Role of peptidoglycan-active enzymes in the assembly of bacterial motility, secretion and efflux systems

肽聚糖（PG）是细菌细胞壁的主要结构成分，但在高等生物中不存在。它是一种坚固的链环栅栏状材料，孔径约为100 kDa。它完全包围着细菌，提供保护，防止内部高压，并有助于形成独特的细菌细胞形状。尽管PG对细菌生存至关重要，并且在几十年前就被确定为最早的抗生素的靶标，但其合成的分子细节仅被部分理解。在某种程度上，这种缺乏理解的原因是大多数细菌具有多种非必需的PG活性酶，这些酶具有看似冗余的活性，这使得将特定功能分配给单个蛋白质具有挑战性。青霉素等抑制PG合成的药物通常同时靶向多种酶，因此很难确定是否一种或多种蛋白质的丢失导致了观察到的杀伤效果。在物理方面，PG是一个单一的，巨大的共价连接聚合物，很难在理解单个蛋白质的贡献所需的分辨率水平上进行表征。我们需要知道的事情包括单个蛋白质的作用，它们在何时何地起作用，以及它们的活动如何影响通过PG层到达细胞外部的大型蛋白质复合物的组装。这些复合物包括那些参与运动、蛋白质分泌和有毒化合物排出的关键过程的复合物。利用缺乏单一PG活性酶的细菌，我们进行了一项长期研究计划，以了解PG代谢和结构的控制如何与细胞壁跨越蛋白复合物的组装相交叉。通过表型筛选，我们已经确定了一种酰胺酶和一种PBP，它们是运动和分泌系统正常组装所必需的；以及一种不同的PBP和一种裂解转糖基酶，它们充当细胞壁损伤的哨兵，并在药物外排泵的组装中发挥作用，这两个过程都会导致抗生素耐药性。我们的研究将带来克服抗生素耐药性的新策略，这是一个日益严重的问题，威胁着加拿大人的健康，因为菌株对所有已知的抗生素都具有耐药性。

Peptidoglycan (PG) is a major structural component of bacterial cell walls but is absent in higher organisms. It is a strong, chain link fence-like material with a pore size of ~100 kDa. It completely surrounds bacteria, providing protection against high internal pressures and contributing to the formation of distinct bacterial cell shapes. Although PG is essential for bacterial viability and was identified decades ago as the target of the earliest antibiotics, the molecular details of its synthesis are only partly understood. In part, this lack of understanding has arisen because most bacteria have multiple, non-essential PG active enzymes with seemingly redundant activities, making it challenging to assign specific functions to individual proteins. Drugs such as penicillin that inhibit PG synthesis often target multiple enzymes simultaneously, making it hard to determine if loss of one or more proteins is responsible for the observed killing effects. On the physical side, PG is a single, gigantic covalently linked polymer that has been difficult to characterize at the level of resolution needed to understand the contribution of individual proteins. Among the things we need to know are what individual proteins do, when and where they do it, and how their activities affect the assembly of large protein complexes that pass through the PG layer to the outside of the cell. Such complexes include those involved in the key processes of motility, protein secretion and efflux of toxic compounds. Using bacteria lacking single PG-active enzymes, we have undertaken a long term research program to understand how the control of PG metabolism and structure intersects with assembly of cell-wall-spanning protein complexes. Through phenotypic screens, we have identified an amidase and a PBP that are required for normal assembly of motility and secretion systems; and a different PBP and a lytic transglycosylase that act as sentinels of cell wall damage as well as having roles in the assembly of drug efflux pumps, both processes that lead to antibiotic resistance. Our research will lead to new strategies to overcome antibiotic resistance, an increasingly serious problem threatening the health of Canadians, as strains resistant to all known antibiotics are already here.