但不再刺激 ATP 酶活性。我们的工作表明，G0507 作为一种化学探针，具有很大的潜力来剖析革兰氏阴性细菌中脂蛋白的转运。
