Investigating new systems for integrity of the bacterial outer membrane

许多危及生命的感染都是由细菌引起的。这些感染大多用抗生素治疗。不幸的是，现在许多细菌对我们常用的抗生素产生了抗药性。这是一个全球性问题，每年造成数十万人死亡。为了解决这个问题，我们必须首先了解细菌如何保护自己免受抗生素的侵害。所有的细菌细胞都被一层膜包围着，这层膜起着保护屏障的作用。有些细菌有第二层“外”膜。这些细菌尤其不受抗生素的影响，因此更难治疗。我的工作重点是理解一组叫做“AsmA”的蛋白质，它有助于维持第二层“外”膜。然而，这些蛋白质尚未得到充分的研究。因此，这项工作将为我们提供关于细菌如何构建外膜的新见解，并有可能使我们能够设计针对该膜的新药，这将有助于我们治疗耐抗生素细菌。细菌利用脂质和蛋白质构建外膜。这些建筑材料必须从细胞内部运输。根据现有数据，我的假设是AsmA蛋白可能在这种运输中发挥作用。然而，我们目前还不了解1)AsmA蛋白如何作为转运蛋白，2)它们究竟在运输什么，或者3)细菌是否在没有AsmA蛋白的情况下存活。为了解决这个问题，我想回答以下问题：1)这些蛋白质是什么样子的？看到AsmA蛋白的三维结构将使我们了解它们是如何工作的。这很有挑战性，因为这些蛋白质比一粒米要小一百万倍。我们将利用一种叫做低温电子显微镜的先进技术来克服这一挑战。我们将把AsmA蛋白放大10万倍，成像，并使用复杂的计算机软件计算出它们的3D结构。2) AsmA蛋白与哪些分子相互作用？了解AsmA蛋白与哪些分子相互作用将使我们能够确定它们究竟在运输什么。为此，我将从细菌中分离出AsmA蛋白，并识别附着在其上的任何其他分子（例如，脂质、蛋白质等）。3)没有AsmA蛋白的细菌表现如何？我将使用成熟的遗传学方法从细菌中去除AsmA蛋白。然后，我将比较有和没有AsmA蛋白的细菌，并提出这样的问题：细菌能在没有AsmA蛋白的情况下存活吗？没有AsmA蛋白的细菌细胞看起来不同吗？当没有AsmA蛋白时，外膜中的脂质/蛋白会发生变化吗？没有AsmA蛋白的细菌对抗生素的耐药性会降低吗？总的来说，这项研究将为AsmA蛋白的工作原理提供令人兴奋的新见解，帮助我们了解细菌如何构建外膜并保护自己免受药物的侵害。这可能会带来治疗危及生命的细菌感染的新方法。

Many life-threatening infections are caused by bacteria. Most of these infections are treated with antibiotics. Unfortunately, many bacteria are now becoming resistant to the common antibiotics that we use. This is a global problem, causing hundreds of thousands of deaths each year. To solve this issue, we must first understand how bacteria protect themselves from antibiotics.All bacterial cells are surrounded by a membrane, which acts as a protective barrier. Some bacteria have a second, 'outer', membrane. These bacteria are especially well-protected from our antibiotics and are therefore harder to treat. My work focuses on understanding a set of proteins called 'AsmA' proteins which help maintain this second, 'outer', membrane. However, these proteins have not yet been well-studied. Therefore, this work will provide us new insights into how bacteria build the outer membrane, and potentially enable us to design new drugs targeting this membrane that will help us to treat antibiotic-resistant bacteria.Bacteria build their outer membrane using lipids and proteins. These building materials must be transported from inside the cell. Based on existing data, my hypothesis is that AsmA proteins may play a role in this transport. However, we do not currently understand 1) how AsmA proteins may act as transporters, 2) what exactly they are transporting, or 3) whether bacteria survive without AsmA proteins. To address this, I want to answer the following questions:1) What do these proteins look like? Seeing the 3D structure of AsmA proteins will allow us to understand how they work. This is challenging, because these proteins are about one million times smaller than a grain of rice. We will overcome this challenge by using a state-of-the-art technique called cryo- electron microscopy. We will magnify the AsmA proteins one hundred thousand times, image them, and use sophisticated computer software to figure out their 3D structure.2) Which molecules do AsmA proteins interact with? Understanding which molecules AsmA proteins interact with will allow us to determine what exactly they are transporting. To do this, I will isolate AsmA proteins from bacteria and identify any other molecules that are attached to (e.g., lipids, proteins etc.).3) How do bacteria without AsmA proteins behave? I will use well-established genetics methods to remove AsmA proteins from bacteria. I will then compare bacteria with and without AsmA proteins asking questions such as: Can bacteria survive without AsmA proteins? Do bacterial cells without AsmA proteins look different? Do the lipids/proteins in the outer membrane change when there are no AsmA proteins? Do bacteria without AsmA proteins become less resistant to antibiotics?Overall, this study will give exciting new insights into how AsmA proteins work, helping us understand how bacteria build their outer membrane and protect themselves against drugs. This could lead to new methods for treatment of life-threatening bacterial infections.