Structural analysis of the inter-membrane bacterial secretosome

蛋白质分泌是生命所必需的。细菌分泌蛋白质用于广泛的膜和细胞外活动，包括包膜生物发生、致病性和抗生素降解。这一过程的主要途径是通过细菌质膜的普遍存在的Sec机制。您的项目将关注这一过程的机制，以及随后通过细菌包膜的下游转运，以及最终革兰氏阴性菌外膜的生物发生。革兰氏阴性菌具有由周质组成的细胞壁，周质具有肽聚糖（PG）层，被外膜包围。细菌细胞如何确保分泌蛋白质的快速和特异性分选，以便折叠到周质中，或递送到外膜，所有这些都是在缺乏能量的情况下完成的？ATP驱动的Sec机制跨内膜运输的机制相对较好地理解。质量控制系统到位，以确保折叠或，如果需要的话，降解驻留周质蛋白。然而，外膜蛋白到β-桶装配机器（BAM）的途径不太清楚。我们已经确定了细菌全转位子（HTL）与周质伴侣和BAM之间的相互作用，形成一个跨越整个细胞包膜的结构。这个巨大的集合体--细菌分泌体--可以形成一个连续的管道，使蛋白质从胞质溶胶有效地传递到外膜。它的存在将对我们理解外膜生物发生产生深远的影响[见我们实验室最近的预印本：Alvira et al. 2019] https：//doi.org/10.1101/589077]。由于细胞壁容易受到攻击，因此它是许多抗生素的靶标，例如β-内酰胺类抗生素。显然，Sec和Bam机制之间的连接对于靶向是普遍的;这种相互作用的破坏将干扰生存所必需的细胞壁生物合成，因此具有所需的抗生素活性。该项目将利用互补技术来探索内膜和外膜的translocons的相互作用。因此，该项目将提供各种生物化学和生物物理技术的培训机会，包括高分辨率电子冷冻显微镜和X线断层摄影术。此外，由于这是一个意想不到的和令人兴奋的细菌生物学新领域，你将有很好的发现前景。这可以通过探索分泌机制的结构和功能的基本特征，以及开发药物开发的新策略来实现。

Protein secretion is essential for life. Bacteria secrete proteins for a wide range of membrane and extracellular activities, including envelope biogenesis, pathogenicity and degradation of antibiotics. The major route for this process is via the ubiquitous Sec machinery of the bacterial plasma membrane. Your project would concern the mechanism of this process and subsequent poorly understood downstream transit through the bacterial envelope, and ultimately the biogenesis of the Gram-negative outer- membrane.Gram-negative bacteria possess a cell wall composed of a periplasm with a peptidoglycan (PG) layer, surrounded by an outer-membrane. How does the bacterial cell ensure rapid and specific sorting of secreted protein for folding into the periplasm, or delivery to the outer- membrane, all done in the absence of energy? The mechanism of ATP driven transport across the inner-membrane by the Sec machinery is relatively well understood. Quality control systems are in place to ensure folding or, if required, degradation of resident periplasmic proteins. However, the route for outer- membrane proteins to the beta-barrel assembly machinery (BAM) is less clear. We have identified an interaction between the bacterial holo-translocon (HTL) with a periplasmic chaperone and BAM, forming a structure that spans the entirety of the cell envelope. This giant assembly -the bacterial secretosome- could form a contiguous conduit for efficient passage of proteins from the cytosol to the outer- membrane. Its existence will have far reaching implications for our understanding of outer-membrane biogenesis [see recent pre-print from our lab: Alvira et al. 2019]https://doi.org/10.1101/589077]. Given that the cell wall is prone to attack, it is a target for many antibiotics, such as the -lactams. Obviously then, the connection between the Sec and Bam machineries is rife for targeting; subversion of this interaction will disturb cell wall biogenesis essential for survival, and hence have the desired antibiotic activity. The project will harness complementary technologies to explore the interplay of the translocons of the inner and outer membranes. Therefore, the project will offer training opportunities in a wide range of biochemical and biophysical techniques, including high-resolution electron cryo-microscopy (cryo-EM) and - tomography (cryo-ET). Moreover, as this is an unexpected and exciting new area of bacterial biology, you will have very good prospects for discovery. This could be through the exploration of fundamental features of the structure and function of the secretory machinery, as well as for the development of new strategies for drug development.