Investigating new systems for integrity of the bacterial outer membrane

许多危及生命的感染是由细菌引起的。这些感染中的大多数是用抗生素治疗的。不幸的是，许多细菌现在对我们使用的常见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。要解决这个问题，我们首先必须了解细菌是如何保护自己免受抗生素的伤害的。所有的细菌细胞都被一层膜包围着，这层膜起到了保护屏障的作用。有些细菌有第二层“外层”膜。这些细菌对我们的抗生素保护得特别好，因此更难治疗。我的工作重点是了解一组名为“ASMA”的蛋白质，它们帮助维持第二层“外膜”。然而，这些蛋白质还没有得到很好的研究。因此，这项工作将为我们提供关于细菌如何建立外膜的新见解，并有可能使我们能够设计针对这一膜的新药，这将有助于我们治疗耐药细菌。细菌利用脂质和蛋白质建立它们的外膜。这些建筑材料必须从牢房内部运输。根据现有的数据，我的假设是ASMA蛋白可能在这种运输中发挥了作用。然而，我们目前尚不清楚1)ASMA蛋白如何作为转运蛋白，2)它们到底运输的是什么，或3)细菌是否在没有ASMA蛋白的情况下生存。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？观察ASMA蛋白的3D结构将使我们了解它们是如何工作的。这很有挑战性，因为这些蛋白质大约比一粒大米小一百万倍。我们将通过使用被称为冷冻电子显微镜的最先进技术来克服这一挑战。我们将把ASMA蛋白放大十万倍，将其成像，并使用复杂的计算机软件来确定其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到底运输的是什么。为此，我将从细菌中分离出ASMA蛋白，并确定附着在细菌上的任何其他分子(例如，脂类、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来去除细菌中的ASMA蛋白。然后，我会比较有没有AsmA蛋白的细菌，问一些问题，比如：没有AsmA蛋白的细菌能生存吗？没有ASMA蛋白的细菌细胞看起来不一样吗？当没有ASMA蛋白时，外膜中的脂质/蛋白质会发生变化吗？没有ASMA蛋白的细菌对抗生素的耐药性会降低吗？总体而言，这项研究将为ASMA蛋白的工作方式提供令人兴奋的新见解，帮助我们理解细菌如何建立外膜，并保护自己免受药物的伤害。这可能导致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.