研究目标及意义：抗生素的不当使用甚至滥用加速了细菌耐药性的发展，并导致了临床上细菌多重耐药性（Multi-drug Resistance, MDR）的产生，俨然成为了威胁人类健康的重大公共卫生问题之一。据2020年临床上对病原菌的分离数据显示，革兰氏阴性菌占比高达71.1 %，远远高于革兰氏阳性菌。同时，革兰氏阴性菌所具备的外膜（Outer Membrane, OM）结构也赋予了其对多种抗生素的低渗透性，使得临床上对革兰氏阴性菌感染的处理要更为棘手。鉴于阴性菌OM上独特的β-桶状外膜蛋白（Outer Membrane Proteins, OMPs）在病原菌的免疫原性、致病性及耐药性等方面发挥至关重要的作用，OMPs目前已成为开发新型抗生素的理想作用靶点之一。然而，由于OMPs的生物发生机制仍不清楚，目前临床上尚没有靶向OMPs的抗生素在使用。因此，亟待开展β-桶状OMPs的功能及其生成演化机理的研究，从而为新型抗生素的开发奠定理论基础。目前，已知β-桶状OMPs的正确组装均依赖于β-桶组装机械（β-Barrel Assembly Machinery, BAM）复合物的功能，但对于该复合物发挥组装功能的确切分子机制，特别是BAM每个蛋白组分在此过程中的具体功能尚知之甚少，缺乏比较直接的生化证据。另外，生物信息学分析表明，不同种属的细菌中BAM复合物的组成差异较大。因此，鉴于BAM复合物功能的高度保守性，有必要研究这些含有不同蛋白组分的BAM复合物在其组成与功能上的潜在联系。为此，对BAM复合物最小功能单元的鉴定以及对BAM脂蛋白功能的解析，构成了阐释BAM复合物组装OMPs的具体分子机制，以及理解BAM复合物在不断进化的过程中对每个蛋白组分需求变化的基础科学要求。

研究方法：本文拟使用一种离体重组系统，包括两个部分：一个是由大肠杆菌（Escherichia coli, E. coli）BAM复合物中不同的纯化蛋白组分（即BamA-E）组合所形成的特定蛋白脂质体，另一个是能够实现模式底物（即E. coli外膜蛋白A（Outer Membrane Protein A, OmpA））过表达及分泌的原生质球系统。在特定的温度条件下，将成功分泌的OmpA多肽链分别与上述不同的特定蛋白脂质体混合以进行OmpA的插膜实验，研究并比较E. coli BAM复合物不同蛋白组分的组合对OmpA插膜情况的影响，以鉴定E. coli BAM复合物的最小功能单元形式，并探究BAM脂蛋白（即BamB-E）的具体功能。同时，利用另一种OMP（即E. coli BAM复合物中的必需蛋白BamA）作为模式底物对上述结果进行验证。此外，对于遗传鉴定的、可在细菌体内无BAM脂蛋白存在的情况下，依旧维持细菌最基本生长的BamA功能性突变体（即BamA^E470K），探究其单独是否具有组装OMPs的功能。

研究结果与结论：本论文证明BAM复合物中的单个蛋白组分（即BamA/B/C/D/E）均无法有效组装底物蛋白OmpA，且BAM复合物中的任意两个蛋白组分的组合（即以BamA为核心的BamAB/BamAC/BamAD/BamAE组合、以BamB为核心的BamBC/BamBD/BamBE组合、以BamC为核心的BamCD/BamCE组合，以及以BamD或BamE为核心的BamDE组合）也无法形成一个有效的BAM功能单元以组装OmpA蛋白。但是，在由BAM复合物三个蛋白组分形成的所有组合中，有且只有BamADE成功且高效地组装了OmpA蛋白，表明BamADE是E. coli BAM复合物的核心组分，构成了E. coli中组装OMPs的最小功能单元。另外，利用BAM复合物四个蛋白组分的组合进行探究并统计学分析后发现，BamADEC对OmpA的组装效率要高于BamADEB，甚至高于BamABCDE。由此，本论文认为在BAM复合物最小功能单元（即BamADE）的基础上，脂蛋白BamB与BamC存在明显的冗余功能，且BamC具有比BamB更好的促进作用。此外，基于BamABCD和BamABCE能有效组装OmpA的事实，本论文认为最合理的解释可能是BamB与BamC之间可以相互协作以形成一个有效的功能单元，从而代替脂蛋白BamD或BamE的相应功能，以保持BAM复合物最小功能单元（即BamADE）的完整性。而且，上述实验结果均利用另外一种底物蛋白（即BamA）得到了验证。最后，本论文对BamA^E470K突变体蛋白进行研究发现，其单独无法有效组装OmpA，同时加入脂蛋白BamD和BamE时，就可以高效组装OmpA蛋白。因此，本论文认为无论是野生型BamA还是突变体BamA^E470K，均需要BamD和BamE的共同协助才能有效组装OMPs，进而维持BAM复合物最小功能单元（即BamADE）的完整性。

