Investigating new systems for integrity of the bacterial outer membrane

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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.