大肠杆菌β桶外膜蛋白生成必需因子BamA的作用机制及其作为抗菌靶点的研究

β桶外膜蛋白广泛分布于革兰氏阴性菌、线粒体和叶绿体的外膜上，发挥多种生物学功能，它们的生成和质量控制需要多个蛋白的协同参与，其中BAM(β-barrel assembly machine)复合物在此过程中发挥核心作用，但其详细作用机制并不清楚。申请人近10年在大肠杆菌外膜蛋白生成领域取得了一些进展；本项目拟在此基础上，深入研究BAM复合物中必需因子BamA在β桶外膜蛋白生成过程中的作用机理。我们将使用新的生化研究手段，如非天然氨基酸随机插入、活细胞光交联等，在活细胞中鉴定BamA和底物蛋白、膜间质分子伴侣SurA相互作用的氨基酸残基；在此基础上，结合BamA的三维结构，设计破坏BamA功能的自抑制多肽，达到杀菌目的。初步实验结果表明，针对BamA结构设计的自抑制多肽具有极强的杀菌活性。本研究不仅有助于加深对细菌外膜蛋白生成分子机制的理解，也将为靶向设计新型抗生素、杀灭耐药菌提供新思路。

β-barrel outer membrane proteins (OMPs) are widely present on the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts, and play important functions in a variety of biological processes. Their biogenesis and quality control involve many protein factors, among which the BAM (β-barrel assembly machine) complex plays a key role but the underlying mechanism is poorly understood. In the past 10 years, the applicant has investigated the biogenesis and quality control of E. coli β-barrel OMPs and made some progress; the present project is designed to study the detailed mechanism of action of BamA, an essential subunit of the BAM complex. We will apply new biochemical research tools (e.g., random insertion of unnatural amino acids and in vivo photo-crosslinking) to identify the amino acid residues that mediate the interaction of BamA with substrate proteins or other key quality control factors (e.g., periplasmic chaperone SurA) in living E. coli cells. This study, in conjunction with the reported three-dimensional structure of BamA in the BAM complex, will guide us to rationally design self-inhibitory peptides that kill E. coli cells by disrupting the structure and function of BamA. Preliminary experimental results indicate that a newly designed peptide targeting BamA has extremely strong bactericidal activity. Our study will advance our understanding of the mechanism of β-barrel OMP biogenesis and may open an avenue for target-based design of new antibiotics for killing antibiotic-resistant pathogens.