Research goals and significance: The improper use or even abuse of antibiotics has accelerated the development of bacterial resistance and led to the emergence of Multi-drug Resistance (MDR) in clinical practice, thereby becoming one of the major public health issues threatening human health. According to the clinical data on the isolation of pathogens in 2020, Gram-negative bacteria accounted for a high proportion of 71.1 %, far higher than Gram-positive bacteria. Moreover, the outer membrane (OM) structure of Gram-negative bacteria also endows them with low permeability to many antibiotics, making the infectious treatment of Gram-negative bacteria in clinic more challenging. Given the crucial roles of β-barrel outer membrane proteins (OMPs) in the immunogenicity, pathogenicity, and drugs resistance of Gram-negative bacteria, OMPs have become one of the the ideal targets for developing new antibiotics. However, due to the unclear mechanism of OMP biogenesis, there is currently no antibiotics targeting OMPs are used in clinical practice. Therefore, it is urgently needed to study the function, biogenesis and evolution mechanism of β-barrel OMPs, which lays the theoretical foundation for the development of new antibiotics. At present, it is well acknowledged that the correct assembly of β-barrel OMPs depends on the function of the β-Barrel Assembly Machinery (BAM) complex. However, the exact molecular mechanism of OMP assembly by the BAM complex, especially the specific function of each BAM protein component in the process are still poorly understood. Furthermore, bioinformatics analysis showed that the composition of the BAM complex varies greatly among different bacterial species. Therefore, given the highly conserved function of the BAM complex, it is necessary to study the potential connection between the composition and function of the BAM complex containing different protein components. To this end, the identification of the minimum functional unit of the BAM complex and the functional exploration of the BAM lipoproteins are the basic scientific requirements for elucidating the specific molecular mechanism of the BAM complex assembling OMPs, as well as understanding the changes in demand for each protein component of the BAM complex in the process of continuous evolution.

Research methods: This paper intends to utilize an in vitro reconstitution system, which consists of two parts: one part is the defined-proteoliposomes composed of different purified protein components (i.e. BamA-E) of the Escherichia coli (E. coli) BAM complex, and the other part is the spheroplast system capable of over-expressing and secreting the model substrate (i.e. E. coli Outer Membrane Protein A (OmpA)). The experiment was performed by mixing the successfully secreted OmpA polypeptide chains with different defined-proteoliposomes mentioned above, respectively, and under the specific temperature to allow OmpA membrane insertion event to occur. The functional circumstance of different protein components of the E. coli BAM complex was investigated and compared to identify the minimum functional unit of the E. coli BAM complex, as well as to explore the specific function of each BAM lipoproteins (i.e. BamB-E). At the same time, another OMP (i.e. the core protein BamA in the E. coli BAM complex) was also used as the model substrate to validate the above results. In addition, the genetically identified the functional mutant of BamA (i.e. BamA^E470K) that can maintain the most basic growth of bacteria in the absence of BAM lipoproteins was investigated to confirm whether it could assemble OMPs alone or not. 

Research results and conclusions: This paper proved that the single protein component of the BAM complex (i.e. BamA/B/C/D/E ) cannot effectively assemble the substrate protein OmpA, and any combination of the dual protein components of the BAM complex (i.e. BamAB/BamAC/BamAD/BamAE centered around BamA, BamBC/BamBD/ BamBE centered around BamB, BamCD/BamCE centered around BamC, and BamDE centered around BamD or BamE) cannot form an effective functional unit of the BAM complex to assemble OmpA protein. However, among all the combinations formed by the triple protein components of the BAM complex, only BamADE assembled OmpA protein successfully and efficiently, indicating that BamADE is the core component of the E. coli BAM complex, and constitutes the minimum functional unit for OMP assembly in E. coli. In addition, it was found that the assembly efficiency of BamADEC for OmpA is higher than BamADEB, and even higher than BamABCDE, after using the combinations of the four protein components of the BAM complex and conducting statistical analysis. Therefore, this paper believed that there is a significant redundancy function between lipoprotein BamB and BamC based on the minimum functional unit (i.e. BamADE), and BamC has a better promoting effect than BamB. Also, based on the fact that BamABCD and BamABCE can effectively assemble OmpA, this paper speculated that the most reasonable explanation is that BamB and BamC can collaborate with each other to form an effective functional unit, replacing the corresponding function of lipoprotein BamD or BamE, to maintain the integrity of the minimum functional unit of the BAM complex (i.e. BamADE). Moreover, the above experimental results were validated using another substrate protein, namely BamA. Finally, this paper investigated the mutant BamA^E470K protein and found that it cannot effectively assemble OmpA alone. However, when lipoproteins BamD and BamE were added simultaneously, OmpA protein can be efficiently assembled. Therefore, this paper suggested that both wild-type BamA and mutant BamA^E470K require the joint assistance of BamD and BamE to effectively assemble OMPs, thereby maintaining the integrity of the minimum functional unit of the BAM complex (i.e. BamADE).