Yao Jiangwei, Abdelrahman Yasser M, Robertson Rosanna M, Cox John V, Belland Robert J, White Stephen W, Rock Charles O
From the Departments of Infectious Diseases and.
the Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163, and the Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt.
J Biol Chem. 2014 Aug 8;289(32):22365-76. doi: 10.1074/jbc.M114.584185. Epub 2014 Jun 23.
The major phospholipid classes of the obligate intracellular bacterial parasite Chlamydia trachomatis are the same as its eukaryotic host except that they also contain chlamydia-made branched-chain fatty acids in the 2-position. Genomic analysis predicts that C. trachomatis is capable of type II fatty acid synthesis (FASII). AFN-1252 was deployed as a chemical tool to specifically inhibit the enoyl-acyl carrier protein reductase (FabI) of C. trachomatis to determine whether chlamydial FASII is essential for replication within the host. The C. trachomatis FabI (CtFabI) is a homotetramer and exhibited typical FabI kinetics, and its expression complemented an Escherichia coli fabI(Ts) strain. AFN-1252 inhibited CtFabI by binding to the FabI·NADH complex with an IC50 of 0.9 μM at saturating substrate concentration. The x-ray crystal structure of the CtFabI·NADH·AFN-1252 ternary complex revealed the specific interactions between the drug, protein, and cofactor within the substrate binding site. AFN-1252 treatment of C. trachomatis-infected HeLa cells at any point in the infectious cycle caused a decrease in infectious titers that correlated with a decrease in branched-chain fatty acid biosynthesis. AFN-1252 treatment at the time of infection prevented the first cell division of C. trachomatis, although the cell morphology suggested differentiation into a metabolically active reticulate body. These results demonstrate that FASII activity is essential for C. trachomatis proliferation within its eukaryotic host and validate CtFabI as a therapeutic target against C. trachomatis.
专性胞内寄生细菌沙眼衣原体的主要磷脂类别与其真核宿主相同，只是在2位还含有沙眼衣原体合成的支链脂肪酸。基因组分析预测沙眼衣原体能够进行II型脂肪酸合成（FASII）。AFN - 1252被用作化学工具，特异性抑制沙眼衣原体的烯酰 - 酰基载体蛋白还原酶（FabI），以确定衣原体的FASII对于在宿主内复制是否必不可少。沙眼衣原体FabI（CtFabI）是一种同四聚体，表现出典型的FabI动力学，其表达补充了大肠杆菌fabI（Ts）菌株。在饱和底物浓度下，AFN - 1252通过与FabI·NADH复合物结合抑制CtFabI，IC50为0.9μM。CtFabI·NADH·AFN - 1252三元复合物的X射线晶体结构揭示了药物、蛋白质和辅因子在底物结合位点内的特异性相互作用。在感染周期的任何时间点，用AFN - 1252处理沙眼衣原体感染的HeLa细胞都会导致感染滴度下降，这与支链脂肪酸生物合成的减少相关。在感染时用AFN - 1252处理可阻止沙眼衣原体的第一次细胞分裂，尽管细胞形态表明其分化为代谢活跃的网状体。这些结果表明FASII活性对于沙眼衣原体在其真核宿主内增殖至关重要，并验证了CtFabI作为抗沙眼衣原体治疗靶点的有效性。
