Biochemistry of Bacterial Cell Membranes

描述(申请人提供)：病原菌之间不断增加的耐药性是一个主要问题。该项目是一项综合性的尝试，旨在研究细菌为防止抗生素和化疗药物进入细菌细胞内的靶标而使用的两种一般和协同机制。这种药物必须进入细菌并结合到靶标上，才能抑制和杀死细胞。细菌首先建立一层低渗透性的表面膜，从而使这一过程变得困难。一个突出的例子是铜绿假单胞菌或不动杆菌的外膜，具有极低的渗透性通道或孔道。使通道蛋白表现出如此低通透性的分子机制似乎是在这些孔蛋白的特殊折叠过程中，该项目将研究这一过程的细节。另一个例子是分枝杆菌的外膜或细胞壁，它具有独特的脂质成分，这使得脂类药物显然很难进入。该项目将以定量的方式检测该膜的脂成分，以及脂成分对通透性的影响。防止药物外流的第二种机制是主动外排。因此，细菌可以过度生产具有惊人广泛特异性的多药外排泵，以便在药物到达靶点之前主动将药物泵出。该项目将检查大肠埃希菌的AcrB泵，该泵能泵出除氨基糖苷类以外的大多数常用抗生素。它将试图阐明药物分子在大的AcrB转运体中遵循的路径，以及AcrB与其附属蛋白AcrA和TolC在周质和外膜相邻隔室中相互作用的细节。还将研究药物存在对AcrB与AcrA和TolC组装的影响。许多研究将利用共价连接的三聚体形式的AcrB，这样每个组分单体就可以固定在代表运输过程中有限步骤的精确构象中。希望这些研究能够对革兰氏阴性菌和分枝杆菌的药物进入(和排出)过程有一个比较完整和定量的了解，并将有助于设计更好的抗菌剂和更好的外排泵抑制剂。 与公共卫生相关：细菌病原体的抗药性在迅速和持续增加，它们往往对大量药物产生抗药性，有时甚至对大多数可用的药物产生抗药性。这种现象背后的一个主要机制是药物的主动外排(泵出)与外膜通透性障碍相结合，这限制了药物的流入。这项专注于这两种成分的分子机制的研究有望对预防细菌的耐药性做出重大贡献。

DESCRIPTION (provided by applicant): The increasing drug resistance among pathogenic bacteria is a major problem. This project is an integrated attempt to study the two general and synergistic mechanisms that bacteria use in order to prevent the access of antibiotics and chemotherapeutic agents to their targets within the bacterial cell. Such drugs must enter the bacterium and bind to the targets in order to inhibit and kill the cell. This process is made difficult by the bacteria by first building a low permeability surface membrane. An outstanding example is the outer membrane of Pseudomonas aeruginosa or Acinetobacter spp, with their exceptionally low-permeability channels or porins. The molecular mechanisms that make the channel protein to show such a low permeability appear to be in the peculiar folding process of these porins, and the project will examine the details of this process. Another example is the mycobacterial outer membrane or cell wall with its unique lipid composition, which makes the entry of lipophilic drugs apparently quite difficult. The project will examine the lipid composition of this membrane in a quantitative manner, and the effect of the lipid composition on permeability. The second mechanism for preventing drug access is the active efflux. Thus, the bacteria can overproduce multidrug efflux pumps of surprisingly wide specificity to actively pump out drugs before they reach the target. The project will examine the AcrB pump of Escherichia coli, which pumps out most of the commonly used antibiotics, with the sole exception of aminoglycosides. It will try to elucidate the path the drug molecule follows inside the large AcrB transporter, and the details of interaction between AcrB and its accessory proteins AcrA and TolC in the adjacent compartments of periplasm and outer membrane. The effect of the presence of drugs on the assembly of AcrB with AcrA and TolC will also be examined. Many of the studies will utilize a covalently linked trimer version of AcrB, so that each component monomer can be fixed in the precise conformation representing a finite step in the transport process. It is hoped that these studies will result in a rather complete and quantitative understanding of the drug entry (and expulsion) process in gram-negative bacteria and mycobacteria, and will help in the design of better antimicrobial agents and also better inhibitors of the efflux pumps. PUBLIC HEALTH RELEVANCE: There is a rapid and continuing increase in drug resistance among bacterial pathogens, which have often become resistant to a large number of agents, sometimes even to most of the available agents. One major mechanism underlying this phenomenon is the active efflux (pumping out) of drugs coupled with the outer membrane permeability barrier, which limits the influx of drugs. This project focusing on molecular mechanisms of both of these components is hoped to contribute significantly in preventing drug resistance in bacteria.